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In this issue

M
ore than 1,400 bacterial, viral and parasitic species are
known to cause human disease. Understanding how these
pathogens colonize, survive and inflict disease remains
one of the key goals of microbiology. This, the first issue
of 2008, features several Reviews that discuss microbial virulence.
On page 79, John Boothroyd and Jean-Francois Dubremetz discuss
the specialized rhoptry organelle, which exists in apicomplexan parasites
such as Toxoplasma gondii. In T. gondii, recent findings indicate that these
organelles contain the molecular machinery that enables the parasite to
affect host gene expression and co-opt host functions.
▶ cover: ‘A to B’ by George Marshall, inspired by Dealing with bacteria, Samuel Miller and colleagues discuss the
the Review on p28.
mechanisms that enable Salmonellae to interact with, and manipulate, host
cells (page 53). Specifically, they focus on the interplay between various
bacterial and host proteins that enables the invasion of epithelial cells,
stimulation and repression of signalling cascades, sensing of the intracellular
environment and establishment of a niche for intracellular replication.
So, how can we harness our understanding of microbial virulence to stem
david o’connell susan jones the continuing rise in infectious-disease-related morbidity and mortality?
Antibiotic-based strategies, which typically target bacterial viability, have
resulted in widespread resistance — meticillin-resistant Staphylococcus
aureus infections reached epidemic levels last autumn in some parts of the
United States. Perhaps rather than targeting bacteria for eradication, we
sheilagh molloy sharon Ahmad
should focus instead on de-clawing pathogens by inhibiting the virulence
mechanisms that promote infection or that cause disease symptoms. On
page 17, Scott Hultgren and colleagues highlight various bacterial virulence
mechanisms that could be targeted and consider the recent efforts towards,
and the remaining challenges that face, antivirulence-based drug discovery.

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nature reviews | microbiology volume 6 | january 2008 | 


© 2008 Nature Publishing Group
Editorial

The value of vaccines


Our ability to control infectious diseases is continuously being eroded by antimicrobial
resistance, the decline in industrial antibiotic development and increasing development
timelines from discovery to market. There has never been a better time to rediscover the
value of vaccination.

As a preventative strategy in the fight against infectious higher prices in developed countries and lower prices in
diseases, vaccination is considered to be the most cost- under-developed countries. It should also be possible to
effective medical intervention. Indeed, in many devel- decrease liability risks (by measures such as the Vaccine
oped countries, measles, mumps, rubella, hepatitis B, Injury Compensation Program in the United States) and
Haemophilus influenzae type b, tetanus, pertussis and protect manufacturers from lawsuits that are related to
diphtheria have been controlled or nearly eliminated the unanticipated adverse effects of a properly manufac-
as chief causes of morbidity and mortality by the use of tured, safe and effective vaccine. Ultimately, however, an
vaccines. Yet, despite their role in delivering many of the economic solution is required. There have been numer-
successes that have been achieved in infectious disease ous demonstrations of the cost-effectiveness of immuni-
control, there seems to be relatively little enthusiasm zation. Attributing the true economic value to vaccines
for vaccine development among those who have the — both real and intangible — will create a self-sustaining
capability for vaccine development and production. system to ensure the development and adequate supply
There are a number of contributing factors to this lack of vaccines to those that need them most.
of enthusiasm, most of which are linked to economics. But how can governments be mobilized into action?
The simple truth is that the direct economic value that is If the ever-increasing threat of a public health calamity
associated with vaccines is negligible compared with that does not provide the necessary incentive, the promise of
of pharmaceutical drugs. Globally, potential vaccine sales success might do the trick. An example that illustrates the
are approximately US$6 billion per year. Although the effectiveness of a global approach to vaccination is the
vaccines sector is out-performing the rest of the industry campaign for the eradication of poliomyelitis. Launched
in terms of revenue growth, it still represents less than in 1985 for South America, it was taken up by the World
2% of the worldwide pharmaceutical market. Because of Assembly, which in 1988 committed to the global eradi-
the low return for their investment, high regulatory costs, cation of poliovirus by the year 2000. Progress towards
uncertain market conditions and exposure to legal liabil- eradication of the virus has been fast. In 1988, 125 coun-
ity, most pharmaceutical manufacturers do not consider tries in 5 continents reported endemic poliovirus but by
the development and production of vaccines as an attrac- 2003 only 6 polio-endemic countries were reported and,
tive business opportunity. For example, over the past officially, only 4 remain: India, Pakistan, Afghanistan and
30 years, the number of companies that distribute vaccines Nigeria. So, although the 2000 deadline passed with the
in the United States has decreased from 30 to 5, a situa- incidence of disease stalled at around 2,000 cases per year,
tion that has directly contributed to serious shortages of and there have been setbacks, including a recent outbreak
influenza, tetanus–diphtheria, measles–mumps–rubella, in Nigeria, the initiative is an unequivocal example of
pneumococcal, meningococcal and other vaccines. Not how a globally coordinated effort can, within a short
surprisingly, there is even less incentive for the vaccine period of time, reduce the incidence of an infectious dis-
the direct industry to develop new vaccines against diseases that ease by more than 99.9%. The elimination of the disease
economic value are largely limited to the developing world. from the remaining endemic regions is now achievable,
that is associated Governments do have a number of well-documented leaving the welcome problem of whether, and how long,
and under-used tools at their disposal to make vaccines vaccination should be continued against a disease that
with vaccines more attractive to industry. These options include: tax no longer exists.
is negligible breaks or subsidies to reduce research and development In their efforts to control infectious diseases, govern-
compared expenses; extending patent protection for intellectual ments and non-governmental organizations need look
with that of property that is related to vaccines of public health impor- no further than the poliomyelitis eradication campaign,
tance; working with industry and others to find ways to both for their inspiration and justification in taking that
pharmaceutical reduce the costs of meeting regulatory requirements; and first important step — placing vaccination back where it
drugs. allowing tiered pricing whereby vaccines can be sold at belongs, at the top of the global public health agenda.

 | january 2008 | volume 6 www.nature.com/reviews/micro


© 2008 Nature Publishing Group
Research highlights

hi v of primary infection. Analysing more


than one region of the HIV-1 genomes

Infection, superinfection
allowed the authors to detect cases in
which the initial and superinfecting
viruses had recombined, a frequent

and (lack of) protection event during HIV-1 replication. By


analysing the proviral sequences from
36 women infected with HIV-1, seven
Superinfection with HIV-1 occurs of the timing of re-exposure and the cases of superinfection were detected,
when an individual who is already relatedness of the virus strains. including 3 cases in which both
infected with one strain of HIV-1 Prior to this study, approximately …this lack viruses belonged to the same HIV-1
acquires a second strain from twenty cases of HIV-1 superinfection of protection subtype; this finding indicates that
a different partner. Because a had been reported in the literature, is largely closely related viruses do not confer
re-infection event suggests that which suggested that natural infec- protection. The authors also showed
independent of
the immune response generated tion does not always generate a pro- that in 5 of the 7 cases, infection with
against the original infection is tective immune response. However, the timing of re- the second virus occurred over 1 year
not sufficient to protect against if researchers are to accurately assess exposure and the after the initial infection, and, in some
later exposures, there are obvious the impact of superinfection on the relatedness of the cases, 5 years after initial infection. In
implications for HIV-1 vaccine success of an HIV-1 vaccine, ques- virus strains. other words, superinfection occurred
design. However, despite the tions need to be answered about after the host immune response to
potential importance of superinfec- the frequency of superinfection and the first HIV-1 strain should have had
tion in HIV disease and vaccine whether this process is restricted time to develop and mature.
development, there is uncertainty to the times in infection when an This study clearly demonstrates
regarding its incidence and timing. HIV‑1-specific immune response has that HIV-positive individuals are at
Now, an in-depth, population-level not yet developed. continued risk of acquiring a second
assessment of HIV-1 superinfection, To address these issues, Piantadosi HIV infection. Indeed, if the lack
published in PLoS Pathogens, con- and colleagues screened a cohort of of protection against re-infection
firms that natural HIV-1 infection high-risk Kenyan women for HIV-1 is shared by at least a proportion
does not always elicit a protective superinfection by comparing 2 partial of all HIV-infected individuals, the
immune response, and that this lack gene sequences (gag and envelope) over potential value of an HIV-1 vaccine
of protection is largely independent a 5-year period, beginning at the time that attempts to mimic the immune
response to natural infection is called
into question. Follow-up studies to
compare the immune responses of
those individuals that become super-
infected with those that do not, and
CTL deciphering the contribution of these
NtAb responses to immune protection, will
HIV-1 RNA be the focus of future research.
David O’Connell

0 1 2 3 4 5 ORIGINAL RESEARCH PAPER Piantadosi, A.


Time (years post-infection) et al. Chronic HIV-1 infection frequently fails to
Superinfection with an HIV-1 virus from a second partner (pink) can occur several years after an initial HIV-1 infection (green). At this time protect against superinfection. PLoS Pathog. 3,
(see arrow), cytotoxic T-cell responses (CTL) and neutralizing antibody responses (NTAb) to the first infection have had time to develop. e177 (2007)
Figure redrawn, with permission, from one kindly provided by Julie Overbaugh, Fred Hutchinson CancerNatureResearchReviews | Microbiology
Center, Washington, USA.

nature reviews | microbiology volume 6 | january 2008


© 2008 Nature Publishing Group
Research highlights

MicroBIAL Physiology

How low can you grow?


Removal of the greenhouse gas
methane by methanotrophic bacteria
is an important climatic process. All
methanotrophs characterized so far
are meso- or thermophilic members
of the phylum Proteobacteria. Now,
two papers published in Nature
report the cultivation, characteriza-
tion and draft genome analysis of
two new methanotrophs from the
phylum Verrucomicrobia that are
the most acidophilic methanotrophs
ever studied.
Some environments that are
rich in methane are also extremely
acidic, so two groups of researchers
set out to investigate whether novel
methanotrophs thrive in these In a parallel study, Dunfield to three similar pmoCAB operons.
conditions. PCR primer sets for et al. analysed 16S rRNA genes of Phylogenetic analyses of the three
the conserved pmoA gene, which bacteria in a sample from Hell’s Gate, pmoA alleles encoded by V4 and
encodes a subunit of the particulate an acidic geothermal site in New those encoded by SolV indicate that
form of methane monooxygenase, Zealand that is rich in methane, and these acidophilic lineages diverged
have been used routinely to sur- identified novel Verrucomicrobia from Proteobacteria a long time
vey numerous environments for sequence signatures that were ago, and exclude the possiblity
methanotrophs. Pol et al. extracted unrelated to any cultured bacteria. that Verrucomicrobia species have
DNA from a hot, acidic, methane- A strain named Verrucomicrobia acquired the pmoA gene by a recent
producing fumarole in the Solfatara, isolate V4 (provisional name horizontal-gene-transfer event.
Italy, and used primers for pmoA to Methylokorus infernorum) was Inspection of the genomes indicated
construct an environmental clone isolated into pure culture from this that both species might also use novel
library. They found two classes sample by supplying methane as the methylotrophic pathways.
of pmoA sequences in the library sole carbon and energy source. Both These studies have provided
— gammaproteobacterial-like SolV and V4 have one striking phe- an important step forward in the
sequences and a divergent set of notype: V4 can grow at pH 1.0 and understanding of the physiology and
pmoA homologues. Intrigued by SolV can grow at pH 0.8. This is the diversity of methane consumption by
these unusual pmoA sequences, first time that extremely acidophilic bacteria in natural environments.
they isolated a strain into pure methanotrophs have been described. Susan Jones
culture that had a divergent pmoA Draft genomes of both new species
sequence that was similar to those were assembled and scrutinized for ORIGINAL RESEARCH PAPERS Pol, A. et al.
present in the clone library and insights into their physiologies; both Methanotrophy below pH1 by a new
a 16S rRNA sequence that was are remarkable in that they have three Verrucomicrobia species. Nature 14 Nov 2007
(doi 10.1038/nature06222) | Dunfield, P. F. et al.
typical of Verrucomicrobia, and pmoCAB operons that are completely Methane oxidation by an extremely acidophilic
named it SolV (provisional name different. Most proteobacterial bacterium of the phylum Verrucomicrobia.
Nature 14 Nov 2007 (doi 10.1038/nature06411)
Acidimethylosilex fumarolicum). methanotrophs typically encode up

nature reviews | microbiology volume 6 | january 2008


© 2008 Nature Publishing Group
Research highlights

RNA

Extending the network of sRNA control


Mechanisms by which the translation by occlusion of the
small non-coding RNAs ribosome. Surprisingly, GcvB
(sRNAs) of Gram-negative binds to sequences upstream
bacteria regulate multiple of the RBS of other regulated
mRNA targets have proved genes, but still prevents the 30S
difficult to pin down, owing ribosome from docking onto the
to the limited complementarity mRNA to initiate translation.
between sRNAs and target mRNAs. Fusion of the 5′-untranslated
In a paper published in Genes & region of gltI, which contains
Development, Jörg Vogel and the GcvB-docking site,
colleagues report that GcvB, a with the unrelated E. coli
200-nucleotide sRNA, directly ompR gene conferred GcvB-
regulates 7 mRNAs that encode mediated repression to ompR,
periplasmic peptide and amino- proving that GcvB can regulate any
acid transport proteins in mRNA that contains a cognate C-
Salmonella enterica serovar and A‑rich R1-binding motif. The
Typhimurium (S. typhimurium). precise mechanism of this
The GcvB sRNA represses novel type of translational
translation of the dppA and oppA pep- control remains unclear.
tide transporter genes in nutrient-rich Other studies have shown
conditions. Intriguingly, gcvB mutants that an sRNA (514 nucleotides)
are pleiotropic, and computational periplasmic-protein profiles of an named RNAIII functions to
analyses indicated that GcvB might S. typhimurium strain that is over- repress multiple virulence factors
have numerous target genes, which expressing gcvB with an in silico in the Gram-positive pathogen
led Vogel and colleagues to investigate screen for binding partners of R1, Staphylococcus aureus by comple-
how the GcvB sRNA functions. Vogel and co-workers identifed five mentary base-pairing with the RBS
RNA-structure probing pinpointed a additional candidate GcvB targets of target-gene mRNAs. Together,
29-nucleotide G- and U-rich linker that encode periplasmic amino-acid these studies show that specific
region in GcvB, named R1, that transport proteins — gltI, livJ, livK, interactions between sRNAs and
interacts with sequences that span the argT and STM4351. The predicted mRNAs in both Gram-positive
ribosome binding site (RBS) of both GcvB-binding sites in these genes and Gram-negative bacteria can
peptide transporter genes. Strikingly, were not conserved, but were rich in regulate multiple genes.
deletion of R1 destroyed the ability of C and A residues. Susan Jones
GcvB to repress dppA and oppA. Biochemical assays revealed that
Bioinformatic searches revealed GcvB functions as a sequence-specific ORIGINAL RESEARCH PAPER Sharma, C. M.,
that R1 is well-conserved among inhibitor of translation, but the details Darfeuille, F., Plantinga, T. H. & Vogel, J.
A small RNA regulates multiple ABC transporter
the gcvB genes of Pasteurella spp., of inhibition vary among the different mRNAs by targeting C/A-rich elements inside
Vibrio spp. and many enterobacte- target mRNAs. GcvB binds to the RBS and upstream of ribosome-binding sites.
ria. By combining scrutiny of the of dppA and oppA mRNAs to repress Genes Dev. 21, 2804–2817 (2007)

nature reviews | microbiology volume 6 | january 2008


© 2008 Nature Publishing Group
Research highlights

Host response

IgA — peacemaker in the gut

Using a novel gnotobiotic mouse thetaiotaomicron, which is a promi- adapted to this response by induc-
model in which the diverse gut nent, obligately anaerobic, Gram- ing genes that are involved in the
microbiota is reduced to a single negative member of the human metabolism of oxidative products
bacterial species, and the antibody distal intestinal ecosystem that also of the host response. The presence
repertoire to a single monoclonal efficiently colonizes the intestines of of IgA, however, reduced intestinal
immunoglobulin A (IgA) antibody adult germ-free C57BL/6J mice. pro-inflammatory signalling (by
that is directed against the bacteri- B. thetaiotaomicron was introduced downregulating signal transducer
um’s capsular polysaccharide, Jeffrey into germ-free, recombination- and activator of transcription 3 and
Gordon and colleagues provide activating-gene-1-deficient (Rag1–/–) interferon regulatory factor 8, for
evidence that supports a role for IgA mice (which lack mature B and T example), and bacterial epitope
in the gut as a mediator of tolerance. cells) or germ-free Rag1–/– mice that expression.
The IgA antibody response has a had been injected under their dorsal Therefore, these results suggest
key role in establishing and maintain- skin with B. thetaiotaomicron-primed that it is the IgA antibody response
ing a non-inflammatory relationship IgA-producing hybridoma cells. that establishes a quiescent relation-
between the host and microbiota An inverse relationship was found ship between B. thetaiotaomicron
in the gut. Germ-free mice that are between IgA antibody levels and and its host. They are also consistent
colonized with normal gut microbiota the levels of its B. thetaiotaomicron with a model in which a set of adap-
develop bacteria-specific IgA antibody epitope in the intestinal lumen tations that involve both the symbi-
responses, but the effects of these — that is, infected mice with IgA ont and its host lead to co-evolved
responses on the biology of the host secreted by the hybridoma cells homeostasis.
and microbiota are not well defined. had lower levels of epitope expres- Sharon Ahmad
Peterson et al. developed a gnotobiotic sion than mice without IgA. In the ORIGINAL RESEARCH PAPER Peterson, D. A.
mouse model to study these effects. absence of IgA, B. thetaiotaomicron et al. IgA response to symbiotic bacteria as a
As their model symbiont, they elicited a more robust innate mediator of gut homeostasis. Cell Host Microbe
2, 328–339 (2007)
chose the bacterium Bacteroides immune oxidative response, and

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© 2008 Nature Publishing Group
Research highlights

in brief
b a c t e r i a l pat h o g e n e s i s
Invasive and adherent bacterial pathogens co-opt
host clathrin for infection
Veiga, E. et al. Cell Host & Microbe 2, 340–351 (2007)
Although many viruses enter host cells by clathrin-mediated
endocytosis, it was thought that clathrin-coated vesicles were
too small to internalize bacteria. So, it was a surprise when
Listeria monocytogenes was shown to enter host cells by a
clathrin-dependent mechanism. Building on this previously
published finding, Veiga et al. investigated whether clathrin
is required for the entry of other pathogens. They found
that bacteria that enter cells after interactions with specific
receptors, such as Staphylococcus aureus, require clathrin for
entry, whereas those that inject effectors into host cells to
facilitate their own entry by altering the host cytoskeleton,
such as Shigella flexneri, do not. Clathrin was also required
to assemble pedestals beneath adherent enteropathogenic
Escherichia coli, which remain extracellular.

molecular ecology
Mutational activation of niche-specific genes provides
insight into regulatory networks and bacterial
function in a complex environment
Giddens, S. R. et al. Proc. Natl Acad. Sci. USA 104, 18247–18252 (2007)
The identification of environment-induced loci (EIL) is hampered
by a lack of discernable phenotypes in the laboratory when EIL
are mutated. Giddens et al. describe a new, broadly applicable
method — SpyVet (suppressor analysis with in vivo expression
technology) — to identify regulatory loci that control EIL in the
rhizosphere bacterium Pseudomonas fluorescens. Promoters of
EIL are fused to dapB (required for lysine biosynthesis), rendering
strains prototrophic in the rhizosphere, where EIL are expressed,
but auxotrophic in minimal growth media, where EIL are not
expressed. Transposon mutagenesis, coupled with a simple
screen for prototrophy in the laboratory, can quickly identify
genes that regulate EIL–dapB fusions. A secondary screen for
other members of the regulatory hierarchy was also feasible,
which enabled these researchers to identify a regulatory
network of seven regulators.

b ac t e r i a l s e c r e t i o n
A minimal Tat system from a Gram-positive organism:
a bifunctional TatA subunit participates in discrete
TatAC and TatA complexes
Barnett, J. P. et al. J. Biol. Chem. 26 Nov 2007 (doi 10.1074/jbc.
M708134200)
The Tat (twin-arginine) secretion system transports fully folded
proteins across bacterial membranes. Gram-negative bacteria
encode three subunits (TatABC) that are essential for secretion.
TatABC form a substrate-binding complex that is thought to
recruit a separate and heterogeneous TatA complex, which can
form a translocation pore. Bacillus subtilis has three TatA and
two TatC variants. However, it lacks tatB, as do all Gram-positive
bacteria. The B. subtilis tatAd gene complemented a tatB mutant
of Escherichia coli, indicating that tatAd can replace TatA and
TatB. Importantly, a B. subtilis TatAdCd system secreted a Tat
substrate upon expression in E. coli, even though the TatAd
complex is discrete, in contrast to the heterogeneous E. coli TatA
complex. Direct experiments in B. subtilis are now needed to
resolve the current controversy over how Tat secretes proteins.

nature reviews | microbiology volume 6 | january 2008


© 2008 Nature Publishing Group
Research highlights

b ac t erial d e v elop m en t

Moving in the right direction


New data published in a recent issue the second model discussed
of Molecular Microbiology might above. However, analysis
have finally solved the controversy of translocation-defective
over the function of SpoIIIE dur- SpoIIIE showed that,
ing sporulation in Bacillus subtilis. in the absence of DNA
During sporulation, B. subtilis translocation, SpoIIIE
undergoes an asymmetric cell complexes formed on
division that generates two cells of both sides of the septum,
unequal size — a small forespore supporting the first
and a larger mother cell — and a model.
chromosome must be accurately To resolve this
segregated into each cell. The asym- conundrum, Becker and
metric-division septum is formed Pogliano moved on to look
before chromosome segregation is at the effects of chromosome
completed, pinching one of the repli- orientation. In a B. subtilis strain
cated chromosomes into a small lobe, in which the chromosome par-
which constitutes ~30% of the genome titioning proteins Soj and Spo0J
and is present in the forespore, and had been deleted, the SpoIIIE
a large lobe, which constitutes ~70% complex could assemble in, and
of the genome and is present in the move DNA out of, the forespore.
mother cell. To ensure successful translocation Finally, using a range of time-lapse
completion of chromosome segrega- complex. In this fluorescence microscopy studies the
tion, the trapped large lobe must be work, Eric Becker and Kit Pogliano authors were able to show that there
translocated across the septum from set out to determine which of these was a direct correlation between the
the mother cell into the forespore. models is correct. polarity of SpoIIIE-mediated chro-
Chromosome segregation during They began by devising a method mosome segregation and the position
sporulation requires the double- that would allow them to tag SpoIIIE of oriC after septation — SpoIIIE
stranded DNA translocase SpoIIIE, specifically in the mother cell and the moves the chromosome into the cell
a protein that is related to the FtsK forespore. To do so, the authors made that contains the replication origin.
translocase of non-spore-forming use of the high-affinity interaction So, it seems that aspects of both
bacteria. In recent years, results from between the leucine zipper domains models of SpoIIIE/FtsK function
different laboratories have supported of cFos and cJun — the leucine zip- were correct — the assembly of a
two models of SpoIIIE/FtsK func- per domain of cFos was fused to the stable SpoIIIE translocation complex
tion. In the first, which is based on carboxyl terminus of SpoIIIE and the is indeed cell-specific, but this spe-
single-molecule studies of FtsK, the leucine zipper domain of cJun was cificity, and the resulting transloca-
proteins function as reversible DNA fused to the amino terminus of green tion polarity, are determined by the
translocases, and the polarity of the fluorescent protein (GFP). Cell- orientation of the chromosome.
translocated DNA is determined by specific promoters were used to Sheilagh Molloy
the DNA substrate. In the second, express this GFP fusion protein
ORIGINAL RESEARCH PAPER Becker, E. C. &
SpoIIIE functions as a DNA exporter, in either the mother cell or the
Pogliano, K. Cell-specific SpoIIIE assembly and
and the polarity of the translocated forespore. The results showed that DNA translocation polarity are dictated by
DNA is determined by the cell- wild-type SpoIIIE forms a focus chromosome orientation. Mol. Microbiol. 66,
1066–1079 (2007)
specific assembly of a stable SpoIIIE only in the mother cell, supporting

nature reviews | microbiology volume 6 | january 2008


© 2008 Nature Publishing Group
Research highlights

V IR U S S T R U C T U RE

One of a kind!
B1
B2
Despite the availability of a
commercial vaccine, the highly N-linked
contagious measles virus remains glycosylation
a substantial health risk for which Asn215
there is currently no specifically
targeted treatment or antiviral
therapy. The structure of the measles B3
Asn200
virus haemagglutinin (MVH), which
the virus uses to bind to host-cell
receptors, has now been revealed in
a recent study published in Nature
Structural & Molecular Biology.
Uniquely, species of Morbillivirus B6
(such as the measles virus) lack
neuraminidase activity and, instead
of binding to the host using sialic
acid, these viruses bind directly to B4 B5
C
the host-cell receptors SLAM and/or
CD46. In this study, Colf and col- N
leagues solved the structure of MVH Cartoon of the structure of the measles virus haemagglutinin. Figure 1b from Colf, L. A., Juo, Z. S. & Garcia,
at a resolution of 2.7 Å. The overall K. C. Structure of the measles virus hemagglutinin. Nature Struct. Mol. Biol. 18 Nov 2007 (doi:10.1038/
nsmb1342).
fold was similar to that of a
β-propeller, with six interconnected
blades (B1–6) — each of which large positional differences were neuraminidase structural fold that
contains four antiparallel β-strands detected, which suggests that, despite is similar to other viruses, such as
— that surround a large cavity. Four having similar folds, the haemag- PIV, but share few other structural
potential N-linked glycosylation sites glutinin structures of the two viruses or sequence similarities. By using
were detected, and the authors were have diverged considerably. Finally, the structure of MVH as a template,
able to model the glycan electron mutagenesis and antibody-blocking structure-based drugs could be
densities of two of these, Asn200 and data were mapped onto the solved designed that target the measles virus
Asn215, so supporting the theory MVH structure and it was found that at its point of entry and bring us one
that asparagine-linked glycosylation the binding surfaces of SLAM and step closer to treating this potentially
enables MVH folding and stablization. CD46 are far apart and distinct from fatal viral disease.
The authors then compared the large cavity that is analogous to Gillian Young
the structure of MVH with those that used by other viruses for sialic
of other viruses and found that it acid binding. Original Research Paper Colf, L. A., Juo, Z. S.
was most similar to the haemag- This study further demonstrates & Garcia, K. C. Structure of the measles virus
glutinin/neuraminidase fold of the the unique features of species hemagglutinin. Nature Struct. Mol. Biol. 18 Nov 2007
(doi:10.1038/nsmb1342)
parainfluenza virus (PIV). However, of Morbillivirus, which retain a

nature reviews | microbiology volume 6 | january 2008


© 2008 Nature Publishing Group
Research highlights

b ac t erial v ir u lence

The cycle of success for Legionella


Two recent papers have provided Rab protein is then recruited back activation of Rab1 by the GEF activ-
an insight into the mechanisms that to the membrane and activated by a ity of DrrA/SidM was enhanced in
Legionella pneumophila uses to sub- guanine nucleotide-exchange factor the presence of a lipid bilayer. They
vert the host-cell vesicular trafficking (GEF). After the active Rab has were then able to follow the associa-
pathway to create its replicative niche. carried out its function, membrane tion of the proteins with the mem-
L. pneumophila must establish cycling is completed when the Rab is brane during nucleotide exchange,
a replicative niche within alveolar inactivated by a GAP protein. which confirmed that once the GDF
macrophages. This process involves L. pneumophila uses a type IV activity of DrrA/SidM has displaced
remodelling of the Legionella- secretion system to secrete effector the Rab-GDI, the free Rab1 protein
containing vacuole (LCV) by exploit- proteins into the host-cell cytoplasm. is inserted in the membrane and can
ing the ability of the host protein Previous work had shown that one then be activated by the GEF activity
Rab1 to recruit material from the type IV effector, DrrA/SidM, is of DrrA/SidM.
vesicular transport pathway between required for the recruitment of Rab1 Both groups were also interested
the endoplasmic reticulum and Golgi to the LCV and has Rab1-specific in other L. pneumophila type
apparatus. Rab proteins achieve this GEF activity. Machner and Isberg, IV effectors that might function
by reversibly associating with lipid and Ingmundson et al. were inter- downstream of DrrA. Machner and
membranes through a process that ested in whether DrrA/SidM also has Isberg showed that L.  pneumophila
is known as Rab membrane cycling. GDF activity. Both groups purified LidA binds GTP–Rab1 that has been
Active GTP-bound prenylated Rab overexpressed, tagged forms of Rab1 activated by DrrA/SidM and sup-
proteins are present in membranes. and Rab-GDI from eukaryotic cells to ports the accumulation of activated
Once inactivated by a GTPase- ensure that they were prenylated and Rab1 on the LCV. Ingmundson et
activating protein (GAP), GDP–Rabs then showed that DrrA/SidM inter- al. found that LepB is delivered into
are removed from the membrane and feres with Rab1–Rab-GDI complex the host-cell cytoplasm shortly after
maintained in an inactive form in formation by displacing the Rab-GDI, bacterial uptake, is present on the
the cytosol through association with thus confirming that DrrA/SidM early LCV membrane and can dis-
a guanine nucleotide-dissociation does have GDF activity. Machner rupt secretory transport in a mam-
inhibitor (GDI). The GDI can be and Isberg went on to look at the malian cell line. They completed
displaced by a GDI-displacement effects of the presence of liposomal their work on LepB by demonstrat-
factor (GDF), and the GDP-bound membranes. They found that the ing that this L. pneumophila effector
has GAP activity towards Rab1 and
propose that this activity completes
the membrane cycling of Rab1 that
is begun by DrrA/SidM.
DrrA/SidM is the first protein to
be identified that has both GDF and
GEF activity. Both groups point out
that this raises the possibility that
there might be eukaryotic GEFs
that have similar dual functions.
Sheilagh Molloy

ORIGINAL RESEARCH PAPERS Machner, M. P.


& Isberg, R. I. A bifunctional bacterial protein
links GDI displacement to Rab1 activation.
Science 318, 974–977 (2007) | Ingmundson, A. et al.
Legionella pneumophila proteins that regulate
Rab1 membrane cycling. Nature 450, 365–369
(2007)

nature reviews | microbiology volume 6 | january 2008


© 2008 Nature Publishing Group
news & analysis
genome watch

A poultry existence
Helena Seth-Smith

The two poultry pathogens discussed in contains CDSs that encode many novel and two cryptic plasmids. Comparative anal-
this month’s Genome Watch are closely adhesins that are presumably specific for the yses revealed that 78% of the CDSs from the
related to well-characterized organisms avian trachea, including several filamentous APEC 01 chromosome are common to all
that infect humans, so scrutinizing haemagglutinins. There are potentially novel sequenced E. coli strains, which means that
their genomes could reveal factors that biosynthetic pathways for both lipopolysac- these CDSs comprise the minimal E. coli
determine host-specificity. charide (LPS) and capsular polysaccharide, backbone. A further 9% of the CDSs are
which would also modify the bacterial-cell shared among all the sequenced ExPECs,
Avian influenza has the potential to ravage surface. There are eleven CDSs that seem which might indicate their importance to the
the poultry industry and be transmitted to to encode fimbriae, which could aid bind- survival of the bacteria outside the intestine.
humans. Bacterial pathogens of poultry are ing of the bacteria to cells of the respiratory These genes include those that code for LPS
also economically important. Bordetella avium tract. The interaction with the host might synthesis, fimbriae, virulence‑associated pili
causes non-lethal respiratory disease, which also be mediated by 2 novel surface proteins, and iron acquisition. The remaining 13% of
can predispose birds (mainly turkeys) to each of which comprises more than 4,000 CDSs are unique to APEC 01 and are pre-
secondary infections. Infection amino acids, and 7 autotransport- dominantly located on 4 APEC‑specific
of the respiratory tract by ers. Bordetella species often syn- pathogenicity islands (PAIs), which are
avian pathogenic Escherichia thesize toxins; B. avium can phage-related and might contain additional
coli (APEC) strains can result produce dermo­necrotic toxin, virulence factors.
in colibacillosis, which can be which has been shown to The differences between the avian and
lethal. be important in causing mammalian Bordetella strains seem to
The genome of B. avium disease in turkeys, and reflect evolution over millions of years
consists of 3,732,255 base tracheal cytotoxin. through the vertical transfer of genes. The
pairs and contains 3,417 Most E. coli strains are E. coli genomes show more evidence of hori-
coding sequences (CDSs)1. associated with the intes- zontal gene transfer, and many of the differ-
The genome of the related tine. Strains that have ences between strains are restricted to PAIs
strain Bordetella bronchisep- strayed from this lifestyle are and plasmids.
tica, which is responsible known as ExPEC (extraintes- Helena Seth-Smith is at the Sanger Institute,
for respiratory disease in tinal pathogenic E. coli) and include Wellcome Trust Genome Campus, Hinxton,
mammals, was sequenced APEC and UPEC (uropathogenic) Cambridge CB10 1SA, UK.
previously. A comparison of strains, of which the UPEC strains are e-mail: microbes@sanger.ac.uk

the two could pinpoint those responsible for urinary-tract infec- doi:10.1038/nrmicro1830
genomic features that are tions in humans. Multiple E. coli 1. Sebaihia, M. et al. Comparison of the genome sequence
important to host specifi- genomes have been sequenced, of the poultry pathogen Bordetella avium with those of
B. bronchiseptica, B. pertussis, and B. parapertussis
city, that is, the infection of including laboratory strains, those reveals extensive diversity in surface structures
either an avian or a mamma- that cause food poisoning and associated with host interaction. J. Bacteriol. 188,
6002–6015 (2006).
lian host. These 2 strains share a other ExPEC strains. It has been pro- 2. Johnson, T. J. et al. The genome sequence of avian
conserved Bordetella backbone posed that UPEC originated from pathogenic Escherichia coli strain O1:K1:H7 shares
strong similarities with human extraintestinal
of 2,380 orthologous CDSs that APEC, and genome comparisons pathogenic E. coli genomes. J. Bacteriol. 189,
constitutes two thirds of the could help us to understand these 3228–3236 (2007).

B. avium genome. The CDSs that evolutionary connections.


are unique to B. avium mainly Strain APEC 01 has a chromosome
DATABASES
encode surface-associated pro- of 5,082,025 base pairs, and comprises Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
teins that might be important 4,467 CDSs2. It also harbours four entrez/query.fcgi?db=genomeprj
APEC 01 | Bordetella avium | Bordetella bronchiseptica
in adhesion and immune-system plasmids: one involved in virulence;
All links are active in the online pdf
evasion. The B. avium genome one involved in antibiotic resistance;

 | january 2008 | volume 6 www.nature.com/reviews/micro


© 2008 Nature Publishing Group
disease watch | in the news
Choose your battles wisely Dandruff on the collar? was unexpectedly ended after preliminary
results showed that the vaccine was not
The genome of Malassezia globosa — a fungus conferring resistance. Further analysis
that causes discomforting and embarrassing suggests that the vaccine — which
dandruff — has been sequenced by an consists of 3 HIV genes and an attenuated
international team led by Thomas Dawson adenovirus 5 vector — may have increased
at Procter & Gamble. Malassezia spp.- HIV susceptibility in recipients. Notably,
associated dandruff affects more than 50% 21 of 392 male vaccine-recipient patients
of humans, and more than UK£3 billion are with pre-existing immunity to adenovirus 5
spent annually on dandruff treatments. acquired HIV infections, compared with
Genome sequencing revealed that the 9 of 386 from the placebo group. As the
M. globosa genome is among the smallest trial was halted abruptly, researchers
of all the free-living fungi analysed so far, cannot determine whether these results
containing as few as 4,285 predicted protein- are statistically significant. Among
coding genes. Of these, approximately 50 other explanations, vaccination may
encode secreted proteins, including many have increased the production of CD4+
lipid-degrading proteins, that are probably T cells — HIV’s favorite infection target
responsible for breaking down hair and scalp, — in adenovirus-5-immune patients. An
thereby causing the irritation, inflammation upcoming trial of a vaccine developed by
and flaking that are hallmarks of dandruff. Not the National Institutes of Health has been
only might this sequenced genome represent delayed to allow further analysis. Nature/
Neil Smith

a gold mine for developers of anti-dandruff New York Times


shampoos, they also have agricultural
implications as M. globosa is closely related
to various common pathogenic plant fungi
HIV numbers revised
Animals, like plants, can respond in that affect corn, wheat and other food crops. The United Nations has lowered their
different ways to parasite infections: they PNAS/The Telegraph estimate of how many people are infected
can mount a full-scale immune response with HIV globally from 39.5 million to
or they can tolerate the invader. Andrew 33.2 million. These revised numbers,
Read and colleagues from the University
Novel MRSA virulence factors however, are attributed to improved
of Edinburgh, UK, introduced rodent identified methods of data collection rather than to
malaria into five strains of laboratory mice Although community-acquired meticillin- a real decline in HIV infection. The number
and monitored parasite load and animal resistant Staphylococcus aureus (CA-MRSA) of HIV carriers is still rising, although
health, as measured by anaemia and body causes the majority of the staphylococcal the global prevalence of HIV seems to
mass. In some strains of mice, parasite load infections that result in trips to emergency be levelling off. An estimated 2.5 million
increased and the mice stayed healthy, rooms, the basic cause of CA-MRSA new infections occurred in the past year
which indicated that the mice could virulence remains unidentified. However, (which works out at a staggering 6,800 new
tolerate the parasite. In other mice strains, a class of secreted cytolytic peptides infections per day), down from a likely peak
however, parasite load decreased, which (so‑called PSMs) that function primarily to in the late 1990s of over 3 million infections
indicated that the mice were resisting destroy leukocytes has now been identified per year. Sub-Saharan Africa remains the
infection. The authors suggest that these by Michael Otto, from the Rocky Mountain hardest hit by HIV, accounting for nearly
findings could have important implications Laboratories, USA, and colleagues. Deletion 50% of the new cases of infection and 68%
for pathogen evolution: pathogens might of PSM genes resulted in a decrease in the of the total number of cases. WHO
not be pushed to evolve increased virulence severity of CA-MRSA in two mouse abscess
if they are tolerated rather than destroyed. and bacteraemia models. Moreover, in vitro
However, tolerated pathogens are also studies indicated that PSM genes caused
more likely to spread. S. aureus to recruit, activate and lyse human
In a somewhat related paper, Lynn Martin, neutrophils. Notably, hospital-acquired
from the University of South Florida, and MRSA strains exhibit a significantly
colleagues report that fast-living strains lower expression of PSM genes than
of mice develop high fevers in response to do CA-MRSA strains, which are
simulated infection, but slow-living strains more virulent. Nature Med.
do not. The authors propose that the ‘live
fast, die young’ mice tolerate the harm
that fever inflicts on bodily tissues, as they
Vaccine may have
already have short lifespans. Slow-living increased HIV risk
mice, however, have more to lose and may In late September, a trial of Merck’s
therefore adopt targeted strategies, such candidate HIV vaccine, which included
as antibody production. Science/Funct. Ecol. over 3,000 people from 9 countries, STOCKBYTE

nature reviews | microbiology volume 6 | january 2008 | 


© 2008 Nature Publishing Group
N e w s & a n a ly s i s

Use condoms! Anti-polio programme gets autoinducers. V. cholerae produces


two autoinducers, autoinducer 2 (AI‑2)
a booster
Although the global prevalence of HIV and CAI‑1. The structure of AI-2 has
is showing signs of levelling off, the been known for many years, and in this
transmission rates for sexually transmitted latest paper Bassler et al. characterized
infections (STIs) in the United Kingdom and synthesized CAI‑1. To assess the
are still on the rise, despite concerted feasibility of controlling V. cholerae by
public health efforts to reverse this trend. manipulating QS the authors showed that
During 2006, report the Health Protection synthetic CAI‑1 can repress production
Agency (HPA), 376,508 STIs were newly of the V. cholerae virulence factor toxin
diagnosed — a 2.2% increase compared co-regulated pilus as well as natural CAI-1.
with the number of new STIs in 2005. The “This work”, the authors argue, “provides
number of STIs diagnosed each year in a demonstration that interference with
the United Kingdom has increased almost quorum-sensing processes in general ... has
continually since the 1990s. In particular, great promise in the clinical setting.” Nature
the sexual health of young adults has
worsened, although the HPA also warns of
a continuing HIV and STI epidemic among
Outbreak news
men who have sex with men. An estimated Rift Valley Fever. An outbreak of Rift Valley
73,000 adults in the United Kingdom are Fever has killed 164 people out of a total
now HIV-positive, although one-third of of 451 who have contracted the disease
these individuals remain unaware that they in Sudan. The majority of these infections
are infected. More funding and education are the result of direct or indirect contact
is needed to tackle these problems, say with the blood and organs of infected
experts. HPA/BBC animals, although some cases are also the
IMAGE SOURCE result of bites from infected mosquitoes.
Sudanese health authorities initially
Can Africa cope with avian The campaign to eradicate polio has sought help to control the outbreak in
influenza? received a much-needed US$200 million mid-October. Rift Valley Fever normally
African nations are unlikely to be able to (£97 million) grant from the Bill and Melinda has a fatality rate of ~1%, but the fatality
achieve the five priorities that have been Gates Foundation and Rotary International. rate is ~50% in patients who develop the
identified by the World Health Organization An initial $100 million will be distributed haemorrhagic form. WHO/Associated Press
(WHO) to fight avian influenza, argue through grants to the WHO and UNICEF to
Robert Webster and colleagues. So far there support mass immunization campaigns in Encephalitis. A ‘mystery’ pathogen
have been only 40 cases of avian influenza polio-affected countries, polio-surveillance that causes encephalitis has resulted in
in Africa, for which there has been a 40% activities, and community-education 21 deaths, mostly children, in a remote
fatality rate. However, the predominant and outreach programmes. Although region of Bangladesh. An additional
strain of avian influenza in Africa, the immunization programmes have made 200 people are affected. Bangladesh’s
Qinghai strain, “has acquired several huge strides over the past 20 years, polio is International Epidemiology Research
troubling properties, including respiratory still endemic in Nigeria, Pakistan, India and Centre and the Centers for Disease
rather than faecal transmission in poultry.” Afghanistan, and approximately 700 cases Control and Prevention in the United
The five priorities of the WHO for the of polio are reported each year. Despite this States are working together to identify the
combat of avian flu are: reduce human generous donation, WHO officials report causative pathogen. Reuters
exposure; strengthen surveillance; intensify that the polio-eradication programme is still
rapid containment; enhance response facing a $650 million shortfall over the next Ebola. WHO officials have confirmed
capacity; and coordinate global research. 2 years. BBC that Ebola virus has killed 16 people and
Owing to a number of factors, including affected an additional 50 in western
inadequate surveillance, the denial of Uganda. The virus belongs to a distinct
outbreaks and the poor communication
Cracking cholera’s lines of subtype that has not been detected before,
of risks to the public, African nations are communications as indicated through tests conducted by
unlikely to achieve these targets in the The Vibrio cholerae signalling molecule national laboratories in Uganda and the US
opinion of some observers. For example, cholera autoinducer-1 (CAI-1) may form Center for Disease Control. Ebola is fatal in
it took 4 weeks to officially confirm the the basis of therapeutic intervention approximately 80% of cases, and as yet there
outbreaks in all of the affected African against V. cholerae infection, reports is no known cure. BBC
countries, apart from Egypt. Webster and Bonnie Bassler and colleagues. V.
colleagues call for each African nation cholerae, and other bacteria, use quorum In the News was compiled with the assistance of
David Ojcius, University of California, Merced,
to realistically assess its status, conduct sensing (QS) to sense and respond to USA. David’s links to infectious disease news
regular active surveillance and be more population density, and this cell-to- stories can be accessed on Connotea (http://www.
forthcoming with data. Lancet Infect. Dis. cell communication is mediated by connotea.org), under the username ojcius.

10 | january 2008 | volume 6 www.nature.com/reviews/micro


© 2008 Nature Publishing Group
Progress
promoting bacterial basolateral entry into

The versatility of Shigella effectors polarized epithelial cells1,4 (FIG. 1).


IpaA binds the amino-terminal head
domain of vinculin, which is a key com-
Michinaga Ogawa, Yutaka Handa, Hiroshi Ashida, Masato Suzuki and ponent of focal adhesions, and stimulates
actin depolymerization5. IpaA also targets
Chihiro Sasakawa
β1-integrin and stimulates the GTPase
Abstract | When Shigella infect the intestinal epithelium, they deliver several activity of RhoA, thereby inducing the loss
of actin stress fibres6. Thus, IpaA might
effectors through the type III secretion system (T3SS) into the surrounding space
facilitate recycling of the free actin pool
and directly into the host-cell cytoplasm, where they can mimic and usurp host by the destruction of stress fibres and
cellular functions or subvert host-cell signalling pathways and the immune contribute to the production of membrane
response. Although bacterial strategies and mechanisms of infection vary greatly, ruffles. The interaction between IpaB and
recent studies of Shigella effectors have revealed that Shigella possess a highly the CD44 receptor, which is present on the
evolved strategy for infection. epithelial cell membrane in areas that are
rich in lipid rafts, stimulates basolateral
invasion7. IpaB is a multifunctional effec-
The intestinal epithelium acts as an intrinsic which eventually leads to entry by macro­ tor2,4,8. As well as being secreted at the tip
defensive barrier against microbial invaders. pinocytosis. As soon as a bacterium is of the T3SS needle and acting as a T3SS
The barrier function has four major ele- surrounded by a membrane vacuole within translocator (with IpaC), it is also involved
ments: the resident commensal bacteria; an epithelial cell, it disrupts the vacuolar in vacuole disruption, activates caspase‑1
the integrity of the epithelial barrier; membrane and escapes into the cytoplasm. in macrophages and modulates cell-cycle
the rapid epithelial-cell turnover; and the Shigella can multiply in the epithelial-cell progression in epithelial progenitor cells
innate immune response. Nevertheless, cytoplasm and move both intra- and inter- (discussed below). IpaC is delivered into
many pathogenic bacteria can circumvent cellularly, and so infection of the intestinal the epithelial-cell cytoplasm and integrates
these defences and exploit the intestine epithelium by Shigella elicits a strong into the membrane, where it is assumed
as a replicative niche, a shelter from host inflammatory response1,2. to have a key role, either directly or indi-
immune surveillance or a port of entry for To proceed through the series of steps rectly, in inducing actin polymerization4.
dissemination into deeper tissues. that are involved in infection, Shigella Although there is no direct evidence as
Shigella species (abbreviated here to secrete many effector proteins through the yet, IpaC might be involved in promot-
Shigella) are human-adapted pathogens that type III secretion system (T3SS)3. TABLE 1 ing Shigella invasion of epithelial cells by
are capable of colonizing the intestinal epi- lists some of the characterized Shigella inducing actin foci through the accumula-
thelium by exploiting epithelial-cell func- effectors and their homologues in other tion of the host non-receptor tyrosine
tions and circumventing the host innate bacterial pathogens. This Progress article kinase Src at the site of bacterial entry4. On
immune response. Shigella have neither highlights recent advances in our under- entry, the phosphorylation of cortactin — a
adherence factors nor flagella. Following standing of the roles of the Shigella effec- cortical actin-binding protein that activates
ingestion by the faecal–oral route, Shigella tors that are delivered into host cells by the the Arp2/3 complex and induces actin
move through the small intestine to the T3SS during intestinal infection. polymerization — occurs in a Src-dependent
colon and rectum, where they cross the epi- manner and Crk (a Src-related tyrosine
thelial barrier through microfold cells that Effectors in the early stages of infection kinase) activates the small GTPase Rac1
overlie solitary lymphoid nodules. When The ability to invade, colonize and trans- (Ref. 4).
the epithelial barrier has been breached, locate across mucosal barriers is an impor- IpgB1 is assumed to have a major
Shigella immediately enter the macrophages tant feature of enteric pathogens. Notably, role in producing membrane ruffles by
that reside within the microfold‑cell pocket. in the case of Shigella, the ability to invade activating Rac1 through ELMO–Dock180,
Once within the macrophages, the infecting epithelial cells and subsequently spread a Rac1 guanine nucleotide-exchange
bacteria disrupt the phagosomal membrane from cell to cell is pivotal in establishing factor9. For example, when IpgB1 is
and disseminate from the phagosome into an intestinal infection. When Shigella ectopically expressed in HeLa cells, large
the macrophage cytoplasm, where they come into contact with epithelial cells, membrane ruffles are produced. Pull-
multiply and induce rapid cell death1,2. they deliver a subset of effectors through down assays have identified ELMO as
Shigella that are released from dead the T3SS both around the bacterial surface the IpgB1-binding partner; IpgB1 binds
macrophages can enter the surrounding and directly into host cells. These effectors, to the amino‑terminal region of ELMO,
enterocytes by inducing the production of which include IpaA, IpaB, IpaC, IpgB1, which also binds RhoG. An in vitro bind-
membrane ruffles at the basolateral surface, IpgB2, IpgD and VirA, are involved in ing assay showed that RhoG binding to

nature reviews | microbiology volume 6 | january 2008 | 11


© 2008 Nature Publishing Group
Progress

Table 1 | Activities of Shigella type III secretion system (T3SS) effectors


T3SS Biochemical Target (or targets) Role in infection Selected homologues Refs
effector activity
IpaA Unknown Vinculin Bacterial invasion Salmonella spp. SipA (also called 9,10
SspA)
IpaB T3SS translocon Caspase-1, CD44 Macrophage apoptosis and Salmonella spp. SipB (also called SspB) 6,34,36
and Mad2L2 cell-cycle arrest and Yersinia spp. YopB
IpaC T3SS translocon Unknown Bacterial invasion Salmonella spp. SipC (also called SspC) 37
IpgB1 RhoG mimic ELMO Bacterial invasion Salmonella spp. SifA and SifB; EHEC, 11,12
EPEC and Citrobacter rodentium Map;
and EHEC EspM1 and EspM2
IpgB2 RhoA mimic mDia and ROCK Unknown
IpgD Inositol phosphate Phosphatidylinositol Bacterial invasion and host-cell Salmonella spp. SopB (also called 7,8
phosphatase 4,5-bisphosphate survival SigD)

VirA Cysteine protease Tubulin Bacterial invasion and intracellular EHEC, EPEC and C. rodentium EspG 13,14,17
spreading and EPEC EspG2 (also called Orf3)

IcsB Unknown Shigella VirG (also Escape from autophagy Burkholderia spp. BopA 20
called IcsA)
OspC1 Unknown Unknown Polymorphonuclear transepithelial Shigella OspC2, OspC3 and OspC4 38
migration and Vibrio parahaemolyticus OspC2
OspE2 Unknown Unknown Intercellular spreading Shigella OspE1; Salmonella spp. 39
EspO1STYM; and EHEC EspO1-1 and
EspO1-2
OspF Phosphothreonine MAP kinases Suppression of innate immune Salmonella spp. SpvC; 27,29,38
lyase responses Pseudomonas syringae HopAI1; and
Chromobacterium violaceum VirA
OspG Serine/threonine E2 ubiquitin- Suppression of innate immune Yersinia enterocolitica YE2447; 26
kinase conjugating responses C. rodentium NleH; and EHEC NleH1-1
enzymes and NleH1-2
IpaH9.8 E3 ubiquitin ligase U2AF35 and yeast Suppression of innate immune Shigella IpaH4.5; Salmonella spp. 24,25
Ste7 responses SspH1, SspH2 and SlrP; Yersinia
pestis YP3416 and YP3418; P. syringae
IpaH7.8 E3 ubiquitin ligase Unknown Escape from endocytic vacuoles of PSPTO1492 and PSPTO4093; and 25,40
phagocyte Rhizobium spp. Y4fR
Chromosomal Possibly E3 Unknown Suppression of innate immune 23
IpaHs ubiquitin ligase responses
EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic E. coli, MAP, mitogen-activated protein.

ELMO is competitively inhibited by IpgB1, are found in enteropathogenic Escherichia Intra- and intercellular motility
implying that IpgB1 mimics the function coli (EPEC), enterohaemorrhagic E. coli Many pathogenic bacteria can invade host
of RhoG in producing membrane ruffles (EHEC) and Shigella. Intriguingly, VirA cells, and some species, such as Shigella,
during Shigella invasion9. IpgB2 is an is delivered into the host-cell cytoplasm Listeria monocytogenes, Mycobacterium
IpgB1 homologue that binds to mDia1, near the site of bacterial entry and induces marinum, Rickettsia conorii and
which facilitates actin nucleation, and the local microtubule (MT) degradation13. As Burkholderia pseudomallei, are capable of
Rho kinase ROCK through its GTPase- degradation of MTs by EspG results in the inducing actin nucleation at one pole of the
binding domains. In this way, IpgB2 release of various MT‑associated proteins, bacterium to gain the propulsive force that is
mimics the activity of RhoA in inducing including GEF‑H1, and GEF‑H1 activates necessary to move through the host cell and
the formation of stress fibres, although RhoA14, VirA activity is assumed to con- into neighbouring host cells. This activity,
its specific involvement in bacterial inva- tribute to ruffle formation during Shigella often called actin-based bacterial motility,
sion remains unclear10. IpgD possesses invasion through the cross-talk between is crucial for establishing an infectious foot-
phosphatidylinositol (4,5) bisphosphate RhoA and Rac1. hold as well as renewing replicative niches.
(PI(4,5)P2) phosphatase activity and Clearly, these studies indicate that Intriguingly, the bacterial proteins that are
catalyses the hydrolysis of PI(4,5)P2 to Shigella exploit the interactions between a involved in mediating actin nucleation vary
phosphatidylinositol 5‑monophosphate subset of effector proteins and their host from species to species. Nevertheless, these
(PI(5)P), thereby promoting local actin target molecules (or target functions). Such proteins share the ability to recruit and acti-
polymerization4,11,12. interactions are capable of stimulating sev- vate the Arp2/3 complex, which is required
VirA, which is a member of the EspG/ eral host-cell signalling pathways that are for actin polymerization near the bacterial
VirA family, shares significant amino-acid involved in inducing actin polymerization surface8. In the case of Shigella, an outer-
homology, as well as functional similarity, and remodelling the architecture of the membrane protein called VirG (also known
with EspG. EspG/VirA family members host-cell surface (FIG. 1). as IcsA) has a key role, as VirG can directly

12 | january 2008 | volume 6 www.nature.com/reviews/micro


© 2008 Nature Publishing Group
Progress

cysteine-protease-like activity17. Consistent


with this observation, a virA mutant that
lacked this activity was found to be less
T3SS capable of moving smoothly within the host
RhoG ELMO IpgB1 Shigella cytoplasm than the wild type and was inca-
T3SS pable of maintaining continuous cell–cell
effectors spreading17.
Although there is no direct evidence
CD44 as yet, the ability to clear a path through
Dock180 IpaB
IpaC the host-cell MTs might not be unique to
IpgB2 Shigella. L. monocytogenes ActA is capable
mDia
IpgB2 of directly recruiting the Arp2/3 complex in
Rock the vicinity of the bacterial surface, and the
RhoA Rac1 Cdc42 destruction of MTs is occasionally detected
along the path of L. monocytogenes move-
ment. Intriguingly, L. monocytogenes ActA
is not a VirA homologue but it indirectly
WAVE IpaC recruits Op18, an MT‑sequestering host
Microtubule protein, near the bacterial surface18. It is
destabilization Major actin tempting to speculate that Op18 facilitates
rearrangements L. monocytogenes movement within epithe-
Arp2/3 Membrane lial cells. The discovery of a novel bacterial
PI(4,5)P2 ruffling activity that destroys MTs demonstrates that,
VirA
although MTs are an obstacle to bacterial
IpgD movement within the host-cell cytoplasm,
IpaA Entry certain bacteria have evolved an activity to
IpaA
Vinculin remove this barrier.

Escape from autophagy


IpaB
α5β1 integrin Autophagy is a ubiquitous degradation
IpaC
system in eukaryotic cells that is required
not only for the cellular response to starva-
tion and stress, and for the removal of
damaged or surplus organelles, but also
T3SS effectors for removing bacterial pathogens that
Figure 1 | A simplified model of membrane-ruffle production in response to the stimulation invade the cytoplasm of host cells. For
of host cellular signalling by Shigella effectors. Upon contact between Shigella and an epi- example, Streptococcus pyogenes (Group A
thelial cell membrane, the bacterium delivers several effectors through the type III secretion Streptococcus) and Staphylococcus aureus are
Nature Reviews
system (T3SS) around the bacterial surface and into the host-cell cytoplasm. | Microbiology
By interacting with capable of invading epithelial cells, and both
their host binding partners the effectors are eventually able to activate the Rac1–WAVE–Arp2/3 pathogens are targeted by the autophagic
pathway, which leads to the protrusion of membrane ruffles around the bacterial entry point. It
machinery and eventually undergo lyso-
should be noted that this model does not rule out any other additional, as yet uncharacterized,
somal degradation. Autophagy is achieved
signalling pathways that may be involved in mediating actin polymerization during Shigella
invasion. See the main text for details. by a series of autophagy-related (Atg)
proteins that are highly conserved from
yeast to humans19. During multiplication
within epithelial cells, Shigella are recognized
interact with N‑WASP (neural Wiskott– The movement of bacteria within host by components of the autophagic pathway.
Aldrich syndrome protein)15. When VirG cells is highly variable and depends on their However, the secretion of IcsB through the
and Cdc42 interact with N‑WASP, N‑WASP location in the host cell. For example, some T3SS on entry into the cytoplasm allows the
becomes activated, which, in turn, recruits motile Shigella suddenly change direction, bacteria to escape autophagic destruction20.
and activates the Arp2/3 complex16. The spin around or stop moving. It has recently Although an icsB mutant is fully invasive
formation of the VirG–N-WASP–Arp2/3 been shown that Shigella movement within and can escape from the phagocytic vacuole
complex at one pole of the bacterium allows the host-cell cytoplasm is severely hindered in epithelial cells, it is ultimately enclosed
Shigella to induce actin nucleation and by MTs, but a motile bacterium can destroy by autophagosomes. Surprisingly, IcsB does
elongation, thus gaining propulsive force surrounding MTs using VirA17 (FIG. 2). not directly inhibit autophagy itself. Instead,
(FIG. 2). During bacterial movement, some As described above, VirA is also used to the VirG protein — which is required for
bacteria impinge on the host-cell membrane promote bacterial entry. Thus, VirA has dual actin-based bacterial motility (FIG. 2) — is
and cause the membrane to protrude and roles in both bacterial invasion and intracel- targeted for autophagic recognition by bind-
penetrate into that of neighbouring cells, lular spreading. Characterization of VirA ing to Atg5 (a protein that is involved in
thereby allowing the bacteria to disseminate activity indicated that the degradation of MTs the elongation of isolation membranes).
into adjacent cells. by VirA depends on its α‑tubulin-specific In in vitro binding assays, both IcsB and

nature reviews | microbiology volume 6 | january 2008 | 13


© 2008 Nature Publishing Group
Progress

MT network (MAPKs) and nuclear factor (NF)-κB and


results in the production of proinflammatory
cytokines22.
Capping To circumvent the innate immune
protein Profilin response, Shigella deliver more than ten
F-actin VirA
VirA
effector proteins, including the IpaH
IcsB
Vinculin proteins, OspG and OspF, into host cells
Arp2/3 through the T3SS2,23 (FIG. 3; TABLE 1).
N-WASP VirG Shigella
G-actin IpaH9.8, a member of the IpaH protein
Cdc42 VirA family, translocates to the nucleus, where
IcsB
VirA it interacts with U2AF35, an mRNA splic-
VirA
ing factor24. Infection of epithelial cells
by an ipaH9.8 mutant increased the levels
of proinflammatory cytokines, and RNA
interference (RNAi)-mediated knockdown
of U2AF35 production decreased these
Figure 2 | Shigella movement within the host-cell cytoplasm requires actin polymerization
levels. In a murine lung-infection model,
and microtubule degradation. Asymmetric distribution of VirG Nature (also known
Reviewsas| Microbiology
IcsA) on the
bacterial surface is essential for the polar movement of Shigella in epithelial cells. The accumu-
the ipaH9.8 mutant induced more severe
lation of VirG at one pole of the bacterium recruits and activates the Arp2/3 complex by the inflammatory responses and greater
interaction between VirG and N‑WASP (neural Wiskott–Aldrich syndrome protein). Motile proinflammatory cytokine production than
Shigella secrete VirA through the type III secretion system and destroy local microtubules (MTs), did wild-type Shigella, which resulted in a
thereby facilitating bacterial movement. 30-fold decrease in bacterial colonization24.
It has recently been suggested that the IpaH
homologues (including IpaH9.8) that are
Atg5 can bind to VirG. Intriguingly, IcsB unclear, recent studies have indicated that produced by plant and animal pathogens,
and Atg5 share the same binding region on these intracellular pathogens are also capa- such as Yersinia species, Salmonella spe-
VirG, and the affinity of IcsB for VirG is ble of certain manoeuvres that allow them cies, Edwardsiella ictaluri, Bradyrhizobium
much stronger than the affinity of Atg5 for to circumvent autophagic recognition. japonicum, Rhizobium species and some
VirG, which suggests that IcsB acts as an Pseudomonas species, share an E3 ubiquitin
anti-Atg5-binding protein. The interaction Modulation of the innate immune response ligase activity in the highly conserved
between IcsB and VirG near the bacte- Major bacterial-cell components, such as carboxy‑terminal region25 (TABLE 1).
rial surface, therefore, seems to provide lipopolysaccharide (LPS), peptidoglycan Although the host target (or targets) of each
a mechanism of escape from autophagic (PGN), and, perhaps, nucleic acids (DNA ubiquitin ligase remains unknown, in a
recognition1,20. L. monocytogenes can also and RNA), are readily released from yeast-cell system, IpaH9.8 was shown to have
escape autophagy during multiplication in multiplying and killed bacteria. These an activity that interferes with the pherom-
the host-cell cytoplasm, but the mechanism components are recognized by Toll-like one response by the ubiquitination of the
that underlies this escape seems to differ receptors and Nod-like receptors, which MAPK kinase Ste7, which is then degraded
from that of Shigella21. Although the precise activate the host innate defence systems, by proteasomes25. These findings suggest
mechanism is still unknown, in contrast stimulate inflammatory signalling cascades that IpaH and IpaH homologues have a
to Shigella VirG, ActA, which is present on and induce cellular and humoral immune central role in dampening host inflam-
the surface of L. monocytogenes and medi- responses. Nevertheless, many bacterial matory-related signals during bacterial
ates actin polymerization at one pole of pathogens that infect the intestinal mucosa infection (FIG. 3).
the bacterium, seems to be involved in the can circumvent the host innate immune OspG can bind to ubiquitinated E2s
escape of L. monocytogenes from autophagic response and colonize their replicative (ubiquitin-conjugating enzymes), such as
recognition. niches2. The mechanisms that are used by UbcH5b, a component of the Skp1–culin–F-
The recognition of intracellular pathogens bacteria to dampen host inflammatory box protein (SCF)β–TrCP complex that is
by autophagy is not limited to pathogens signals have been extensively studied using a involved in phospho‑IκBα ubiquitination
that invade the cytoplasm. As long as the range of animal and plant pathogens. and its subsequent degradation by the
innate immune response of the host is Shigella release LPS and PGN into host proteasome26. Thus, OspG interferes with
intact, intracellular pathogens, such as cells, and these pathogen-associated molecular IκBα degradation, thereby resulting in a
Mycobacterium tuberculosis, Legionella pneu- patterns are the main cause of the strong repression of NF‑κB activation. Consistent
mophila and Coxiella burnetii, which are inflammatory response that is induced by with this observation, the characterization of
sequestered in vacuolar compartments, can Shigella infection2. For example, the LPS that the phenotype of an ospG mutant in in vitro
be also targeted by autophagy. Unless they is released by Shigella as they escape from and in vivo models of infection has shown
are able to modify the vesicles in which phagosomes into the macrophage cytoplasm that OspG is involved in downregulating
they are contained or avoid autophagic activates caspase‑1, which induces the produc- the host inflammatory response to Shigella
uptake at an early stage of infection, they tion of interleukin‑1β and cell death. PGN infection26.
are entrapped by a lamellar membranous released from Shigella that are multiplying OspF also translocates to the host-cell
structure that is associated with LC3, an within epithelial cells activates the Nod1–RICK nucleus. OspF has a specific phosphatase
essential autophagic protein. Although pathway, which stimulates signalling cascades activity that dephosphorylates and inactivates
the mechanisms that are involved remain that involve mitogen-activated protein kinases MAPKs, such as ERK1/2, JNK and p38,

14 | january 2008 | volume 6 www.nature.com/reviews/micro


© 2008 Nature Publishing Group
Progress

thereby blocking the phosphorylation of the


serine 10 residue of histone H3, which is
required for the transcription of a subset of
NF‑κB-regulated genes27–29. Intriguingly, in
contrast to the OspF activity that is proposed Shigella
above, Shigella OspF, Salmonella spp. SpvC
and Pseudomonas syringae HopAl1 have also Nod1/2 T3SS
effectors OspG
been shown to share the ability to dephos-
PGN
phorylate MAPKs through their phospho-
threonine lyase activities, thereby enabling LPS
E2s
these bacterial effectors to interfere with RICK Ub
MAPK activity3,29 (TABLE 1). In rabbit ileal UbcH5b IpaHs
loops and a mouse-lung infection model, β-TrCP
IκB
the ability of a Shigella ospF mutant to
induce inflammatory responses was much IKK p65 p50
MAPK Ub Ub
greater than that of wild-type Shigella. complex IκB Unknown
Although there is some controversy regard- NF-κB
ing the activity of OspF, which might be
attributed to the different experimental
conditions that are used in each study or
the dual activities that are encoded by Proteasome
OspF, these studies have clearly indicated
OspF P
that OspF participates in dampening the
inflammatory response27. P IpaH9.8 U2AF35 Interleukin-8
MAPK MAPK production
The reason for the presence of such a vari-
able number of effectors in Shigella and other
IL-8
pathogens remains a matter of speculation.
As numerous signal-transduction pathways Histone H3 p65 p50
are engaged in inducing the inflammatory
response to bacterial infection, many effec-
tors must be engaged in modulating the
Figure 3 | Shigella and the downregulation of the host inflammatory response. During the
various inflammatory signal pathways at multiplication of Shigella in epithelial cells, the bacteria shed components,Naturesuch as peptidoglycan
Reviews | Microbiology
different levels and different times during (PGN) and lipopolysaccharide (LPS), into the cytoplasm. The recognition of PGN by Nod1 activates the
bacterial infection. nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK)-dependent inflammatory signals.
Shigella delivers a set of effectors, including IpaH9.8, IpaHs (IpaH9.8 homologues), OspF and OspG, into
Slowing of rapid epithelial-cell turnover the host cell through the type III secretion system (T3SS), which enables them to circumvent the host
The intestinal epithelium renews itself inflammatory response and inactivate the innate immune system. See the main text for details.
every several days, which provides an
important intrinsic defence system that
limits bacterial colonization. The rapid Other cyclomodulins, the cytolethal Shigella IpaB is involved in mediating
turnover of intestinal epithelial cells forms distending toxins (CDTs), are produced by the translocation of effectors through the
a crucial physical, as well as functional, Shigella dysenteriae, Campylobacter jejuni, T3SS and also functions as an invasin that
barrier, and renewal is sustained by E. coli and Salmonella enterica serovar interacts with CD44 (Refs 1,4,7). It has
the vigorous proliferation of epithelial Typhi (S. typhi). One of the CDTs that is been shown that IpaB is delivered into the
progenitor cells that migrate upwards produced by C. jejuni possesses a deox- epithelial-cell cytoplasm by intracellular
from the bottom of the intestinal crypts. yribonuclease‑I-like activity and causes bacteria and causes cell-cycle arrest by
Nevertheless, many pathogenic bacteria, limited DNA damage when it is delivered targeting Mad2L2, an anaphase-promoting
including Shigella, are capable of coloniz- into the host-cell nucleus, which leads to complex/cyclosome (APC) inhibitor34.
ing the intestinal epithelium. Recent stud- the activation of ATM (a PI3 kinase) and Cell-cycle progression is stringently con-
ies have indicated that a growing family of eventually results in cell-cycle arrest32. trolled by cell-cycle-specific proteolysis,
bacterial toxins, effectors and small com- Helicobacter pylori VacA, which induces which involves the ubiquitination of
pounds, called cyclomodulins, are capable cellular vacuolation in epithelial cells, is target proteins by two main types of E3
of interfering with the eukaryotic cell also capable of efficiently blocking the ligase complexes: the APC complex and
cycle30,31. Some of the cyclomodulins — for proliferation of T cells by inducing G1/S the SCF complex35. The APC complex is
example, the EPEC protein cycling inhibi- cell-cycle arrest33. Although the biological a multisubunit complex that targets sub-
tor factor (Cif) — inhibit host-cell mitosis importance of each cyclomodulin and strates for degradation only during mitosis
after transfer from the bacteria through its target host cells in bacterial infection and the G1 phase; it also targets mitotic
the T3SS. Cells that have been transformed remains largely unclear, it is expected that cyclin A and cyclin B1, so allowing mitotic
by Cif accumulate 4n DNA and re-initiate some cell-cycle inhibitors will prolong the progression. Cyclin B1 ubiquitination
DNA synthesis without dividing, which pathogen’s presence by interfering with the assays have shown that APC undergoes
results in cells that contain 8–16n DNA31. rapid turnover of epithelial cells. unscheduled activation in response to

nature reviews | microbiology volume 6 | january 2008 | 15


© 2008 Nature Publishing Group
Progress

IpaB interaction with Mad2L2. Indeed, Chihiro Sasakawa is also at the Department of Infectious 24. Okuda, J. et al. Shigella effector IpaH9.8 binds to a
Disease Control, International Research Center for splicing factor U2AF(35) to modulate host immune
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Infectious Diseases, Institute of Medical Science,
Shigella fail to accumulate APC substrates, University of Tokyo, 4‑6‑1, Shirokanedai, Minato-ku,
531–539 (2005).
25. Rohde, J. R., Breitkreutz, A., Chenal, A., Sansonetti,
such as cyclin B1, Cdc20 and Plk1, which Tokyo 108‑8639, Japan and CREST, Japan Science and P. J. & Parsot, C. Type III secretion effectors of the
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16 | january 2008 | volume 6 www.nature.com/reviews/micro


© 2008 Nature Publishing Group
REVIEWS
The biology and future prospects of
antivirulence therapies
Lynette Cegelski*, Garland R. Marshall‡, Gary R. Eldridge§ and Scott J. Hultgren*
Abstract | The emergence and increasing prevalence of bacterial strains that are resistant to
available antibiotics demand the discovery of new therapeutic approaches. Targeting
bacterial virulence is an alternative approach to antimicrobial therapy that offers promising
opportunities to inhibit pathogenesis and its consequences without placing immediate life-
or-death pressure on the target bacterium. Certain virulence factors have been shown to be
potential targets for drug design and therapeutic intervention, whereas new insights are
crucial for exploiting others. Targeting virulence represents a new paradigm to empower the
clinician to prevent and treat infectious diseases.

Bacteria that are resistant to current antibiotics have inhibit protein synthesis or DNA replication. More
Riboswitch
An mRNA control element wreaked havoc in the clinic and are a primary cause of recently, fatty-acid biosynthesis has been proposed as
that changes conformation in death in the intensive-care units of our hospitals world- a viable bactericidal target. Lysine analogues have also
response to the binding of a wide1,2. Currently, most infections are caused by impor- been identified that target lysine riboswitches and inhibit
metabolite (for example, tant bacterial pathogens, such as Staphylococcus aureus, bacterial growth10. Further study of such new targets is
glycine, lysine and
coenzyme B12) and influences
that are penicillin resistant, and up to 50% are resistant an important element in the development of new drugs.
gene expression. to stronger drugs, such as meticillin3. Meticillin-resistant However, all these strategies target bacterial cellular
S. aureus (MRSA) infections have reached epidemic processes that are crucial for microbial survival. In our
Microbiota levels this autumn (2007) in some parts of the United current battle against infectious diseases, clinicians are
The entire collection of
States, and are spreading through many sports centres, limited to the use of antibiotics that stimulate bacte-
microorganisms (bacteria,
archaea, fungi, sometimes schools and gymnasiums, affecting predominantly stu- rial evolution11,12. New tactics and weapons are needed
protozoa and viruses) that are dent athletes, but also younger schoolchildren, and have to combat bacteria that are, owing to evolution and
resident on or in the host. already caused deaths in a matter of weeks. Moreover, as selection, moving targets.
most bacterial strains are becoming resistant to multiple Targeting bacterial virulence is an alternative
antibiotics, including vancomycin (the current drug of approach to the development of new antimicrobials
choice for the treatment of MRSA), there is an urgent that can be used to disarm pathogens in the host13–15.
need for antibiotics that have new modes of action on The overall strategy is to inhibit specific mechanisms
therapeutic targets4,5. that promote infection and are essential to persistence
Currently used antibiotics were derived by screening in a pathogenic cascade (for example, binding, inva-
natural products and compound libraries against whole sion, subversion of host defences and chemical signal-
organisms, which identified compounds that have bac- ling), and/or cause disease symptoms (for example, the
teriostatic or bacteriocidal activity. Analogues of these secretion of toxins). Stripping microorganisms of their
*Department of Molecular parent drugs, which have improved potency, phar- virulence properties without threatening their existence
Microbiology, ‡Center for macological properties and efficacy against resistant may offer a reduced selection pressure for drug-resistant
Computational Biology and strains, have increased the number of antibiotics that mutations. Virulence-specific therapeutics would also
Department of Biochemistry
are clinically available6. However, the commercializa- avoid the undesirable dramatic alterations of the host
and Molecular Biophysics,
Washington University, Saint tion of new classes of antibiotics over the past 20 years microbiota that are associated with current antibiotics.
Louis, Missouri 63110, USA. has not met expectations, and current pharmaceuti- Indeed, the microbial cells that comprise the microbiota
§
Sequoia Sciences, Saint cal pipelines lack new, broad-spectrum antibiotics7–9. of a healthy human outnumber human cells by tenfold16;
Louis, Missouri 63114, USA. The antibiotics that are available today are primarily they colonize distinct sites throughout the body and
Correspondence to S.J.H.
e-mail: hultgren@borcim.
variations on a single theme — bacterial eradication confer numerous advantages to the host, some of which
wustl.edu based on different modes of action at the molecular are only beginning to be understood. The balance of
doi:10.1038/nrmicro1818 level. Some target cell-wall biosynthesis, whereas others bacterial populations in the gut, for example, influences

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Table 1 | Representative adhesive fibres, fibre classification and disease association


Adhesive fibre Assembly proteins Adhesin Organisms Associated diseases
Fibres that use the chaperone–usher pathway
Type 1 pili FimC and FimD FimH Escherichia coli, Klebsiella pneumoniae and Cystitis
Salmonella species
P pili PapD and PapC PapG E. coli Cystitis and pyelonephritis
Prs pili PrsD and PrsC PrsG E. coli Cystitis
S pili SfaE and SfaF SfaS E. coli Urinary-tract infection and
newborn meningitis
Hif pili HifB and HifC HifE Haemophilus influenzae Otitis media and
meningitis
Type 2 and 3 pili FimB and FimC FimD Bordetella pertussis Whooping cough
Pef pili PefD and PefC Unknown Salmonella enterica serovar Typhimurium Gastroenteritis
(S. typhimurium)
Long polar LpfB and LpfC Unknown S. typhimurium Gastroenteritis
fimbriae
MR/K(type 3) pili MrkB and MrkC MrkD K. pneumoniae Pneumonia
Myf fimbriae MyfB and MyfC Unknown Yersinia enterocolitica Enterocolitis
Alternate chaperone pathway
CS1 pili CooB and CooC CooD E. coli Diarrhoea
Extracellular nucleation-precipitation pathway
Curli CsgB (nucleator), CsgE CsgA (major subunit) E. coli Sepsis
and CsgF (assembly) and
CsgG (secretion)
Tafi AgfB (nucleator) AgfA (major subunit) Salmonella enterica serovar Enteritidis Mouse typhoid
General secretion pathway
Type 4 pili General secretion PilC Neisseria gonorrhoea Gonorrhoea
apparatus
Pilin protein Pseudomonas aeruginosa, Vibrio cholerae, Cholera
Mycobacterium bovis and Dichelobacter nodosis
Gram-positive pili
Pili LPXTG-motif-mediated Unknown Corynebacterium diphtheriae, Streptococcus Pneumoccocal
export and covalent mutans and Streptococcus pneumoniae diseases
polymerization
Tafi, thin aggregative fimbriae.

energy harvest and caloric intake through complex niche, as bacteria replicate and respond to host defences.
interbacterial metabolic networks17. The microbiota As the coordinated regulation of virulence-gene expres-
is dynamic, and shifts in the balance of microorgan- sion is dynamic and location specific in the host, it
isms that alter the sizes of different bacterial popula- should be possible to identify and target vital genetic or
tions — for example, the use of traditional antibiotic molecular bottlenecks — that is, to target the Achilles’
therapy — can lead to the loss of symbiotic benefits heel of a pathogen during infection18–20. Systems biol-
Pilus and the proliferation of disease-causing opportunistic ogy is engaged in mapping the genetic control networks
A non-flagellar filamentous pathogens. and molecular correlates of pathogenesis (ideally, using
appendage that is formed on A commitment to develop therapeutics that target parameters obtained from relevant in vivo models) to
the surface of many bacteria.
virulence requires a serious change in our perspective for drive the discovery of these bottlenecks and identify
Quorum sensing treating infectious diseases. Some elements of virulence new drug targets.
(QS). The process by which do seem to be fundamental for many pathogens, and Vaccination and other immunomodulatory strategies
bacteria use signalling drugs that target these elements should exhibit broader- are additional crucial avenues that are being pursued to
molecules to monitor bacterial
spectrum activity. However, many antivirulence drugs combat infectious disease and antibiotic resistance, both
density and coordinate gene
expression in a population- could be designed to target specific pathogens and the in immunocompetent and immunologically compro-
density-dependent manner. virulence factors that are unique to their pathogenic mised hosts. The complex relationship between host
cascades. In addition, virulence-gene expression is a immunity and microbial pathogenesis, the balance
Adhesin function of time and space throughout infection, in between protective immunity and immunopathology
The surface-exposed bacterial
molecule that mediates
which factors such as pili could function early to medi- and strategies to exploit the many networks that are
specific binding to a receptor ate adhesion, and others, such as toxin production and involved in antimicrobial strategies have been exten-
or ligand on a target cell. quorum sensing (QS), could operate later, and in a different sively reviewed21–24 and will not be discussed in detail

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Binding Pathogens are capable of presenting multiple adhesins


that can be expressed differentially to permit binding
in specific sites and at particular times over the course
of a complex infectious cycle. Thus, it may be difficult
Superficial to develop a universal class of anti-adherence drugs.
facet cell
Nevertheless, several specific pathogenic adhesive strat-
egies have emerged as hallmark requirements for viru-
Spread to new cells lence in certain infectious diseases that are immediate
Invasion and replication
targets for drug discovery and development.
Some bacteria present non-fimbrial adhesins
on their surface. These are expressed as monomeric
proteins or protein complexes that assemble at the
cell surface — for example, the Dr family of adhesins
Underlying that are expressed by Escherichia coli and are impor-
transitional cell
tant for adhesion in the intestine and urinary tract.
Adhesive autotransporters represent a class of afimbrial
adhesins that are expressed by various unrelated micro­
organisms, including species of Rickettsia, Bordetella,
Neisseria and Helicobacter and many members of the
Biofilm formation family Enterobacteriaceae. Haemophilus influenzae, a
causative agent of sinusitis, bronchitis and otitis media,
IBC expresses an adhesive autotransporter called Hap
that mediates binding to components of the host-cell
extracellular matrix.
Most adhesins, however, are incorporated into hetero­
polymeric extracellular fibres called pili or fimbriae.
Biomass dispersion Many distinct virulence fibres have been described
and cell exit in Gram-negative organisms25. Pili are also produced by
Figure 1 | Multi-step pathogenic cascade of uropathogenic Escherichia coli (UPEC). Gram-positive organisms (reviewed in REF. 26) and have
UPEC coordinate highly organized temporal and spatial events to Reviews
Nature colonize| Microbiology
the urinary been linked to virulence in Streptococcus pneumoniae27.
tract. UPEC bind to and invade the superficial facet cells that line the bladder lumen, Although bacterial fimbriae have diverse functions,
where they rapidly replicate to form a biofilm-like intracellular bacterial community many seem to be crucial to the binding and persistence
(IBC). In the IBC, bacteria find safe haven, are resistant to antibiotics and subvert of pathogenic microorganisms in the host (TABLE 1). In
clearance by host innate immune responses. UPEC can persist for months in a the following subsections, we review the importance of
quiescent bladder reservoir following acute infection and challenge current
pilus-mediated adhesion by E. coli in the pathogenesis
antimicrobial therapies. Quiescent bacteria can re-emerge from their protected
intracellular niche and be a source of recurrent urinary-tract infections. Insight into the
of urinary-tract infection (UTI) and discuss strategies
processes that accompany IBC formation and biofilm dispersal, as well as the factors to inhibit pilus biogenesis. We also describe the need for
that drive bacteria into the reservoir, may aid the design of preventive or therapeutic new approaches to prevent and treat UTI and examine the
strategies for recurrent infections. market considerations for antivirulence therapeutics.

Denying access to uropathogens. Uropathogenic E. coli


(UPEC) are the major causative agents of UTI and
here. In this Review, we highlight the diversity of the engage in a coordinated and regulated genetic and mol­
bacterial virulence mechanisms and consider their ecular cascade to assemble type 1 and P pili, which are
consequences in infectious disease. We review recent associated with infections of the bladder and kidney,
Autotransporter
A large family of secreted efforts towards antivirulence-based drug discovery in respectively (reviewed in REF. 28). Virtually all clinical
proteins in Gram-negative the framework of marketable drugs, and discuss the UPEC isolates express type 1 pili29. These are required
bacteria that harbour three challenges that remain and factors that are crucial to to bind mannose-containing host receptors, invade
functional domains — the developing the antivirulence therapeutic approach. host bladder-epithelial (urothelial) cells 30,31 and
amino-terminal signal peptide,
the secreted mature protein
initiate a pathogenic cascade that involves several
(passenger domain) and a Microbial attachment and invasion distinct phases as examined in the mouse cystitis
carboxy-terminal translocator The physical interaction between the pathogen and the model32,33 and human UTIs34 (FIG. 1). Within urothe-
domain — to allow secretion of host is crucial to the pathogenesis of virtually every lial cells, bacteria first replicate rapidly to form dense
the passenger protein.
bacterial disease. Specific proteins called adhesins biofilm-like communities and are protected from the
Biofilm mediate the adhesive interactions between the patho- flow of urine and host defences33,35. UPEC eventually
A community of cells that are gen and a cell-surface ligand on the host cell that is detach and then disperse, or flux, from the intracel-
attached to a surface or required for host colonization. The ability to impair lular bacterial community (IBC) to initiate new rounds
interface or to each other, and bacterial adhesion represents an ideal strategy to com- of IBC formation in other cells. Some fluxing bacte-
are imbedded in a self-made,
protective matrix of
bat bacterial pathogenesis because of its importance ria form filaments, evade neutrophil phagocytosis
extracellular polymeric early in the infectious process, and it is also suitable for and facilitate bacterial survival33,35. Even after acute
substances. implementation as a prophylactic to prevent infection. infection is resolved, bacteria can remain within the

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REVIEWS

Uroplakin E. coli

Type 1 pilus

Superficial facet cell


b
No compound Plus pilicide

N O CO2Li

Pilicide
Figure 2 | Targeting microbial adhesion. a | Pathogenic Escherichia coli use type 1 pili to bind to hexameric uroplakin
protein arrays on the surface of superficial facet cells that line the bladder lumen. Type 1 pili mediate the binding to, and
subsequent invasion of, these cells. b | Pyridone-based pilicides inhibit pilus biogenesis by disrupting chaperone–usher
Nature Reviews | Microbiology
protein interactions and dramatically reduce piliation levels. Image on the left in panel a reproduced, with permission,
from REF. 102  (1995) National Academy of Sciences. Image on the right in panel a reproduced, with permission, from
REF. 35  (1998) American Association for the Advancement of Science. Images in panel b reproduced, with permission,
from REF. 45  (2006) National Academy of Sciences.

bladder for many days to weeks, regardless of standard also necessary for the assembly of extracellular adhesive
antibiotic treatments, and can be implicated in recur- organelles in a wide range of other pathogens, includ-
rent infection36. It might be possible to target several ing species of Salmonella, Pseudomonas, Haemophilus,
factors in the pathogenic cascade to inhibit virulence. Klebsiella and Yersinia. Therefore, inhibitors of the
Type 1 pili represent an attractive drug target because the chaperone–usher system may serve as broader-range
bottleneck of invasion and IBC formation selects for fit- therapeutics, an attractive feature that would enhance
ness in the urothelium and type 1 pili are required for the marketability of an effective drug. Pilicides are a
both events. class of pilus inhibitors that target chaperone function
Two general strategies have emerged to inhibit and inhibit pilus biogenesis43,44. A new class of pilicides,
pilus-mediated function. The first is adhesion spe- based on a bi-cyclic 2‑pyridone scaffold, inhibit both
cific and involves physically precluding pathogen type 1 and P pili assembly in E. coli by targeting con-
binding to host cells. Carbohydrate derivatives of served regions on chaperones that are ubiquitous in the
host ligands, for example, have showed efficacy chaperone–usher pathways45 (fig. 2). These pilicides also
in blocking the adhesive properties of both type 1 inhibit biofilm formation in E. coli. The synthetic pili-
and P pili in biophysical and haemaglutination cides disrupt an essential protein–protein interaction
assays 38–40. This approach can be readily extended between the chaperone and usher at a site that has been
to other adherent organisms by tailoring the anti- identified by x‑ray crystallography. Thus, pilicides have
adhesive compounds to their receptor specificities the opportunity to work either at the level of attachment
in vivo. The goal of the second strategy is to inter- and invasion or the level of bacterial aggregation once
rupt pilus assembly, which also blocks pilus-mediated they are inside the superficial facet cells that line the
adhesion as well as invasion and intracellular biofilm bladder lumen.
formation.
Type 1 and P pili are both assembled by the chaperone– Therapeutic outlook for UTIs. The urinary tract is a com-
usher system41. Although the fim and pap operons, which mon site of infection in humans, and UTIs result in more
Chaperone–usher system are associated with type 1 and P pili, respectively, are than 8 million outpatient visits per year in the United States
A system that facilitates the
folding, transport and ordered
the best studied of these systems, 17 additional chaper- and, in the year 2000, expended costs of US$3.5 billion
assembly of pilus subunits at one–usher operons have been identified in sequenced for evaluation and treatment 46,47. Women are the
the cell surface. E. coli genomes42. The chaperone–usher systems are most frequent sufferers — a female has a 50% chance

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REVIEWS

of developing an acute UTI during her lifetime — and a Toxin transcription


many experience recurrent infections. With advancing V. cholerae
age and co-morbidities, UTI becomes progressively
more common among both women and men, particu-
larly in the context of urinary catheterization. Limited
treatment options are available for patients with chronic Transcription
and recurrent UTIs. Although these patients can be initiator
treated using prolonged courses of antibiotics, this
Bacterial
radically disrupts the symbiotic host–microorganism toxin
balance and may be accompanied by the evolution of
drug-resistant organisms in the urinary tract48. In addi-
tion, UTIs have a strong causal correlation with systemic b Toxin trafficking and function
infection and sepsis if antibiotic therapy is ineffective49.
Because infection of the bladder is not limited to extra-
cellular colonization, and can involve both the invasion
of host cells and the subversion of host defences and
other specific mechanisms, such as filamentation, to
promote persistence and re-invasion, it may be essential
to target key bottlenecks, such as type 1 pili expression
and/or assembly. Current research efforts are underway
to identify other candidate nodes in the network of these
host–pathogen interactions.
The treatment of recurrent or chronic UTIs, in
which intracellular bacterial reservoirs already exist,
might require a synergistic therapy, such as combining Receptor
mimic
a pilicide, for example, with a stimulator of the immune
response or epithelial-cell renewal (to drive bacteria
out of intracellular niches). Scientific research and new Host
membrane
drug development for UTIs accompany a clear clinical
demand, and are associated with large patient popula-
tions for clinical-stage development and the potential
long-term profits that effective therapies will produce. Host toxin
We anticipate that drug-discovery and development receptor
efforts for UTIs and other chronic infections will
increase markedly over the next 5–10 years.

Bacterial toxins Intracellular


Toxin-powered pathogens can exert a devastating effect toxin target
from a distance. For many infectious diseases, the clinical
Intracellular
symptoms and cause of tissue damage can be attributed inhibitor
to the action of secreted bacterial toxins. Botulinum, Figure 3 | Targeting toxin-powered pathogens. a | The
anthrax, diphtheria, tetanus, cholera and Shiga toxins inhibition of toxin transcription, as described
Nature Reviews for Vibrio
| Microbiology
are produced by distinct bacterial pathogens, and each cholerae, is one way to inhibit the consequences of toxin-
is a major cause of cellular malfunction and morbidity in mediated virulence. b | Neutralizing toxins, or preventing
afflicted individuals50. In addition, their extreme toxicity their trafficking and/or enzymatic activity, at cellular
makes these toxins potential weapons for use in biologi- targets is an alternative strategy to inhibit toxin damage to
cal warfare or a terrorist attack. Multiple opportunities the host.
exist to prevent toxin damage to the host (FIG. 3).

Targeting a toxin transcription factor. Vibrio cholerae is particularly needed. The small molecule virstatin
infection is characterized by severe diarrhoea and dehy- was discovered by screening for inhibitors of toxT gene
dration, which becomes life threatening if effective treat- expression. ToxT is a transcription factor that activates
ment of the symptoms is delayed. Cholera toxin provides the gene transcription of both cholera toxin and another
V. cholerae with its hallmark virulence and triggers the V. cholerae virulence factor, the toxin-co-regulated pilus52.
debilitating symptoms of the disease by interacting with Thus, virstatin inhibits cholerae-gene expression, the
G proteins and cyclic AMP in the intestinal lining to earliest step in toxin production.
interrupt proper ion transport, which results in mas- Selective inhibition of gene expression is a general
sive fluid loss51. The potent toxin has been recognized strategy that can be implemented for many virulence
by Hung and colleagues52 as an ideal candidate for drug factors, as expression is controlled by the environment
development, which, as resistance has emerged to the that is sensed by the organism. In the clinic, however,
antibiotics of choice, ciprofloxacin and azithromycin, such a therapy may have a short window of opportunity

nature reviews | microbiology volume 6 | january 2008 | 21


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REVIEWS

to work. On the one hand, after an extended time, bac- for antibody neutralization. The best defences against
teria may reach high numbers and produce sufficient toxin-empowered pathogens that are encountered in the
toxin to overwhelm the host, such that inhibition of toxin community, or as potential bioweapons, might require
production alone may be too late. On the other hand, a better understanding of pathogenesis and toxin bio-
effective and selective therapeutics could be useful chemistry. For many toxins, new research is needed to
prophylactically in limiting epidemics. provide higher-resolution models of toxin trafficking
than that presented in FIG. 3 (Ref. 55). In this regard,
Neutralizing toxins using antibodies. An antivirulence chemical genetic approaches are driving the discovery
strategy has been the treatment of choice for the post- of toxin-trafficking inhibitors and, in turn, these small
exposure treatment of tetanus, diphtheria and botulinum molecules are being used as tools, or molecular scalpels,
toxins. Antibodies against the toxins are administered to to dissect the mechanistic details of toxin trafficking.
patients to attempt to neutralize toxins while infection Selective luciferase-based screening assays, for example,
is cleared53. In addition to simply neutralizing toxins, have identified lead compounds that inhibit the actions
benefit to the host may be ascribed, in part, to a more of E. coli’s Shiga toxin once it has gained access to the host
sophisticated host response that includes the antibody- cell, and therefore permit protein synthesis56,57. These
mediated recognition and potential clearance of patho- compounds stop the toxin in real time along its path
gens and their toxic cargo, as reviewed elsewhere22–24. The from the cell surface to the endoplasmic reticulum, and
neutralizing antibodies are typically obtained from the provide a unique opportunity to examine toxin-transport
sera of immunized horses or humans, but the produc- processes in more detail. The ability to target toxins after
tion of monoclonal antibodies is an attractive alternative. they have entered host cells, but before they exercise their
Many monoclonal-antibody therapies are already in use function, provides an additional opportunity in time and
for preventing transplant rejection and oncological treat- space to control the sequelae of toxin action.
ment; an antiviral monoclonal antibody, palivizumab, Improved models of the assembly and function of
which prevents respiratory syncytial virus in children, anthrax toxin, produced by the Gram-positive Bacillus
was licensed in 1998. anthracis, provide several distinct checkpoints that can be
Currently, adult cases of botulism, a rare infectious targeted for interception55. Peptides and small-molecule
disease that is caused by Clostridium botulinum, are inhibitors have been identified that bind to lethal factor
treated with an antitoxin that contains horse antibod- (one of the three anthrax toxin components) and inhibit
ies raised against type A, type B and/or type E strains the in vitro enzymatic activity that is linked to patho-
of botulinum neurotoxins. This drug is available from genesis in vivo58–60. Several molecules have been reported
the Centers for Disease Control and Prevention (CDC), to block endosomal acidification, and so inhibit the
United States, if specially requested by a physician. It is conformational changes that are required for pore for-
listed in the Official Monographs of the United States mation at the host-cell surface, and anthrax-toxin entry.
Botulism Pharmacopeia, even though it has never been examined Having multiple ways to target toxin-powered pathogens
A rare, but serious illness that in controlled human clinical trials. Owing to the equine encourages the development of new and diverse thera-
is caused by a nerve toxin,
antitoxin’s potentially serious side effects, it has not typi- peutics that promise to treat a large number of infectious
botulinum, that is produced by
the bacterium Clostridium cally been used to treat infants suffering from botulism. diseases.
botulinum. However, a landmark in antivirulence therapy occurred
on 23 October 2003. The Food and Drug Administration Focus on bioterrorism. The dangers that are associated
Chemical genetics in the United States licensed an antivirulence drug that with an anthrax bioterrorism threat were made clear in
The strategy of using small
molecules to alter and
contained antibotulinum-toxin antibodies produced 2001 when B. anthracis was disseminated throughout the
interrogate biological from humans to the California Department of Health United States postal system leaving five dead and many
processes. The small-molecule Services, based on their seminal clinical research that ill. Anthrax toxins, along with botulinum toxins, are
tools of dissection in this spanned approximately 15 years. A placebo-controlled classified in the highest risk category of biological war-
approach harbour the precious
clinical trial consisting of 122 infants with botulism fare agents by the CDC, and nearly $6 billion have been
chemical scaffolds that may
lead directly to new demonstrated a profound clinical benefit for patients allocated by Project BioShield to develop and stockpile
therapeutics. treated with human botulism immune globulin (BIG), antibiotics, antitoxins and vaccines for these and other
subsequently named BabyBIG54. Clinical results showed high-threat agents61. Vaccination efforts are not generally
Project BioShield a reduction in the length of hospital stay, duration of promoted, however, because the chance of exposure to
The Project BioShield Act was
incorporated into law by the
intensive care, duration of mechanical ventilation, dura- any particular bioterrorism agent is remote.
United States government in tion of tube or intravenous feeding and hospital charges PharmAthene was founded in 2001 to develop
July 2004. Through Project per patient. This remarkable clinical evidence strongly technologies and products to address these biosecu-
BioShield, $5.6 billion will be supports a treatment strategy of neutralizing bacterial rity needs. Their new antivirulence drug, Valortim,
invested by 2013 in the
toxins post-infection. is a human monoclonal antibody that has demon-
development of new
technological and therapeutic strated prophylactic and, separately, post-infection
countermeasures against Targeting toxin trafficking and function. Antibody- protection against B. anthracis toxins in rabbit and
potential bioterrorism agents neutralization strategies will fall short for some patients non-human primate models 62. It has successfully
and to purchase and stockpile and infectious diseases. Toxins that are delivered completed Phase I safety and pharmacokinetic evalu-
effective therapeutics to
prevent and treat the illnesses
through the type III secretion system (T3SS; discussed ation. In 2006, Cangene Pharmaceuticals received a
that are related to these below), for example, are delivered directly into host cells, 5-year, $362-million contract from Project BioShield
threats. do not circulate globally and are not ideal candidates to develop and produce 200,000 doses of a potentially

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improved botulinum antitoxin, a heptavalent antitoxin Type III secretion


that is derived from horses (containing antibodies to The Shiga and cholera toxins discussed above are
7 toxin subtypes), because of its potential use as a exported by type II secretion, a general secretion system
bioterror agent. Its initial order was formally received through which extracellular enzymes are also secreted65.
into the United States Strategic National Stockpile in However, other pathogens deliver their virulent cargo
September 2007. using the T3SS. This system orchestrates the export and
delivery of virulence factors, or effector proteins, from
Scientific leaps and commercialization hurdles. the bacterial cytoplasm across the inner membrane, the
Antibody-based toxin neutralization (discussed above), peptidoglycan and outer membrane and, finally, through
could be considered to be a simple approach to target the host-cell plasma membrane, directly into the host-
toxin-based virulence that does not require a high- cell cytosol in the manner of a molecular syringe66.
resolution roadmap detailing toxin trafficking and sub- The T3SS machinery is used by many Gram-negative
cellular targets. However, toxin-associated diseases are pathogens, such as E. coli, Salmonella enterica serovar
often not that simple. They can be extremely complex, Typhimurium, Shigella flexneri, Pseudomonas aeruginosa
with cascading symptoms that may or may not lead to and species of Yersinia and Chlamydia. Yersinia species,
life-threatening complications. Consequently, the Anti- such as Yersinia pestis, the causative agent of plague,
Infective Drugs Advisory Committee meeting held on inject effector proteins called YOPs (Yersinia outer pro-
12 April 2007, when considering the operational details of teins) through the T3SS into host cells to inhibit the host
executing clinical trials to demonstrate the effectiveness immune response and help them to evade a potent
of a new antivirulence therapy, emphasized the pitfalls host response and subsequent clearance67,68.
and hurdles that might impede the commercialization The conserved elements in the T3SS machinery that
of any antivirulence therapy that targets small patient allow the interchange of T3SS components among some
populations63. At this meeting, research results were bacteria69,70 suggest that it may be possible to design
presented for two separate monoclonal antibodies that broad-range T3SS inhibitors that are effective in treat-
are under development to treat Shiga-toxin-producing ing disparate pathogens, regardless of the unique effec-
bacterial infections. Shiga toxin is produced by Shigella tor proteins that they deliver. Small-molecule inhibitors
dysenteriae and Shiga-toxin-producing E. coli (STEC), of the T3SS have been described in Yersinia pseudotu-
which includes the prototype strain E. coli O157:H7 that berculosis71,72. More recently, the discovery that small-
has caused several food-borne outbreaks around the molecule T3SS inhibitors in Yersinia spp. inhibit the
world. The toxin is secreted into the host cell and inhibits T3SS in Chlamydia trachomatis supports the notion of
host protein synthesis, which can lead to multiple clinical developing broader-range T3SS inhibitors. Interestingly,
manifestations, such as gastrointestinal disease, bloody the role of the T3SS in the pathogenesis of Chlamydia
diarrhoea, the destruction of red blood cells and plate- spp. infection is not well understood, owing, in part, to
lets, and haemolytic uraemic syndrome64. Treatment using the inability to manipulate Chlamydia spp. genetically.
antibiotics is controversial owing to the bolus of toxin However, in the previously mentioned study, the small-
that could be released as bacteria die, which, potentially, molecule inhibitors were recruited using chemical
could overwhelm the patient’s defences. genetics to assess the potential importance of the T3SS in
Even though evidence of efficacy from animal models C. trachomatis infection. Treatment with the T3SS
was presented at this meeting, representatives from com- inhibitor did inhibit the in vivo pathogenic cascade
panies and the physicians on the advisory committee and resulted in a decrease in secreted effector proteins,
were unable to successfully construct an appropriate suggesting that the T3SS was inhibited and is essential
clinical-trial design to evaluate these potential drugs. to the C. trachomatis infectious cycle73–75. These T3SS
The two main challenges for the successful completion inhibitors will be a useful tool for further study of the
of clinical trials seem to be, first, that primary end- T3SS in Chlamydia spp., and might represent target
points for this specific disease in humans are unclear leads for future therapeutics.
and, second, that achieving statistical significance may
not be attainable because the low incidence of this dis- Biofilms and chronic infections
ease prevents adequate patient enrolment. The findings Biofilms are complex, organized bacterial assemblies
of this meeting strongly suggest that researchers should that are highly resistant to antibiotics and host defences.
target the virulence mechanisms that are involved in Biofilms can form on abiotic surfaces, such as surgical
infectious diseases with clear and measurable clinical implants and catheters, and result in persistent infec-
end-points in sufficiently large patient populations. tions that are difficult to treat, thereby leading to further
Haemolytic uraemic These realities of commercialization seem to make health complications and longer hospital stays. Bacteria
syndrome
developing drugs against STEC and other complicated in chronic wounds, which are particularly prevalent in
A disease that primarily affects
infants and children and is diseases that affect small patient populations less attrac- the elderly and diabetic populations, form biofilms that
characterized by the loss and tive compared with diseases that affect large patient prevent proper healing76. Indeed, the ability of bacteria
destruction of red blood cells. populations, such as UTIs and chronic ear and sinus to persist robustly in biofilm communities in the human
Occurs most commonly in infections. These hurdles will accompany the develop- host and the environment, even against antibiotic pres-
children after a gastrointestinal
infection or upper respiratory-
ment of future antivirulence products, and imminent sure, is a fundamental observation that extends across the
tract infection and can lead to decisions surrounding the potential Shiga drugs will field of microbiology and poses serious challenges to
kidney failure. influence the course of future endeavours. the control and treatment of chronic infectious disease.

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Table 2 | Selected two-component response systems (TCRSs) involved in virulence


Histidine-kinase sensor and Organisms Associated virulence property
response regulator
AgrC; AgrA Staphylococcus aureus and Control of most aggressive virulence factors
Staphylococcus epidermidis
AlgD; AlgR Pseudomonas aeruginosa Alginate
RcsC; RcsB Escherichia coli Capsule
BvgS; BvgA Bordetella pertussis Toxins, adhesins and colonization factors
PhoQ; PhoP P. aeruginosa, Salmonella Divalent cation sensing, modification of
enterica serovar Typhimurium, lipopolysaccharide and resistance to antimicrobial
Yersinia pestis and Vibrio peptides
cholerae
TCRSs involved in the regulation of resistance to antibiotics
VanS; VanR Enterococcus faecalis Vancomycin resistance
VncS; VncR Streptococcus pneumoniae Vancomycin resistance
RprX; RprY Bacteroides fragilis Tetracycline resistance

The intracellular biofilms that are formed by UPEC Quorum sensing


in the urinary tract after their invasion of the bladder QS is the illustrative term that is used to describe the
epithelium and smaller numbers of quiescent bac- chemical signalling that takes place among bacteria to
teria that are harboured in underlying reservoirs keep track of their cellular density. QS is mediated by the
are implicated in the aetiology of recurrent UTIs36. production and subsequent recognition of small molecules
Clinical studies of recurrent infection of the middle- called autoinducers, and is used to coordinate gene expres-
ear tissue in children indicate that chronic otitis sion and regulate the numerous processes that are involved
media stems from a persistent biofilm following the in community behaviour and virulence, for example,
first infection77. Helicobacter pylori biofilms have been motility and biofilm formation (reviewed in Refs 82,83).
documented in the biopsied gastric mucosa of patients In nature, the red seaweed Delisea pulchra produces
suffering from gastric ulcers, and it has been suggested chemical compounds called furanones that intercept QS
that the ulcers are manifestations of these biofilms 78. signals and prevent microbial colonization84,85. This natural
In addition, patients with cystic fibrosis are threatened phenomenon has inspired the search for compounds that
by P. aeruginosa biofilm-mediated chronic lung infec- selectively inhibit QS, biofilm formation and the virulence
tions, which are responsible for the high morbidity and of human pathogens during infection86.
mortality of patients with cystic fibrosis79,80. P. aeruginosa is frequently encountered in noso-
Several strategies have emerged to inhibit biofilm comial infections and lung infections in patients with
formation and eradicate established biofilms. These cystic fibrosis, and produces more than 30 QS‑regulated
include, but are not limited to: preventing the initial virulence factors. Numerous studies, predominantly car-
adherence of bacteria to either a surface or another ried out over the past 5 years, strongly support the notion
bacterium (discussed above); interrupting QS mech- that QS inhibitors, including isolated and synthetic
anisms that are required for the gene expression of furanones, can be effective in treating bacterial infec-
biofilm components (discussed below); inhibiting the tions in vivo87,88. Notably, P. aeruginosa biofilms exhibit
biosynthesis of the integral polysaccharide and pro- increased susceptibility to sodium dodecyl sulphate
teinaceous extracellular components of the biofilm and the antibiotic tobramycin if treated with a synthetic
matrix81; and identifying or tailoring enzymes that furanone, compound C‑30 (Ref. 87). In a mouse pulmo-
can degrade the biofilm matrix, thereby rendering nary infection model, treatment with compound C‑30
bacteria accessible to traditional antibiotics and/or resulted in increased bacterial clearance87. Thus, inhibi-
immune clearance. tion of QS can increase the susceptibility of biofilm bac-
If we examine the anti-infective marketplace, the teria to host defences and antibacterial agents. Givskov
health and economic impact of multidrug-resistant and colleagues87 have suggested that direct targeting of
organisms, particularly in the hospital setting, is QS might be effective as an early prophylactic treatment
driving the primary market for drug development. of individuals who have P. aeruginosa infections in the
However, markets that encompass recurrent and lungs, on implants or in wounds.
chronic bacterial infections consist of significantly Other examples underscore the challenge of interrupt-
larger patient populations that have considerable ing the right signals at the right times to reduce bacterial
unmet clinical needs and provide attractive opportu- virulence in the host. A non-native N‑acylated‑l-homo-
nities for drug-development efforts. Effective biofilm serine lactone, for example, was recently discovered that
inhibitors could dramatically change treatment regi- can either inhibit or strongly induce QS in the marine
mens for many infectious diseases and benefit large symbiont Vibrio fischeri, depending on the molecule
patient populations. concentration89. Results from a panel of compounds in the

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AIP their environment91. TCRSs can control host invasion,


drug resistance, motility, phosphate uptake, osmoregula-
tion, nitrogen fixation and other functions. More than
4,000 TCRSs have been identified in approximately 400
AgrC 1 sequenced bacterial genomes92,93. Selected TCRSs that
regulate virulence are provided in TABLE 2.
The essence of signal transduction lies in the recogni-
tion and interpretation of environmental signals that are
AgrB related to host infection, and conversion of those signals
into specific protein–protein interactions and tran-
Cytoplasm scriptional activation94. Bacterial transduction systems
Histidine- 2 generally consist of receiver domains that are covalently
kinase P linked to effector domains (FIG. 4). Specifically, each
sensor 3
TCRS is composed of a histidine kinase that is activated
by extracellular signals in the host environment and a
response regulator that, in turn, transmits the signal to
AgrA Response regulator
the intracellular target to modulate the gene expression
of virulence factors. The interfaces between response
regulators and their protein modulators can be targeted
Nucleus
to prevent phosphorylation and/or activation, as well
4 as the interfaces of homodimer formation of activated
P
response regulators that are required for the control of
gene expression. Broad-spectrum inhibitors could non-
AgrA selectively target the TCRSs that are common to many
Gene RNAIII agrB agrD agrC agrA bacteria. More selective inhibitors could target the inter-
regulation
action of specific phosphorylated response regulators to
prevent expression of a specific virulence gene95.
Figure 4 | Pairing quorum sensing and two-component signalling in the A TCRS is an integral component of the QS system in
Nature Reviews | Microbiology
staphylococcal agr system. Staphylococcus aureus uses a two-component response Gram-positive bacteria that responds to bacterial density,
system (TCRS) to mediate quorum sensing (QS). The regulation of QS involves the and is named agr for accessory gene regulator. The global
production of an autoinducer and an increase in its concentration, expression of RNAIII regulator agr controls the expression of most virulence
and the subsequent regulation of QS genes. S. aureus produces an autoinducing peptide genes in staphylococci and is activated by secreted
(AIP) that accumulates extracellularly and activates the TCRS. The TCRS involves signal
auto­inducing peptides (AIPs) called thiolactones that
recognition by a histidine kinase (AgrC) (1), followed by histidine phosphorylation (2) and
phosphotransfer to a response regulator (AgrA) (3), which then binds to the RNAIII
comprise 7–10 amino acids96. AgrC and AgrA are the
transcript that encodes a small RNA that functions to modulate gene expression of histidine-kinase sensor and response regulator, respec-
S. aureus genes (4). tively, of the TCRS. AIP thiolactones have been suggested
as leads for the development of S. aureus therapeutics that
are urgently needed, owing to the fact that nosocomial
same family elicited varied QS responses, which implies and community-acquired MRSA infections are on the
that the rational design of modified QS antagonists or rise. The agr system also functions to downregulate fac-
agonists may not be straightforward; new insights are tors that are important in biofilm formation, and agr
needed to fully understand these responses. In addition, dysfunction is associated with increased biofilm produc-
redundancies among QS systems can render inhibitors tion97. As discussed above, a more detailed understanding
of a single system ineffective. Indeed, two of the three of the organism’s control networks is needed to identify
parallel QS systems in V. cholerae are dispensable for host the ideal genetic or molecular checkpoints that need to
colonization and the production of two hallmark viru- be targeted to reduce virulence in the host.
lence factors, cholera toxin and the toxin co-regulated The prevention of virulence mechanisms that promote
pilus90. QS circuits are being examined in numerous antibiotic tolerance could also improve the efficacy of cur-
models to determine if similar redundancies exist and to rent antibiotics, and, particularly, slow down the processes
what extent individual chemical signals influence multi- that lead to drug resistance. Several TCRSs are responsible
ple QS pathways. The generation of QS circuit diagrams for inducing the gene expression that confers bacterial tol-
may reveal viable drug targets and small molecules erance to antibiotics. Vancomycin resistance, for example,
that may prove useful as scalpels to probe the roles of QS is triggered by the VanR–VanS TCRS. VanS detects the
in different systems. glycopeptide antibiotic and VanR activates the expression
of the enzymes VanA, VanH and VanX, all of which are
Two-component response systems required for resistance98,99 (reviewed in REF. 11). The TCRS
A central requirement of bacterial virulence is the ability proteins and the three enzymes cooperate to synthesize an
to express subsets of genes in response to signals that are altered peptidoglycan framework that is tolerant to vanco-
specific for a particular environment. Two-component mycin exposure, which is a hallmark of the vancomycin-
response systems (TCRSs) seem to be the dominant resistant enterococci and vancomycin-resistant S. aureus
mechanism by which bacteria and fungi respond to that have emerged in the clinic.

nature reviews | microbiology volume 6 | january 2008 | 25


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Conclusions and perspectives In battling infection, disabling microbial virulence


This era may come to be remembered as one in which in vivo could shift the advantage to the host and render
infectious diseases made a dramatic worldwide resurgence, bacteria impaired in defeating host defences. Alternatively,
owing to the rise of antibiotic resistance and emergence of antivirulence drugs could be used in combination therapy,
new diseases. We must increase the number of available in which bacterial clearance is mediated by standard anti-
therapeutics, protect the effectiveness of current antibiot- biotics and the symptoms of virulence are suppressed. The
ics and, importantly, decrease further pressure for the evo- effective deployment of antivirulence drugs will require
lution of new drug-resistance mechanisms. Neutralizing rapid diagnosis in the clinic of the organism (or organ-
bacterial toxins by using antibodies has emerged as the isms) that is responsible for infection and may include
most-pursued antivirulence therapeutic strategy in indus- profiling of its virulence gene or genes100. Such routine
try, with at least six candidates undergoing clinical trials. and rapid diagnosis would also improve the use of stand-
The initial successes of these antitoxins seem to provide ard antibiotics, and represents a necessary investment as
empirical evidence that supports increased research into medicine improves and becomes more personalized.
other antivirulence approaches. Therefore, it is impera- We must succeed in this continuous war against
tive that we determine the mechanisms of virulence and infectious disease. A recent review of the antibacterial
the consequences of host–pathogen interactions from drug-development efforts by GlaxoSmithKline reveals, in
both the pathogen and host perspective. Insights regard- essence, that we have underestimated our opponent and
ing bacterial virulence and pathogenesis have emerged, that arrogance has dominated the search for new antibi-
yet many crucial questions remain unanswered. Major otics101. Regaining our competitive advantage will require
breakthroughs will require multi-disciplinary tools us to see beyond what we currently accept as dogma. It
from bacterial and host genetics, structural biology and will also depend on the removal of perceived obstacles to
in vivo imaging, animal models, cell biology, immunol- infuse the industry with new opportunities. We need to
ogy, biochemistry, chemical genetics, functional genom- accept that a one-drug-fits-all strategy will probably fail.
ics and systems biology. Atomic-level details of the We also need to consider each specific disease, pathogen
structural interactions at host–pathogen interfaces are and virulence mechanism, and combine the strengths
vital for the discovery of effective antivirulence drugs by of synergistic therapies to minimize the evolution by
structure-based drug design for proposed targets. pathogens of resistance.

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58. Tonello, F., Seveso, M., Marin, O., Mock, M. & Rev. Microbiol. 4, 249–258 (2006).
Montecucco, C. Screening inhibitors of anthrax lethal A holistic perspective of the potential roles of QS Competing interest statement
factor. Nature 418, 386 (2002). signals, from a cooperative to the battleground. The authors declare competing financial interests: see web
59. Panchal, R. G. et al. Identification of small molecule 83. Bassler, B. L. & Losick, R. Bacterially speaking. Cell version for details.
inhibitors of anthrax lethal factor. Nature Struct. Mol. 125, 237–246 (2006).
Biol. 11, 67–72 (2004). A creative composition and a lively review of the
60. Turk, B. E. et al. The structural basis for substrate and business of bacterial communication. DATABASES
inhibitor selectivity of the anthrax lethal factor. Nature 84. Kjelleberg, S. et al. Do marine natural products Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
Struct. Mol. Biol. 11, 60–66 (2004). interfere with prokaryotic AHL regulatory systems? entrez/query.fcgi?db=genomeprj
61. Russell, P. K. Project BioShield: what it is, why it is Aquat. Microb. Ecol. 13, 85–93 (1997). Bacillus anthracis | Chlamydia trachomatis | Clostridium
needed, and its accomplishments so far. Clin. Infect. 85. Manefield, M. et al. Evidence that halogenated botulinum | Escherichia coli | Haemophilus influenzae |
Dis. 45, S68–S72 (2007). furanones from Delisea pulchra inhibit acylated Helicobacter pylori | Pseudomonas aeruginosa | Shigella
62. US Securities and Exchange Commission (SEC). homoserine lactone (AHL)-mediated gene expression by dysenteriae | Shigella flexneri | Staphylococcus aureus |
Pharmathene, Inc. Form 10-Q, Quarter Ended displacing the AHL signal from its receptor protein [in Streptococcus pneumoniae | Vibrio cholerae | Vibrio fischeri |
September 30, Commission File Number 001-32587. process citation]. Microbiology 145, 283–291 (1999). Yersinia pestis | Yersinia pseudotuberculosis
SEC web site [online], http://www.sec.gov/Archives/ 86. Higgins, D. A. et al. The major Vibrio cholerae
edgar/data/1326190/000110465907083136/a07- autoinducer and its role in virulence factor production. FURTHER INFORMATION
29212_110q.htm#Item2_103948 (2007). Nature 14 Nov 2007 (doi:10.1038/nature06284). Scott J. Hultgren’s homepage: http://hultgren.wustl.edu/
63. Food and Drug Administration Center for Drug 87. Hentzer, M. et al. Attenuation of Pseudomonas public
Evaluation and Research. Summary Minutes of aeruginosa virulence by quorum sensing inhibitors. All links are active in the online pdf
the Anti-Infective Drugs Advisory Committee on EMBO J. 22, 3803–3815 (2003).

nature reviews | microbiology volume 6 | january 2008 | 27


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Getting organized — how bacterial


cells move proteins and DNA
Martin Thanbichler* and Lucy Shapiro‡
Abstract | In recent years, the subcellular organization of prokaryotic cells has become a focal
point of interest in microbiology. Bacteria have evolved several different mechanisms to target
protein complexes, membrane vesicles and DNA to specific positions within the cell. This
versatility allows bacteria to establish the complex temporal and spatial regulatory networks
that couple morphological and physiological differentiation with cell-cycle progression. In
addition to stationary localization factors, dynamic cytoskeletal structures also have a
fundamental role in many of these processes. In this Review, we summarize the current
knowledge on localization mechanisms in bacteria, with an emphasis on the role of polymeric
protein assemblies in the directed movement and positioning of macromolecular complexes.

Bacteria are among the most successful and widespread physiological requirements of the bacterial cell. In this
organisms on the Earth. In the course of several billion Review, we highlight key mechanisms of cellular organi-
years of evolution, they have adapted to almost every zation in bacteria that reflect the striking momentum
possible biological niche and developed an astonishing that bacterial cell biology has gained in recent years.
range of metabolic pathways, life cycles and cell mor- Specifically, we will discuss pathways that mediate the
phologies. As a result of continuous selection for fast assembly and positioning of localized protein com-
reproduction rates, they have adopted highly stream- plexes and set the foundation for temporal and spatial
lined architectures and small genomes that have high regulatory processes within bacterial cells. In this
coding densities. Their straightforward organization context, emphasis will be placed on the growing number
has frequently been regarded as indicative of a primitive of dynamic cytoskeletal elements that have been iden-
cellular state. Consequently, advanced features, such as tified in bacteria and their pivotal roles in subcellular
cytoskeletal structures, intracellular transport proc- organization, DNA segregation and cell division.
esses and spatially regulated transcription and protein
localization, were traditionally thought to be restricted to Subcellular organization in bacteria
eukaryotic cells. However, this simplistic view was difficult The realization that bacteria use specifically localized
to reconcile with the complexity of many processes that protein complexes to orchestrate cellular processes led to
are found in bacteria. Notably, regulation of cell shape, cell a surge of research on the mechanisms that structure the
polarization and asymmetric cell division are common bacterial cell (BOX 1). Among the many proteins with une-
*Max Planck Institute for phenomena in bacterial development that are unlikely to ven subcellular distribution that have been identified in
Terrestrial Microbiology, occur without dedicated temporal and spatial regulatory recent years, two major classes can be distinguished. One
Karl-von-Frisch-Straße,
35043 Marburg, Germany.
systems or dynamic cytoskeletal elements. group forms largely stationary complexes that localize to

Department of Recent work, driven by technological advances precisely defined subcellular positions, even though their
Developmental Biology, that have facilitated the resolution of structural details subunits might be in rapid exchange. Proteins belonging
Stanford University School of within bacterial cells, has revealed that bacteria have to the second class, by contrast, are part of highly dynamic
Medicine, Beckman Center
indeed evolved mechanisms to actively control cell-cycle interaction networks with components that continuously
B300, 279 Campus Drive,
Stanford, California 94305, progression and morphological differentiation. Some change their subcellular location in a temporally and
USA. of the factors that are involved in these processes are spatially controlled manner.
Correspondence to M.T. homologues of well characterized eukaryotic proteins,
e-mail: thanbichler@ but they are frequently used outside of their established Assembly of stationary protein complexes. Most sta-
mpi-marburg.mpg.de
doi:10.1038/nrmicro1795
functional contexts. Other factors are found exclusively tionary complexes that have been characterized so far
Published online in bacteria and constitute novel regulatory and struc- are based on integral membrane proteins. They usu-
3 December 2007 tural systems that have evolved to meet the specific ally assemble at the new pole (resulting from the most

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Forespore recent division), the old pole, the cell-division site or the Diffusion and capture. During B. subtilis sporula-
Precursor of the spore; a forespore septal membrane in the case of Bacillus subtilis tion many proteins localize differentially to the septal
resting cell that is highly sporulation, where they mediate the establishment of membrane, which provides an amenable system for
resistant to a number of cellular organelles, perform localized catalytic activities investigating the principles that underlie directed pro-
environmental stresses, such as
heat, ultraviolet irradiation and
or serve regulatory functions. But how do these proteins tein positioning within the cell (FIG. 1a). SpoIVFB, a pro-
desiccation. assume a defined intracellular position? Evidence has protein-processing enzyme, is synthesized in the mother
been presented that at least two localization mechanisms cell, but must be localized in the septal membrane to
Single-molecule tracking operate in bacteria: diffusion and capture; and targeted carry out its function. Rudner and colleagues1 reasoned
Microscopic analysis of the
membrane insertion. During diffusion and capture, that SpoIVFB is either targeted to the septal membrane
movement of individual
fluorescently labelled
newly synthesized proteins are first inserted randomly directly upon synthesis or is first inserted randomly
molecules within a cell. throughout the cytoplasmic membrane and then, after around the mother-cell cytoplasmic membrane and
diffusion, they are captured by an interaction with a pre- then, upon diffusion, captured at the septal mem-
viously localized membrane complex1,2. Targeted mem- brane. To distinguish between these two possibilities,
brane insertion, by contrast, describes a process whereby SpoIVFB was expressed from an inducible promoter
a protein is delivered directly to its destination by trans- in vegetatively growing cells, in which it was found to
location to a given cellular site. In all cases, a crucial issue be randomly positioned in the cytoplasmic membrane.
is the identity of the determinant that is responsible for During initiation of sporulation in these cells, and in the
the localization of the membrane protein and how this absence of the inducer, the existing SpoIVFB accumu-
determinant is positioned in the first place. lated at the septum, demonstrating that diffusion and
capture is the probable mode of SpoIVFB localization
(FIG. 1b). Additional evidence that SpoIVFB diffuses to
Box 1 | Model systems in bacterial cell biology its site of action was provided by taking advantage of
the fact that, when engulfment occurs, the membrane
Although an increasing number of species are studied with respect to their cell biology,
surrounding the forespore is separated topologically
most information on cellular organization in bacteria is currently based on studies in
Escherichia coli, Bacillus subtilis and Caulobacter crescentus. The characteristics of these from the mother-cell cytoplasmic membrane from
model systems are listed below. which it is derived1. SpoIVFB that was synthesized after
engulfment was specifically enriched in the mother-cell
E. coli
• Long history as a bacterial model organism. cytoplasmic membrane, but was absent from the outer
forespore membrane, again suggesting that diffusion
• Large collection of genetic tools, mutant strains and methods.
and capture, rather than targeted insertion, is the mode
• Extensive body of knowledge on many aspects of its physiology. of SpoIVFB localization.
B. subtilis Evidence for the diffusion-and-capture mode of pro-
• Sporulation as a model for cellular differentiation. tein localization also comes from single-molecule tracking
• Well-studied physiology. of the Caulobacter crescentus membrane histidine kinase
• Large size, which facilitates the resolution of cytoskeletal structures. PleC, which localizes dynamically to the pole that is
opposite the stalk at specific times in the cell cycle2.
C. crescentus Following the movement of a PleC–green fluorescent
• Asymmetric cell division (see the figure). protein (GFP) fusion protein over a timescale of seconds
• One round of chromosome replication per cell division. revealed two diffusion coefficients: a high coefficient for
• Abundance of dynamically localized regulatory protein complexes. molecules that were without directional bias and a lower
• Synchronizability. coefficient for molecules that were targeted to the cell
In the course of its life cycle, C. crescentus differentiates from a mobile, flagellated pole. However, the components that mediate the sink at
swarmer cell into a sessile stalked cell (see the figure). Subsequently, it undergoes an the cell pole have not yet been identified.
asymmetric cell division that regenerates the stalked cell and creates a new swarmer Diffusion and capture might be a widespread mecha-
cell. The stalked cell immediately enters a new round of cell division, whereas the nism that operates in several different systems. For
swarmer cell needs to develop into another stalked cell to start the next division cycle. example, many components of the bacterial cell-division
The single polar flagellum is shed during the swarmer-to-stalk-cell transition and apparatus are known to assume an even subcellular dis-
re-established at the pole opposite the stalk in the predivisional cell. tribution unless a defined precursor complex has assem-
bled to capture them at mid-cell3. The same principle may
also apply to soluble proteins, which can be tethered to
a pre-localized targeting factor after three-dimensional
Swarmer cell diffusion through the cytoplasm or periplasm.

Targeted membrane insertion. Whereas SpoIVFB and


Stalked cell other proteins that are synthesized in the mother-cell
region of sporulating B. subtilis cells seem to find their
septal destination by diffusion and capture, the septal
Stalk
Flagellum localization of the SpoIIQ protein, which is synthesized
in the forespore, is achieved by direct insertion and
capture4. SpoIIQ was found to act as a localization fac-
Nature Reviews | Microbiology
tor that recruits another mother-cell protein, SpoIIIAH,

nature reviews | microbiology volume 6 | january 2008 | 29


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a B. subtilis sporulation b Diffusion and capture c Zipper model domain exposed to the host cytoplasm. During synthe-
sis, it is directly targeted to the region around the cell
pole. Concomitantly, proteolysis of IcsA occurs over the
A
whole-cell perimeter, in effect clearing away any protein
Q
that has diffused away from the pole9,10. IcsA localizes
Forespore independently of its export from the cytoplasm to the
FB
outer membrane10. It has been proposed that a region
within the amino‑terminal domain interacts with an
A Q
as-yet-unidentified factor in the polar region of the cell,
FB thereby targeting IcsA to its defined site of secretion
A Q
before translocation occurs11.
Stationary complexes usually have locally confined
activities or serve as regulatory landmarks. By contrast,
global processes, such as morphogenesis and macromo-
Q lecular transport, require the assembly of scaffolds that
extend throughout the cell. These structures are formed
A by dynamic cytoskeletal filaments that adopt specific
Q overall arrangements but are highly variable on a short-
Outer forespore membrane
term scale. They provide force as well as directionality,
Figure 1 | Protein localization to the septal membrane during Bacillus subtilis and serve as tracks for the localization of other proteins. In
sporulation. During sporulation (a), B. subtilis undergoesNature Reviews | Microbiology
an asymmetric cell division the following section, we will focus specifically on protein
that generates two offspring of unequal size and developmental fate — a larger filaments that are involved in the determination of bacte-
mother cell (green) and a smaller forespore (blue). The mother cell subsequently rial-cell shape; other dynamic structures are discussed in
engulfs the forespore in a phagocytosis-like process, thereby surrounding it with a BOX 2 and in the later sections of this article.
second membrane, the so-called outer forespore membrane. During this process,
several proteins specifically localize to the interface between the two compartments Dynamic protein scaffolds and cell shape
(b). In several cases, their positioning is mediated by diffusion and capture, as
A major role in subcellular organization has recently
exemplified by the recruitment of the freely diffusible mother cell protein SpoIVFB
(FB; purple spheres) by a pre-localized complex including the forespore protein been attributed to actin-like proteins that belong to the
SpoIIQ (Q). The interaction of proteins across the mother cell and forespore MreB family. They are found in many bacteria, in which
membranes involves a zipper-like mechanism (c). Using this principle, SpoIIQ engages they assemble consistently into spiral-like structures that
the mother cell factor SpoIIIA (A), which, in turn, is responsible for the septal line the inner face of the cytoplasmic membrane. MreB
localization of other proteins, among them SpoIVFB. homologues play an important part in the regulation
of cell shape and have been implicated in cell polarity
and chromosome segregation12–18. Their function in
to the forespore septal membrane5. Based on the direct morphogenesis is dependent on the presence of another
interaction between the extracytoplasmatic domains of scaffold that is formed by the extracytoplasmic protein
SpoIIQ and SpoIIIAH, a zipper-like mechanism has been MreC and is modulated by accessory factors, such as the
proposed as a method that is used to capture SpoIIIAH4–6 intermediate filament protein crescentin.
(FIG. 1c). This interaction subsequently recruits additional
factors that are synthesized by the mother cell (including The bacterial actin-like cytoskeleton. In eukaryotes, the
SpoIVFB)6,7, which yields a protein network that anchors actin cytoskeleton consists of a highly dynamic network
localized proteins at the septum. However, the zipper of filaments, the assembly of which is regulated by the
mechanism alone is not sufficient to direct the forespore binding and hydrolysis of ATP and is further modulated
Protofilament protein SpoIIQ to its septal position. Interestingly, it has by an abundance of accessory factors. Actin protofila-
The basic polymeric unit of a
been shown that all of the membrane proteins that are syn- ments possess an intrinsic polarity, whereby monomers
filamentous structure; consists
of a linear row of monomers. thesized in the forespore are initially targeted to the septal are preferentially added to one end and released from
membrane, from which they slowly diffuse to the rest the other in a phenomenon that is called treadmilling19.
Fluorescence recovery after of the forespore membrane, unless they are retained by a Although bacterial and eukaryotic actin homologues
photobleaching specific localization factor4. In agreement with this obser- share minimal sequence identity (~15%), studies on
(FRAP). A method used to
study the dynamics of
vation, a component of the protein translocation com- MreB from Thermotoga maritima revealed that their
polymeric structures. A region plex, FtsY, is specifically localized to the forespore septal three‑dimensional structures and protofilament organi-
within a filament that has been membrane in sporulating B. subtilis cells8. Thus, certain zation are strikingly similar20,21 (FIG. 2a). Nevertheless,
assembled from fluorescently forespore proteins, such as SpoIIQ, appear to be directly the overall assembly properties of T. maritima MreB
labelled monomers is bleached
targeted to their site of action by localized translocation. deviate from the scheme that has been established for its
by illumination with a high-
intensity laser. Subsequently, Another example of directed protein targeting is pro- eukaryotic relatives with respect to several biochemical
fluorescence microscopy is vided by the intracellular pathogen Shigella flexneri. To parameters22.
used to monitor the kinetics facilitate movement within the host cell, this bacterium MreB cables are highly dynamic in vivo and assume
of fluorescence recovery that positions the protein IcsA at its old cell pole, where it several different architectures, comprising spiral-like
results from the substitution of
bleached by unbleached
directs polymerization of host actin into a tail-like assemblies of varying curvature and length as well as
subunits in the course of structure, thereby generating a pushing force. IcsA is short coils, arcs and rings12,14,15,23. The mechanisms
filament turnover. an outer membrane protein that has its amino-terminal and cellular cues that are responsible for these diverse

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Box 2 | Linear arrangement of subcellular compartments — magnetosomes mreB gene, this Gram-positive bacterium synthesizes
three actin homologues, MreB, Mbl and MreBH, which
There are many examples of a interact with each other and assemble into a single
intracellular membrane helical filament12,13,26,27. Fluorescence recovery after photo­
compartments in bacteria,
bleaching (FRAP) studies revealed a rapid exchange of
including the chlorosomes
and chromatophores of non-
subunits within Mbl cables that occurred uniformly
oxygenic photosynthetic and without any evidence of polarity with respect to
bacteria and the filament growth23. Furthermore, MreB and Mbl spirals
magnetosomes of were observed to rotate in the cell, with one full turn
magnetotactic bacteria. It is b every 50–60 seconds13,26. Additional support for the
largely unknown how these rapid turnover of MreB cables comes from a study that
structures are established and analysed the movement of single MreB–GFP molecules
maintained at their proper in live cells of C. crescentus28. The MreB subunits were
subcellular position. In the found to migrate directionally along arc-like trajectories,
case of magnetosomes,
which is suggestive of treadmilling of individual subunits
however, a clearer picture of
the underlying mechanisms is starting to emerge.
through stationary MreB filaments. However, the average
Nature Reviews | Microbiology length of these trajectories was significantly shorter than
Magnetosomes are vesicles — formed by invagination of the cytoplasmic membrane123
— that are filled with biomineralized magnetite or greigite and allow bacteria to orient the MreB cable as a whole, and no common directionality
themselves in the Earth’s magnetic field124. The best-studied magnetotactic organisms, was observed relative to the overall polarity of the cell. In
Magnetospirillum magneticum and Magnetospirillum gryphiswaldense, contain 15–20 C. crescentus, and possibly other bacteria, the actin-like
magnetosomes, which are arrayed in linear order parallel to the longitudinal axis of the cytoskeleton is therefore probably composed of numer-
cell and approximately centred at mid-cell123,125. This arrangement generates a compass ous short, polarized and highly dynamic protofilaments
needle-like structure that has an overall magnetic dipole moment that equals the sum of that associate laterally but in random orientation to each
the magnetic dipole moments of the individual vesicles126. As magnetosomes have an other. The overall dynamics of this structure might be
inherent tendency to agglomerate to reduce their magnetostatic energy, the cell must
based on the rapid exchange of protofilament bundles
possess specific mechanisms to stabilize this linear assembly and anchor it within the cell.
Electron cryotomography analyses unveiled filamentous structures that run
rather than individual protein monomers (FIG. 2c).
alongside magnetosome chains123,125 (see the figure). The protein that is responsible
for their formation has been identified as MamK (purple in the figure), a bacterial Regulation of cell-wall biosynthesis by actin-like proteins.
actin homologue that belongs to an evolutionarily distinct group of proteins that is The arrangement of actin-like proteins into large fila-
clearly separated from the MreB family123. It is conserved in all magnetotactic ments and their dynamic positioning within the cell are
bacteria and assembles into linear polymers that extend from one cell pole to crucial for the spatial regulation of peptidoglycan biosyn-
another123,127. In agreement with a role for MamK in magnetosome alignment, thesis in most rod-shaped bacteria (BOX 3). This is due, in
mutants that lack the protein are still able to produce mature magnetosomes, but part, to a direct role in the positioning of the enzymes that
fail to arrange them into a linear array. Recent work in M. gryphiswaldense has are involved in cell-wall formation. In B. subtilis, MreBH
identified a protein, MamJ, that might be involved in attaching magnetosomes to
was shown to interact with the peptidoglycan hydrolase
the MamK filament (green in the figure)125. It is an acidic protein, with a repetitive
primary sequence which localizes as a linear structure that extends from pole to
LytE27. A LytE–GFP fusion is normally found in the
pole. In a mutant strain that lacks a cluster of genes that are involved in form of distinct bands at the division septa and in heli-
magnetosome biogenesis (among them mamK) this linear arrangement is abolished, cal patterns along the lateral cell wall. Upon deletion of
and MamJ is dispersed throughout the cytoplasm. Its cooperation with MamK in the mreBH gene, however, these lateral helical structures
vesicle positioning is supported by the fact that deletion of mamJ largely reproduces are lost. As strains that lack LytE have the same defects
the phenotype of a mamK mutant, leading to the dispersal of magnetosomes in cell shape as mreBH mutant cells, localization of the
without affecting their maturation. As a result, MamK appears to function as a track hydrolase along the helical tracks of MreBH appears to
that recruits vesicles with the help of MamJ, thereby defining the overall be essential for its proper function. So far, this is the only
orientation of the magnetosome chain. It is thought that empty and immature proven example of an immediate influence of actin-like
vesicles are initially randomly distributed along the MamK filament (see the figure,
proteins on peptidoglycan biosynthesis. In many cases,
part a). The accumulation of magnetite (beige spheres in the figure) results in
magnetostatic interactions between individual vesicles, which leads to their
their effect seems to be more indirect and due to a role
aggregation into densely packed chains (see the figure, part b) that are in the localization of MreC, a protein that is frequently
subsequently stabilized by additional factors (blue in the figure )125. Figure adapted, encoded in the same operon as mreB.
with permission, from Nature REF. 125  (2006) Macmillan Publishers Ltd.
The role of MreC in bacterial morphogenesis. MreC
is a membrane protein that is composed of a single,
patterns are unclear. In C. crescentus15,16, and its relative amino‑terminal transmembrane helix and a large extra-
Rhodobacter sphaeroides24,25, the transitions between the cytoplasmic domain in Escherichia coli and B. subtilis29,30,
different MreB structures were found to be synchro- but is a soluble periplasmic protein in C. crescentus31.
nized with cell-cycle progression (FIG. 2b). By contrast, Crystallographic data indicate that it might have the
in bacteria from other phylogenetic groups, the actin- potential to form polymeric structures30. MreC is part of
like cytoskeleton seems to be largely unaffected by the a complex that includes the poorly characterized proteins
developmental state of the cell. MreD and RodA, and is essential for survival in most
The first insights into the turnover kinetics of actin- bacteria that have been investigated; its inactivation
like filaments were provided by studies on B. subtilis. results in a loss of cell shape and subsequent lysis29,31,32.
Whereas most Gram-negative bacteria possess one In support of a role in cell-wall biosynthesis, MreC

nature reviews | microbiology volume 6 | january 2008 | 31


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REVIEWS

a Model for MreB treadmilling of a multi-enzyme peptidoglycan biosynthetic com-


(+) (–)
**
plex, thereby organizing the formation of new cell-wall
material.
MreB–ATP The spatial arrangement of MreC is dependent on the
MreB–ADP
**
positioning of actin-like cables within the cell, although
the underlying mechanisms seem to vary among dif-
ferent bacteria. In E. coli, MreC interacts directly with
MreB and seems to be required for the proper assembly
**
of MreB into helical filaments29. Similarly, MreC forms
helical structures33 that colocalize and physically associate
with Mbl cables in B. subtilis26. The functional relevance
** of this interaction is underscored by the observation that
both Mbl and MreC depletion results in the same type
of morphological defects33,34; these are caused by the
b MreB dynamics in C. crescentus disruption of peptidoglycan biosynthesis in the longi-
tudinal parts of the cell33. In C. crescentus, an extremely
different situation is observed. Here, MreC assembles
into helical structures that do not associate with MreB
cables but, on the contrary, seem to avoid them35. The
mechanisms that underlie this effect are still unknown.
In accordance with biochemical-interaction studies31,
PBP2 was shown to colocalize with MreC in wild-type
C. crescentus cells31,35. However, the depletion of MreC
does not disrupt the already existing spiral-like structures
c Model for the architecture of MreB cables of PBP2, although it does specifically affect the position-
ing of newly synthesized molecules35. Thus, MreC seems
to guide peptidoglycan-synthesizing enzymes to the sites
of active cell-wall growth, where they might subsequently
be retained by interaction with other proteins or nascent
peptidoglycan strands.

Crescentin. Recent work in C. crescentus identified a


protein, named crescentin, that shares the structural
Figure 2 | Dynamics of actin-like filaments in bacteria. MreB Nature Reviews
and | Microbiology
its orthologues characteristics of intermediate filament proteins and acts
polymerize into bundles of two or more protofilaments that are arranged in parallel to as a modulator of cell shape36. Although poorly conserved
each other (a). Their assembly follows the same principles that have been observed for on the primary-sequence level, these proteins share a
actin. Polymerization occurs exclusively in the ATP-bound state (blue spheres). However, common architecture that comprises a long central
soon after incorporation into the filament, MreB hydrolyses the nucleotide, thereby
coiled-coil region that is flanked by variable head and
changing into its ADP-bound conformation (purple spheres). Subunit exchange is
tail domains. Their polymerization occurs spontaneously
restricted to the ends of the filament, and the addition of new monomers occurs
preferentially at the positive-end, that is, the cap which consists of MreB subunits that are and does not require nucleotides or exogenous nucleation
still associated with ATP. Subunit release, by contrast, is largely restricted to the negative- factors37. Similar to its eukaryotic homologues, crescentin
end of the filament, which is composed of MreB–ADP. In the steady state, assembly and assembles into long filaments in vitro, which illustrates
disassembly proceed at equal rates, resulting in the apparent sliding of individual its tendency to associate laterally into small bundles36.
subunits (marked by asterisks) through the filament while the overall polymer length Localization studies revealed that the protein is positioned
remains constant — a phenomenon that is called treadmilling. In Caulobacter crescentus, specifically at the inner curvature of the crescent-shaped
MreB cables exhibit a highly dynamic localization pattern (b). Newborn cells contain C. crescentus cell. Upon inactivation of crescentin, the cells
spiral-like cables that extend between the two poles. Upon initiation of cell division, lose their typical crescentoid morphology and grow in the
these structures are lost and MreB condenses into a ring at the future division site. Later,
form of straight rods, which indicates that crescentin is
as cell constriction progresses, the zone of MreB localization gradually expands again,
responsible for establishing cell curvature36. The mecha-
leading finally to the establishment of new spirals in the two incipient daughter cells.
Biochemical evidence suggests that actin-like cables consist of numerous treadmilling nism that allows this cytoskeletal structure to redirect
filaments that are arranged side by side in a random orientation (c). peptidoglycan biosynthesis remains to be established.

Morphogenetic function of the cell-division apparatus. In


addition to the systems discussed so far, the cell-division
interacts directly with the peptidoglycan synthase penicillin- apparatus has an important role in the regulation of cellu-
binding protein (PBP) 2, and several other high- and low- lar morphology. Not only does it comprise peptidoglycan
molecular-weight PBPs (BOX 3), in C. crescentus31. Similar synthases and hydrolases that mediate the formation of
results have been obtained in B. subtilis, although MreC the division septum3, but, in C. crescentus, it also directs a
appears to associate preferentially with high-molecular- division-independent mode of peptidoglycan biosynthe-
weight PBPs in this organism30. These findings suggest sis that leads to zonal growth around the cell centre38. As
that the protein serves as a scaffold for the formation MurG, the enzyme that catalyses the last cytoplasmic step

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Box 3 | Cell-wall biogenesis

Transglycosylase

GlcNAc MurNAc GlcNAc MurNAc GlcNAc MurNAc


4 1 4 1 4 1 4 1 4 1 4 1

Transpeptidase

GlcNAc MurNAc GlcNAc MurNAc GlcNAc MurNAc


4 1 4 1 4 1 4 1 4 1 4 1

The shape of a bacterium is determined by the architecture of its cell wall (peptidoglycan) —Nature
a rigidReviews
meshwork| Microbiology
that is
formed by linear glucan strands that are crosslinked by short peptide bridges128 (see figure). Peptidoglycan biosynthesis
initiates in the cytoplasm with the assembly of a lipid-bound disaccharide–pentapeptide precursor. This basic building
block comprises the two aminosugars N‑acetylglucosamine (GlcNAc) and N‑acetylmuramic acid (MurNAc), which are
connected by a β‑1,4-glycosidic bond and a conserved five-amino-acid peptide that is attached to the lactyl group of the
MurNAc unit. Once completed, the precursor is transported across the cytoplasmic membrane and, after release from its
lipid carrier, incorporated into the peptidoglycan superstructure. This task is accomplished by a group of enzymes that are
called penicillin-binding proteins (PBPs), which include transglycosylases and transpeptidases. Transglycosylases catalyse
the formation of another β‑1,4-glycosidic bond between the disaccharide moiety of the precursor and the end of an
existing glycan strand. As a result, linear polymers of alternating GlcNAc and MurNAc units are generated, which are all
arrayed in parallel to each other. The pentapeptides, which protrude perpendicularly from the sugar backbone,
subsequently function to interconnect neighbouring glycan strands. To this end, they are linked in a pair-wise manner by
peptide bonds that are formed with the help of transpeptidases. High-molecular-weight PBPs exhibit both
transglycosylase and transpeptidase activity, whereas low-molecular-weight PBPs (such as PBP2) function as
transpeptidases only. Growth of the bacterium requires continuous remodelling of the peptidoglycan envelope. Bacteria,
therefore, also possess a series of autolytic enzymes, such as glycosidases, peptidases and amidases, that selectively
cleave the molecular meshwork and thereby facilitate the insertion of additional cell-wall material.
Peptidoglycan
Meshwork of highly crosslinked
glycan strands that constitutes of peptidoglycan formation, was found to be associated Plasmid segregation. The first indication that replicated
the bacterial cell wall.
with the cell-division apparatus throughout the period DNA might be inherited in a non-random way came
Penicillin-binding protein of zonal growth38,39, medial cell-wall extension might be from work on low-copy-number replicons. Localization
A protein involved in promoted by restricting the delivery of peptidoglycan studies revealed that the copies of these plasmids are
peptidoglycan biosynthesis precursors to the mid-cell region. However, future studies actively separated from each other and positioned in
that is targeted and inactivated are necessary to determine whether this process is addi- the incipient daughter-cell compartments before cell
by the antibiotic penicillin and
its derivatives.
tionally supported by the recruitment of a specific set of division occurs41–46. In all cases that have been inves-
peptidoglycan synthases from the periphery to the cell tigated in detail, partitioning relies on the activities of
Centromere centre. MurG is also enriched at the cell-division site of three different plasmid-encoded factors — a centro-
A region of a DNA molecule E. coli40, which suggests that mid-cell growth zones could meric sequence, a centromere-binding protein and an
that is attached to the
be a common phenomenon in bacteria. ATPase that interacts with the centromeric nucleopro-
DNA-segregation apparatus.
tein complex. Despite this common theme, the nature
Walker ATPase Bacterial DNA segregation of the individual factors and the segregation apparatus
This ATPase is characterized by Evidence has accumulated that, in addition to protein varies considerably among different plasmids. The most
the presence of two conserved complexes, plasmids and chromosomes are also actively important difference concerns the ATPase component,
sequence motifs (Walker A and
Walker B motif), which form
positioned within bacterial cells. As for morphogenesis, which can be either a Walker ATPase (type I partitioning
parts of the nucleotide-binding dynamic cytoskeletal filaments have emerged as essential system), a member of the actin superfamily (type II plas-
pocket. factors in the underlying localization mechanisms. mid-partitioning system) or a tubulin homologue.

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a b
ParM–ATP

Assembly ParM–ADP

ParR

GTP hydrolysis

Catastrophe Stabilization

Figure 3 | Plasmid segregation by the actin homologue ParM. Unlike actin polymers, which eventually reach a steady
Nature Reviews
state that is characterized by an equilibrium between subunit addition and dissociation (treadmilling), | Microbiology
ParM filaments
continuously cycle between phases of rapid growth and complete disassembly (a). Biochemical analyses showed that
only the ATP-bound form of ParM (blue spheres) is able to polymerize efficiently. However, owing to the cooperative nature
of its ATPase activity, ParM rapidly hydrolyses its bound nucleotide once it has become integrated into a filament
(purple spheres). Nevertheless, the overall structure remains stable and continues to grow as long as its ends remain
capped by ATP-bound subunits. Once these are lost, for example, owing to a shortage in the supply of ParM monomers, the
filament starts to depolymerize rapidly (catastrophe). Stabilization of the caps, by contrast, prevents disassembly and
allows the polymer to continue elongation. ParR nucleoprotein complexes recruit filaments that have formed
spontaneously in the cytoplasm and promote their extension into long polymeric structures. As a consequence, plasmids
are pushed apart and moved to opposite ends of the cell (b).

Plasmid segregation by actin-like proteins. The best- surface and, owing to their dynamic instability, went
studied partitioning system that is based on an actin-like through cycles of rapid growth and shrinkage. However,
ATPase is that of the conjugative resistance plasmid R1. as soon as two of these beads came into proximity, the
Its centromeric region (parC) is bound by the DNA- filaments between them suddenly started to grow and
binding protein ParR47. This complex is then recognized push them apart. Elongation occurred exclusively at the
by ParM, an actin-like protein, the ATPase activity of interface between ParM and the centromeric nucleo-
which is essential for plasmid stabilization48,49. protein complex and was based on the stabilizing effect
In vitro, ParM polymerizes in an ATP-dependent of the ParR–parS complex on the filament ends, which
manner, forming filaments that are reminiscent of prevented the spontaneous disassembly of the polymer.
F‑actin50,51. Despite this similarity, its assembly kinetics These data suggest that the three-component partition-
have several distinct features. For example, the nuclea- ing system of R1 is sufficient to place plasmid copies at
tion of ParM is a rapid and spontaneous process51,52, opposite cell poles without the help of additional host
with filaments elongating in a symmetrical bidirectional factors (FIG. 3b).
fashion that is contrary to the polarized growth of actin52. Interestingly, the B. subtilis plasmid pBET131 was
In addition, ParM filaments never reach an equilibrium found to encode a novel actin-like partitioning ATPase
between association and dissociation, as is typical for named AlfA that lacks dynamic instability and has polym-
treadmilling actin polymers, but instead continuously erization kinetics that are similar to those of MreB55. Thus,
cycle between phases of rapid growth and complete dis- actin homologues have adopted different molecular
assembly52,53 (FIG. 3a). This behaviour, which is known as mechanisms to mediate the movement of sister plasmids
dynamic instability, has previously only been observed into the two daughter-cell compartments.
for eukaryotic tubulin54.
Localization studies have revealed that ParM can Plasmid segregation by Walker-type ATPases. Most
form axial filaments in E. coli51. Each filament is flanked non-actin-based partitioning ATPases are members of
Walker A cytoskeletal by two copies of plasmid R1 that appear to be pushed the so-called Walker A cytoskeletal ATPase (WACA) fam-
ATPases
apart towards the cell poles by the polymerization of ily (type I partitioning systems)56. Analogous to the R1
(WACA). A group of Walker
ATPases that possess a distinct ParM between them48. The biochemical mechanism partitioning system, each of these factors interacts with
version of the Walker A motif of this segregation process has recently been unravelled a DNA-binding protein that associates cooperatively
that deviates from the universal by reconstituting the R1 partitioning system in vitro53. with a cluster of conserved sites on its cognate plasmid.
consensus. These proteins When a DNA fragment that contained the R1 parS Despite differences in their primary sequences, overall,
share structural similarity with
P‑loop GTPases and are
region was attached to beads and mixed with ParR and WACA ATPases show similar behaviour in vitro. They
recognized as members of the ParM, the addition of ATP induced the formation of possess weak cooperative ATPase activity and are capable
GTPase superfamily. numerous short filaments that extended from the bead of polymerizing in an ATP-dependent manner, forming

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massive protofilament bundles that measure up to sev- a ori

eral micrometres in length. However, detailed analyses of


their polymerization dynamics and interactions with the
centromeric nucleoprotein complex have not yet been
performed. The biology of these systems has recently
been described in several excellent reviews57–59.
Although type I and type II partitioning systems ter
share some common principles, their modes of action are
fundamentally different. Whereas ParM assembles into b
stable axial filaments that push plasmids to opposite cell
poles48,53, WACA ATPases exhibit a highly dynamic locali-
zation pattern in vivo. SopA and ParA, which are encoded ori
ter ori
by the E. coli plasmids F and pB171, respectively, were
shown to oscillate back and forth across the nucleoid at
~20 minute intervals60,61. However, the molecular mecha- ter
nisms that couple the dynamic assembly of partitioning
proteins to plasmid stabilization remain to be elucidated. C. crescentus E. coli
Studies on the localization of plasmids F and pB171 Figure 4 | The arrangement of chromosomal DNA in
revealed that at the beginning of their division cycle most bacteria. Consecutive segments of chromosomal DNA are
Nature Reviews | Microbiology
cells contain a single plasmid cluster that is positioned at folded into supercoiled domains that are stacked on top of
each other and arranged into a circular superstructure (a).
the cell centre. Later on, this cluster is duplicated, and the
Consequently, the subcellular position of a locus correlates
newly generated copies are moved to the quarter positions
directly with its location on the circular chromosomal map.
of the cell. Cytokinesis then occurs halfway between the In Caulobacter crescentus, the origin of replication (ori) is
two clusters, resulting in daughter cells that each carry localized to the flagellated pole of the swarmer cell,
a single cluster at the mid-cell42,43,45. If the copy number whereas the terminus (ter) is found at the opposite pole (b).
of pB171 is increased artificially, cells accumulate up to Therefore, the left and right arms of the chromosome are
seven plasmid clusters that are evenly distributed along oriented in parallel to the long axis of the cell. In
the long axis of the cell62. This finding indicates that type I Escherichia coli, by contrast, ori and ter are both located at
partitioning systems do not move plasmids to defined mid-cell, such that the two arms of the chromosome flank
subcellular positions, as is the case for the R1 partition- the transverse cellular axis. Panel a adapted, with
permission, from REF. 77  (2006) Elsevier Science.
ing system, but rather distribute them to clusters that are
arranged in regular arrays within the nucleoid.
Interestingly, the cytoplasmic chemotaxis sensory
clusters of R. sphaeroides have a localization pattern that Arrangement of chromosomal DNA. Bacterial genomes
is strikingly similar to that of plasmids that are segregated are usually organized into circular chromosomes (one or
by type I partitioning systems. The proper positioning several) that are up to several megabase pairs in size. It has
of these clusters was found to be dependent on a pro- long been unclear how the cell packages these enormous
tein, called PpfA, that is homologous to WACA plasmid molecules into the confined space of its cytoplasm and
partitioning ATPases63, which suggests that bacteria can concomitantly ensures their faithful replication and par-
use similar mechanisms to arrange proteins and DNA titioning during each cell-division cycle. With the advent
within the cell. of site-specific labelling methods, it has become possible
to probe the arrangement of chromatin in a systematic
Plasmid segregation by a tubulin homologue. A third fashion66,67. An early study in B. subtilis determined the
class of plasmid-segregation systems is based on the subcellular location of four chromosomal loci and found
activity of tubulin homologues. Recent work showed that their arrangement followed a reproducible pattern68.
that plasmid pBtoxis from Bacillus thuringiensis serovar Conclusive evidence that chromosomal DNA is not dis-
israelensis encodes a protein — designated TubZ — that tributed randomly within the cell but in fact has a highly
shows significant similarity to tubulin64,65 and is essential conserved organization was provided by a large-scale
Nucleoid for its stable partitioning. TubZ assembles into highly analysis that investigated the subcellular positioning of
A distinct region within the
cytoplasm that harbours
dynamic filaments that translocate rapidly through the 112 chromosomal loci in C. crescentus69 (FIG. 4). This work
the chromosomal DNA. cell65. FRAP analyses revealed that filament migration showed that the origin of replication is invariantly found
is achieved by an actin-like treadmilling mechanism. at the flagellated pole of the bacterium and the terminus
Origin of replication However, it is unclear how the translocation of TubZ is located at the opposite end of the cell. Other loci are
A chromosomal site that serves
filaments is related to plasmid stabilization. arranged between these two fixed points in exactly the
as the starting point of the
bidirectional DNA-replication The unusual characteristics of TubZ, a tubulin homo- same order as on the circular chromosomal map. A simi-
process. logue with actin-like dynamics, and ParM, an actin lar correlation between the subcellular and chromosomal
homologue with tubulin-like dynamics, demonstrate positions of loci was revealed in E. coli. However, unlike
Terminus that bacterial and eukaryotic cytoskeletal proteins have C. crescentus, E. coli places the origin and terminus regions
A chromosomal region in which
the two replication forks meet
diverged considerably with respect to their function and at the cell centre, and the two arms of the chromosome
towards the end of DNA polymerization mechanisms, even though they share the (replichores) form separate domains that are located on
replication. same evolutionary origin and overall structures. opposite sides of its transverse axis70,71. The chromosomal

nature reviews | microbiology volume 6 | january 2008 | 35


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REVIEWS

architecture of other organisms has not been analysed in These individual complexes then aggregate into a single
detail yet, but it is likely that the linear ordering of loci is centromere-like superstructure in a ParA-dependent
a common principle among bacteria. manner86. Chromosomally encoded partitioning systems
So, how is this conserved arrangement established? are able to functionally replace plasmid-borne type I
Studies on the movement of newly duplicated chromo- partitioning systems and confer stability to intrinsi-
somal regions showed that the specific localization of each cally unstable plasmids, indicating that they indeed
site is established during the DNA-segregation process. constitute active segregation machineries87,88. In agree-
Immediately after the initiation of replication, the newly ment with this finding, the ParAB system was shown
synthesized origin regions are separated and rapidly to be required for proper transfer of the chromosomal
moved to their conserved positions in the incipient origin region into the forespore compartment at the onset
daughter cells69,72–75. Subsequently, the remaining bulk of of sporulation in B. subtilis89. In doing so, it cooperates with
the chromosome is duplicated progressively from the ori- a sporulation-specific protein, named RacA, that serves to
gin to the terminus region. Similar to the origin regions, tether the origin-proximal region of the chromosome to
the two new copies of each locus are quickly partitioned. the cell pole89–91. Moreover, vegetative cells of B. subtilis
They are condensed immediately after emergence from that are deficient in ParA or ParB fail to separate their
the replisome, segregated into the daughter-cell com- origin regions after duplication, although the bulk of the
partments and added successively to the newly replicated chromosome is left unaffected92.
DNA that has already been deposited there69,70,76. Unlike Evidence for a direct role of ParAB in origin seg-
eukaryotes, therefore, bacteria segregate their DNA while regation has come from the study of Vibrio cholerae, a
replication is in progress, with the two sister nucleoids bacterium that possesses two circular chromosomes, of
growing in layers that reflect the succession of loci on which each encodes its own type I partitioning system.
the chromosome. The mechanisms that mediate bacte- The smaller chromosome (ChrII) has the typical segre-
rial-chromosome dynamics are unclear, and several dif- gation pattern that is shared by low-copy-number plas-
ferent models have been proposed to account for the rapid mids, such as F and pB171, and the larger chromosome
and ordered movement of loci during the chromosome- (ChrI) exhibits dynamics that are similar to those of the
segregation process77. The current understanding is that C. crescentus chromosome80,93. Interestingly, ParBI, which
the two copies of the origin are actively separated from binds to 3 parS sites that are approximately 65 kb to the
each other and positioned in the incipient daughter cells, right of the replication origin of the large chromosome,
so that they serve as landmarks for the establishment of the is consistently localized closer to the cell poles than to
new sister nucleoids. The bulk of the chromosome then the origin itself and moves ahead of the origin during
follows as a consequence of DNA condensation, probably the segregation process94. These findings suggest that
supported by the activities of other factors. Finally, the two the ParBI–parS complex defines the chromosomal cen-
sister chromosomes are decatenated and actively cleared tromere and mediates segregation and polar positioning
from the cell-division site to allow cytokinesis to occur78. of the origin regions. ParBI and the origin regions lose
their polar localization in mutants that lack ParAI and
Chromosome segregation. The existence of an active are instead found near the cell centre before the start of
mechanism that mediates origin segregation is supported S phase and around the quarter positions in cells that
by the fact that, in all bacteria investigated, movement have initiated DNA replication. Therefore, ParAI seems
of the two origin copies occurs in a highly reproducible to be necessary for correct positioning of the ParBI–parS
and directed manner and is significantly faster than cell complex. Time-lapse analyses indeed showed that the
elongation69,79,80. One of the factors that has been impli- protein assembles into a dynamic polymeric structure
cated in this partitioning process is the cytoskeletal protein that seems to pull the moving ParB I–parS complex
MreB. Its overproduction, depletion16,34,81 or inactivation from the old pole towards the new pole94. In doing
by the antibiotic A22 (Refs 17,18) results in misplacement so, the ParAI structure first stretches throughout the
of the origin regions, and chromosome-segregation defects cell and then progressively retracts in the direction of
in various bacteria. Moreover, MreB was shown to inter- the new pole, with its lagging edge colocalizing with
act, directly or indirectly, with a chromosomal region that the moving ParBI–parS complex. Thus, a mitotic-like
flanks the origin of replication in C. crescentus17. However, mechanism that is based on type I partitioning systems
it has remained controversial whether these results actually seems to mediate origin segregation in V. cholerae and,
reflect a direct involvement in DNA segregation. presumably, other bacteria.
Other studies have provided evidence of a role for V. cholerae cells that lack ParAI only have minor
type I partitioning systems in chromosome segrega- growth and overall chromosome-partitioning defects94,
tion. In most bacteria, an operon encoding the WACA and similar observations have been made for most other
ATPase ParA and its cognate centromere-binding protein bacteria in which the type I chromosome-partitioning
ParB is located in the vicinity of the replication origin, systems had been inactivated82. These findings indicate
whereas conserved ParB-binding sites (parS) are scattered that active segregation of the newly synthesized origin
throughout the origin-proximal region of the chromo- regions is not necessarily required for the establish-
some82. Recent studies in B. subtilis demonstrated that, ment of two fully separated sister nucleoids. As a result,
upon binding to individual sites83, ParB spreads into the chromosome segregation might rely, in part, on other
flanking chromosomal regions, forming nucleoprotein mechanisms, the precise nature of which remains to be
complexes that cover up to 20 kilobases (kb) of DNA84,85. determined.

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a E. coli b C. crescentus The Min system. The first mechanism that was shown to
MinE control placement of the bacterial division site was dis-
MipZ
MinD FtsZ Nucleoid
Origin covered in E. coli. It is based on the activity of three pro-
teins, encoded by the minCDE operon, that cooperate to
establish a fascinating, self-contained oscillatory system98
FtsZ (FIG. 5a). MinD is an ATPase of the WACA family and
ParB interacts with MinC to form a membrane-associated, top-
MinC Release of MinCD Origin segregation ologically unspecific inhibitor of FtsZ-ring formation99,100.
Owing to the activity of MinE, however, the MinCD com-
plex is normally restricted to the polar regions of the cell,
thus only allowing cell division to occur close to the cell
centre101,102. MinCD continuously oscillates between the
two cell halves in 40–50 second intervals103–105. In doing
Assembly of a new patch Displacement of FtsZ so, it first assembles into a large polymeric patch at one
end of the cell. Subsequently, this cap-like structure starts
to shrink, gradually losing subunits from the pole-distal
end, until it has completely disappeared. Concomitantly,
a new MinCD assembly forms at the opposite pole, and
Start of the next cycle Z-ring formation the cycle starts again. MinE forms a circular structure that
follows the retracting edge of the MinCD patches101,106. In
its absence, the oscillatory behaviour of the system is lost,
and the MinCD complex is evenly dispersed throughout
the membrane103. This mechanism is widely used among
bacteria, albeit frequently in modified forms. B. subtilis,
Figure 5 | Positioning of the cell-division plane. In Escherichia for example, lacks MinE and positions the MinCD com-
Naturecoli, assembly
Reviews of the FtsZ
| Microbiology
ring is restricted to the mid-cell by nucleoid occlusion and the MinE-driven pole-to-pole plex statically at its cell poles107,108. Details on the molecular
oscillation of the cell-division inhibitor MinCD (a). By contrast, division-plane localization function of the Min proteins are provided in two recent
in Caulobacter crescentus is mediated by MipZ, an inhibitor of FtsZ polymerization that comprehensive reviews96,109.
forms a complex with the DNA-binding protein ParB at the chromosomal origin of
replication, thus exploiting chromosome dynamics for its subcellular positioning (b). Nucleoid occlusion. In mutants that lack a functional
Panel b adapted, with permission, from REF. 113  (2006) Elsevier Science. Min system, cell division occurs either close to the poles
or between nucleoids, but never on top of regions that
contain chromosomal DNA110. Recent studies identi-
To generate offspring that contain equivalent DNA, fied two unrelated proteins, Noc from B. subtilis111 and
the cell must not only provide a mechanism for proper SlmA from E. coli112, that mediate this so-called nucleoid
chromosome segregation, but also ensure that cytokinesis occlusion effect (FIG. 5a). In their absence, Min-deficient
occurs precisely between the two newly formed sister cells accumulate numerous FtsZ clusters that are ran-
nucleoids. The core machinery that mediates cell divi- domly distributed throughout the cytoplasm. These
sion is conserved in most bacteria. Nevertheless, several clusters frequently overlap with the nucleoids and, under
independent mechanisms have evolved to determine the certain conditions, promote cell-division events that lead
subcellular site of its assembly. to dissection of the chromosome. However, the inactiva-
tion of NocA or SlmA has little effect on FtsZ localization
Division-site placement in cells that have a functional Min system, which suggests
Formation of the bacterial-cell-division apparatus, called that nucleoid occlusion might serve as a fail-safe mecha-
the divisome, occurs in a multistep process that is initi- nism that ensures proper cell division under conditions
ated by assembly of the tubulin homologue FtsZ into a of unbalanced growth. However, how do Noc and SlmA
ring-shaped structure at the future division site. This function? Both proteins possess a helix–turn–helix DNA-
cytoskeletal element mediates the recruitment of all other binding motif that allows them to interact nonspecifically
divisome components, both directly and indirectly, and with chromosomal DNA and therefore to colocalize with
plays a crucial part in the subsequent constriction proc- the nucleoid. SlmA was shown to mediate the recruitment
ess3. Although the three-dimensional structures of FtsZ of FtsZ to the nucleoid if expressed at high levels and has
and tubulin are remarkably similar95, the two proteins a strong bundling effect on FtsZ filaments in vitro112.
show distinct polymerization behaviour. The assembly Nucleoid occlusion factors might, therefore, function by
dynamics of FtsZ have been discussed extensively in out-competing other cell-division proteins that bind to
several recent reviews56,96,97. Most importantly, FtsZ has and stabilize the FtsZ ring, thereby efficiently preventing
a high propensity to polymerize in vivo, forming spiral- the assembly of a functional divisome.
and arc-like structures that are in a constant process of
polymerization and disassembly. Their consolidation at Regulation of cell division by MipZ. There are several
Nucleoid occlusion
The inhibitory effect of the
the cell centre, which results from the destabilizing effect organisms that lack MinCDE homologues and nucle-
nucleoid on the formation of of negative regulators in the polar regions of the cell, is oid occlusion but yet still manage to divide properly in
the septal FtsZ ring. thought to provide the basis of septal-ring formation. the mid-cell region, which suggests the existence of

nature reviews | microbiology volume 6 | january 2008 | 37


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REVIEWS

alternative regulatory mechanisms. Recent work in regulatory complexes that define the cell-division plane.
C. crescentus has indeed identified a novel system that This astonishing range of mechanisms has been identi-
couples the assembly and positioning of the FtsZ ring to fied almost exclusively in a handful of established model
the initiation of chromosome replication and the bipolar organisms that represent only a small fraction of the bac-
positioning of the duplicated origin regions113 (FIG. 5b). terial world. Therefore, novel systems that are involved in
Its central component is MipZ, an essential ATPase the dynamic temporal and spatial organization of bacteria
that has weak similarity to ParA-like DNA-partitioning might await discovery in the future.
proteins and is widely conserved among alphaproteo- Although several different systems that mediate the
bacteria. MipZ directly interacts with the chromosome- spatial organization of bacteria have been identified, their
partitioning protein ParB113, which, in turn, binds to a function is often poorly understood. Notably, the factors
cluster of sites (parS) that are approximately 15 kb away that mediate the specific positioning of protein complexes
from the chromosomal origin of replication114,115. Together at the bacterial cell poles remain obscure. Recent work in
with the origin region, the resulting complex is positioned C. crescentus identified a membrane protein, TipN, that
at the old pole in newborn cells113. Initiation of DNA rep- interacts with the cell-division apparatus and remains
lication then generates two copies of the parS-containing attached to the new poles after cytokinesis, so serving as
segment, both of which are immediately decorated with a landmark that regulates the assembly and positioning
ParB and MipZ. During the subsequent segregation proc- of the flagellum in the two daughter cells117,118. However,
ess, one of these segments stays at the original position, even though this system provides an elegant way to
while the second copy moves rapidly across the cell to the pass on positional information to the next generation
opposite pole. MipZ interacts with ParB in a highly dynamic and to differentiate between the old and new cell pole,
manner that is regulated by ATP binding and hydrolysis113. the primary determinants that retain TipN at its polar
Consequently, it is not stably tethered to the origin regions position are still unknown. One of the structures that
but rather is distributed in a gradient, with its concentra- might help to define the cell poles is peptidoglycan. Its
tion progressively increasing towards the polar ParB–parS architecture at the curved polar regions is probably differ-
complexes. In vitro studies demonstrated that MipZ acts as ent from that in the straight longitudinal sections of the
an inhibitor of FtsZ polymerization113. Consequently, FtsZ cell, thus providing a specific target for peptidoglycan-
is consistently found in the subcellular region that exhib- binding proteins. In addition, there are data that
its the lowest concentration of MipZ. In newborn cells, it suggest a role for cardiolipin in polar protein localiza-
initially forms a focus at the new pole, opposite the tion. This minor lipid species was reported to accumu-
single MipZ–ParB complex that is located at the old pole. late, possibly owing to a self-organizing process119, in the
After duplication and segregation of the origin regions, polar regions of the cytoplasmic membrane in E. coli120
the polar FtsZ aggregate is disassembled and a new one and B. subtilis121, and it has been suggested that it medi-
is formed at the cell centre. Synthesis of the other cell- ates polar positioning of the osmosensory transporter
division proteins, which occurs later in the C. crescentus ProP in E. coli122. However, although both the peptidog-
cell cycle116, might then stabilize FtsZ into a septal ring lycan- and cardiolipin-mediated localization mecha-
that can establish a functional divisome and initiate nisms are appealing, future work is required to test their
cytokinesis. This mechanism ensures that the division site validity.
is positioned between the two nascent sister nucleoids. Aside from polar targeting, little information is avail-
Based on its homology to ParA-type WACA ATPases, and able on the biochemical mechanisms that are responsible
its interaction with ParB, MipZ is probably derived from for the assembly of bacterial actin homologues, tubulin
a plasmid-partitioning protein that later adopted a role in homologues and WACA ATPases into dynamic cytoskele-
the spatial regulation of cell division. tal filaments. The same applies to the temporal and spatial
regulation of these structures as well as their interplay with
Concluding remarks other cellular factors. Finally, further studies are required
Flexibility in the use of dynamic protein structures is to fully understand the molecular details that underlie the
a common theme in bacteria. Tubulin filaments are regulation of division-site placement by nucleoid occlu-
elementary parts of the cell-division apparatus as well as sion and MipZ. As demonstrated by FtsZ positioning in
mediators of plasmid segregation. Similarly, actin cables C. crescentus, different spatial regulatory systems can be
function in DNA partitioning, cell-shape determination tightly interconnected. It will, therefore, be a major task
and protein and vesicle localization. Comparable diversity to elucidate the interdependence of dynamic processes in
is also observed for WACA ATPases, which form filaments the cell and merge the knowledge of individual processes
that are involved in DNA segregation as well as dynamic into a global concept of cellular function.

1. Rudner, D. Z., Pan, Q. & Losick, R. M. Evidence that 3. Goehring, N. W. & Beckwith, J. Diverse paths to in adjacent daughter cells mediates protein
subcellular localization of a bacterial membrane midcell: assembly of the bacterial cell division localization. Genes Dev. 18, 2916–2928 (2004).
protein is achieved by diffusion and capture. Proc. Natl machinery. Curr. Biol. 15, R514–R526 (2005). Demonstration of a zipper-like interaction between
Acad. Sci. USA 99, 8701–8706 (2002). 4. Rubio, A. & Pogliano, K. Septal localization of proteins from the forespore and mother cell that
2. Deich, J., Judd, E. M., McAdams, H. H. & forespore membrane proteins during engulfment allows the assembly of protein complexes at the
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Drugs versus bugs: in pursuit of the


persistent predator Mycobacterium
tuberculosis
James C. Sacchettini*, Eric J. Rubin‡ and Joel S. Freundlich*
Abstract | Tuberculosis (TB) claims a life every 10 seconds and global mortality rates are
increasing despite the use of chemotherapy. But why have we not progressed towards the
eradication of the disease? There is no simple answer, although apathy, politics, poverty and
our inability to fight the chronic infection have all contributed. Drug resistance and HIV-1
are also greatly influencing the current TB battle plans, as our understanding of their
complicity grows. In this Review, recent efforts to fight TB will be described, specifically
focusing on how drug discovery could combat the resistance and persistence that make TB
worthy of the moniker ‘The Great White Plague’.

Many think of tuberculosis (TB) as the scourge that for 6 months. As patients feel better within 1–2 weeks,
devastated Europe in the seventeenth century and they have little motivation to complete therapy. Thus,
rapidly became a leading cause of death worldwide current World Health Organization guidelines call for
before being virtually eliminated (BOX 1). TB has been treatment to be directly observed. The required infra-
brought back to our thoughts, however, by the recent structure for drug-delivery and treatment supervision
reports of outbreaks of drug-resistant disease. In fact, can be difficult to provide in much of the world, particu-
TB never ‘went away’, but has remained the ‘Captain larly in areas that are afflicted with poverty and unstable
of Death’ throughout much of Asia and Africa. TB governments.
mortality rates are once again on the rise — this has For TB, drug resistance is due to genetic muta-
been attributed to the HIV-1 epidemic, which has pro- tions that result in a heritable loss of susceptibility to
duced a new and highly susceptible population, and the antibiotics. These mutations generally occur either in
inconsistent use of antibiotics, which has led to a new the target or the activator of the drug. TABLEs 1,2 sum-
epidemic of drug-resistant disease in many parts of the marize the most common mutations for current first-
world. Given that investment in antibiotics in general, and second-line drugs, and refer to their respective
including antitubercular drugs, has waned over recent mechanism (or mechanisms) of action and half-lives.
years, we have found ourselves unable to respond to the Slow-acting drugs, combined with poor health-care
resurgence of TB. systems, have led to incomplete treatment, relapse
Two factors, persistence (BOX 2) and resistance, and the emergence of resistant mycobacteria. Strains
have made the treatment of the causative organism, of M. tuberculosis that are resistant to at least one of
Mycobacterium tuberculosis, difficult. The term persist- the first-line antibiotics (TABLE 1) have become com-
ence describes the survival of M. tuberculosis despite the mon, and strains that are resistant to two or more are
*Department of Biochemistry use of antibiotics (rather than latency, which refers to not uncommon. Although resistance to single agents
and Biophysics, Texas A&M the ability of apparently dormant bacteria in asympto- generally still permits cure (albeit by using even more
University, College Station,
matic infected individuals to activate as much as decades extended courses of therapy), strains that are resistant
Texas 77843, USA.

Department of Immunology after the initial infection). Little concrete information is to multiple agents cause disease that is far more dif-
and Infectious Diseases, available on the cellular or metabolic status of persistent ficult and costly to treat. This is particularly true for
Harvard School of Public mycobacteria. As a consequence of persistence, drug multidrug-resistant (MDR) strains that are resistant
Health, Boston, treatment is extended, and current antibiotics (BOX 3) to the first-line drugs isoniazid (INH) and rifampicin.
Massachusetts 02115, USA.
Correspondence to J.C.S.
require long courses of treatment to cure patients and Some strains carry far greater levels of drug resistance,
e‑mail: sacchett@tamu.edu prevent relapse. Currently, even the most effective regi- including extensively drug-resistant (XDR) bacteria,
doi:10.1038/nrmicro1816 mens require a combination of at least 3 drugs and last which are MDR and also resistant to fluoroquinolones

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Box 1 | Waksman’s vision


In his speech at the Nobel Banquet in December 1952, Selman Waksman said “The Great White Plague, which only
10 years ago was thought to be immune to drug therapy, is gradually being eliminated…streptomycin pointed a way.
Later supplemented with PAS and more recently with isoniazid, it has brought the control of this disease within sight.”
In 1943, Selman Waksman, a microbiologist at Rutgers University, purified a compound from the soil bacterium
Streptomyces griseus that could kill a wide spectrum of bacteria, including Mycobacterium tuberculosis, in culture and in
animals125,126. Unlike the discovery of penicillin 15 years earlier, Waksman’s discovery of streptomycin was not accidental.
His student, Albert Schatz, conducted a directed screen of 10,000 cultures of soil bacteria to identify those that inhibited
the growth of co-cultured Gram-negative bacteria. He found only ten cultures that could significantly block the growth
of the test bacteria. The Nobel-Prize-winning discovery was heralded by most as the ‘beginning of the end for
tuberculosis (TB)’, although the prize itself has been controversial as some have argued that it should have been shared
with his student, or with Jorgen Lehmen, who discovered the TB drug para-aminosalicylic acid (PAS) in the same year as
the discovery of streptomycin127. The drug was quickly approved by the United States Food and Drug Administration, and
within 2 years Merck was producing 25,000 kg per day of streptomycin. Although the discovery of streptomycin proved
that a bacterium was the cause of the disease and led to almost miraculous responses in patients infected with TB, it
required repeated injection and was associated with significant toxicity. Worse, strains of M. tuberculosis that were
resistant to the drug were discovered within a few months of use.

and at least one injectable antibiotic. These infections define its target. In practice, however, this has not been
are extraordinarily difficult to treat using the current so simple, and we still do not know the targets of many
agents. existing antitubercular drugs that are in clinical use.
Clearly, our current drug armamentarium (TABLES 1,2) Recently, two systematic approaches have emerged
has not been sufficient to control the TB epidemic to define the targets of compounds that have activity
(BOX 4) . New antibiotics, particularly those that are against M. tuberculosis. Expression analysis provides a
derived from new chemical classes, are more likely to method to profile cellular responses to perturbations,
have activity against many drug-resistant strains. The such as small molecules or environmental stress. Several
path to creating antibiotics that act against persistent groups have collected a large number of datasets from
organisms and produce more rapid clearance of infec- DNA microarray experiments that were performed
tions is less clear. Certainly, a vital part of drug develop- under various conditions. In particular, Boshoff and
ment is the understanding of the physiology of growing colleagues9 have evaluated bacterial gene expression in
and persistent organisms, an area in which little infor- response to a number of drugs, toxins and environmental
mation is available, as reviewed elsewhere1–3. A greater conditions. Although these results do not define a single
knowledge of persistence could lead to a directed strat- molecular target, they can be used to identify susceptible
egy for the development of more rapidly effective anti- pathways that may contain drug targets.
biotics. However, until a clear picture of persistence and The recent availability of affordable whole-genome
its role in infection is achieved, researchers will have to sequencing also potentially provides a rapid way of
rely on more empirical approaches, as demonstrated by finding targets. For example, Andries and colleagues10
the discovery of TMC207 (discussed below). selected for mutants that were resistant to the drug
Here, we review recent and ongoing efforts to pro- TMC207 (discussed below) and sequenced their entire
duce new antitubercular drugs, and the properties of chromosomes. All resilient strains contained mutations
current investigational agents. This Review seeks to in a single gene that encoded a subunit of ATP synthase.
complement other recent discussions of TB research, However, this success story is not always easily replicated.
such as those by Janin4, Ginsberg and Spigelman5, and
Williams and Duncan6. We will follow a ‘drug timeline’,
beginning with a discussion of discovery technologies Box 2 | Bacterial persistence and treatment failure
that were designed to provide new clinical antitubercular
Why does antibiotic treatment fail even when bacteria are
candidates, before considering compounds that are cur- not genetically drug resistant? This phenomenon, often
rently in trials and, finally, already approved drugs that termed bacterial ‘persistence’, might be explained in a
are sought to be used as TB therapies. number of ways, given that Mycobacterium tuberculosis
induces a chronic inflammatory response in which
Drug-discovery methods bacteria are sequestered from drugs in tissue. The local
Genetic approaches to target identification. Ideal TB concentration of antibiotics in lesions, such as granulomas,
drug targets should have three characteristics: they might not be adequate to cause bacterial death, or some
should be required for bacterial growth and persistence bacteria might adopt a physiological state that renders
(that is, they must be expressed and essential during them less susceptible to antibiotics. This could be a
stochastic process, in which a subpopulation of cells adopt
the time that treatment occurs); it should be possible to
a physiological state that renders them drug insensitive.
inhibit their activity using small molecules (that is, the Alternatively, an environmental condition, such as low
target should be ‘druggable’)7,8; and they should be acces- oxygen or carbon starvation, might induce the persistent
sible to these modulatory compounds. Theoretically, the state.  All of these hypotheses could be true. However, as
simplest way to find targets that have these ideal char- yet, we do not know if any of them have an important role
acteristics is to discover an active compound and then in treatment failure.

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Box 3 | Lessons from current antitubercular drugs


There is an excellent chance that patients who have tuberculosis (TB) can be cured using currently available drugs if
they are able to complete the required course of therapy. But what characteristics should new drugs have to improve
on current treatment?
Oral bioavailability
• Streptomycin is a highly potent drug but is rarely used, partly because it must be injected.
Good tolerance
• Para-aminosalicylic acid (PAS) was largely abandoned early on because of gastrointestinal intolerance.
Usability in multiple populations
• Fluoroquinolones are not recommended for the treatment of pregnant women and young children — two populations
that are at risk for disease.
• Thiacetazone is associated with life-threatening reactions in patients infected with HIV-1.
Compatibility with antiretrovirals
• Rifampicin alters the metabolism of multiple drugs, particularly protease inhibitors.
Infrequent dosing
• Prospective antibiotics, such as linezolid, might not be useful if they require more than a single dose each day.
Activity against drug-resistant strains
• Drugs such as PAS have been revived owing to the increase in multidrug-resistant and extensively drug-resistant disease.
Activity that is not necessarily in vitro
• Pyrazinamide is a highly effective drug for patients even though it has poor activity under standard laboratory
conditions.
Rapid clearance of chronic infection
• All available drugs, with the exception, possibly, of rifampicin, have limited efficacy in chronic infection. This is
particularly true of agents such as isoniazid that act on the cell wall.
Affordability
• Drugs that are used for other purposes, such as linezolid, are extraordinarily expensive. At current prices, it would be
impossible for them to be used in most areas of the world in which TB is prevalent.

In the case of PA‑824, sequencing the genome of resistant dirty drugs) and, in fact, there is evidence that some
mutants using a DNA microarray method showed that current drugs, such as INH19,20 and para-aminosalicyclic
mutations in a conserved hypothetical gene produced acid21, are capable of inhibiting multiple proteins.
resistance11. However, the encoded protein was not the However, even in these situations, it is unclear if this
target of PA‑824, but instead was a nitroreductase that provides a strong advantage as resistance can still arise
was responsible for the activation of PA‑824. from mutations that seem to affect only single putative
Genetic analysis might prove to be an alternative targets22,23, perhaps because these represent the points of
method for defining new TB drug targets. Using a range greatest vulnerability. In any case, for drugs that hit either
of methods, investigators have found several gene prod- single or multiple targets, it might be difficult to identify
ucts that, if inhibited, could decrease bacterial growth targets using systematic approaches.
or increase host survival after infection12–14. Genes that
are required for growth in vitro are difficult to define High-throughput screening. High-throughput screening
using traditional genetic methods, as mutations in these against target proteins has an important role in modern
genes, by definition, result in clones that are unable to drug discovery24,25. For M. tuberculosis, biochemical
grow. However, negative screens that identify genes that high-throughput screening has contributed to the find-
cannot be mutated have yielded a set of several hundred ing of two clinical drug candidates, TMC207 (Ref. 10)
candidate genes that are required for in vitro growth14,15. and SQ109 (Ref. 26), and has been used to investigate the
Of course, these are simply screens and do not repre- viability of small molecules as modulators of a number
sent proof that individual gene products are useful as of mycobacterial targets.
drug targets. The extension of these screens to in vivo Two different approaches — the targeting of whole-
animal models of TB would be a giant step forwards in cell growth and enzyme inhibition — have been used
target identification. However, to validate putative tar- individually and together to apply high-throughput
gets in the absence of a known inhibitor it is useful to screening to TB drug-discovery efforts. Whole-cell
construct conditional mutants. Fortunately, recent work
has provided conditional promoters that can be used to
construct these informative strains16–18. Box 4 | Gates’ vision
Is it truly important to define single targets for poten- Bill Gates commented at the 2005 World Health Assembly:
tial antibiotics? After all, it might be difficult to evolve “Today, we have tuberculosis drugs you have to take for
resistance to drugs that hit multiple targets (so-called 9 months. Why can’t we find one that works in 3 days?”

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Table 1 | Mechanism of action, resistance and half-life of current first-line antituberculosis agents
Antibiotic Chemical structure Mechanism and target Mutations Half-life Refs
associated with in humans
resistance (hours)
Isoniazid O
H
N Inhibits mycolic acid synthesis; katG (required for 1–3 74
NH2
primary target is InhA and drug activation); inhA
secondary targets are KasA and (promoter mutations);
N
DfrA and others

Rifampicin O HO Inhibits transcription; RNA rpoB 2–3 74


polymerase β-subunit
O
OH O
OH OH
NH
O
H3C N
O N
OH N
O CH3
O

Ethambutol OH Inhibits arabinogalactan embB 3–4 74


H
N synthesis; possibly EmbB
N
H
HO

Pyrazinamide O Unknown (possibly inhibits FAS-I pncA (required for 10 74


N
NH2 or alters membrane energetics) drug activation)
N

Streptomycin H2N NH2 Inhibits protein synthesis; 30S rpsL and rrs 2–3 74
O
N
CHO ribosomal subunit
O
HO OH NHCH3
O OH
OH
H2N N O
OH OH
NH2
OH

screening against either Mycobacterium smegmatis or pharmacokinetic profiles and the target is truly essential.
M. tuberculosis allows searching for the ultimate goal There are many examples of small molecules that have
— potent growth inhibition or killing. As this type of excellent enzyme inhibition but poor whole-cell potency,
screen is not target based, there is a considerable risk possibly owing to their failure to permeate the myco-
of finding compounds that have generalized toxicity, and bacterial cell wall. A variant of this strategy relies on
the lack of information on the target will certainly com- a functional assay in which the inhibition of an entire
plicate the optimization. This risk might be decreased by pathway can be screened for. For example, a luciferase
pre-filtering compound libraries27,28 or designing more reporter can be used to measure the transcription of the
targeted screens. iniBAC operon, which is induced by a diverse set of
High-throughput screening is being pursued at a mycobacterial cell-wall biosynthesis inhibitors, includ-
number of facilities, both in industry and academia. ing ethambutol and INH30. Barry and colleagues26 used
One large National Institutes of Health-funded effort, this technology to discover SQ109 (FIG. 1), an ethambutol
the Tuberculosis Antimicrobial Acquisition and analogue for which the discovery and clinical progress
Coordinating Facility (see Further information), offers will be discussed below.
a free service to investigators for testing candidate GlaxoSmithKline and the Global Alliance for TB Drug
compounds. The facility has evaluated over 79,000 com- Development (TB Alliance; see Further information) have
pounds from more than 9,600 researchers for the inhibi- recently conducted a million-compound screen for new
tion of M. tuberculosis growth29. Of these compounds, inhibitors of an M. tuberculosis enoyl-acyl carrier protein
130 have demonstrated in vitro efficacy against both reductase, InhA, which is the target of INH. They found
drug-sensitive and drug-resistant strains. Such broad a high hit rate, probably because the crystallographically
screening efforts hold promise for uncovering new characterized active site can accommodate hydrophobic
leads for antituberculars that, from the outset, display groups of varying dimensions, which is consistent with its
Pharmacokinetic profile mycobacterial growth inhibition. acceptance of C16–C56 fatty-acid thioester substrates31,32.
A quantitative description of High-throughput screening has also been used to Importantly, these inhibitors should be active against most
the fate of a drug from the identify inhibitors of a targeted enzyme in a cell-free of the INH-resistant strains, as they do not require activa-
moment the treated subject is environment, through either a binding or functional tion by the catalase–peroxidase enzyme KatG33, which is
dosed with the compound to
the moment when it (and/or its
assay. High-throughput screening has the potential to required for the activation of INH. Similar screens car-
derivatives) is expelled from identify hits that could be potent inhibitors of myco- ried out by GlaxoSmithKline and the Novartis Institute
the subject. bacterial growth if the compounds have acceptable for Tropical Diseases for inhibitors of isocitrate lyase, an

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Table 2 | Mechanism of action, resistance and half-life of selected second-line antituberculosis agents
Antibiotic Chemical structure Mechanism and target Mutations Half-life Refs or
associated with in humans sources
resistance (hours)
Fluoroquinolones O O Inhibits DNA gyrase gyrB Moxifloxacin: 12; 129
F
OH
Gatifloxacin: 8
R N
O
H3C

Ethionamide S NH2 Inhibits mycolic acid ethA (required 2 130


synthesis; InhA for drug
activation) and
inhA (promoter
N
mutations)
Cycloserine H2N
O Inhibits peptidoglycan alr 10 DrugBank
synthesis by blocking (overproduction) (see Further
NH
O
the synthesis and use of and ddl information)
d‑alanine (Ala); Ala racemase (overproduction)
and d-Ala-d-Ala ligase
Para‑aminosalicylic NH2 Inhibits folate metabolism; thyA 0.75–1 131
acid possibly dihydropteroate
synthase
OH

HO O

Capreomycin NH2 Inhibits protein synthesis; tlyA and rrs 4–6 The Merck
R
O methylated nucleotides in Manuals
NH HN O
both ribosomal subunits Medical
R = H, OH
H
N NH2
Library (see
HN O HN Further
H H
H2N
N N
O
N information)
NH2 O O
HN

H 2N O

Kanamycin OH
OH Inhibits protein synthesis rrs 2 132
HO O HO OH
O
H2N HO OH NH2
O O
H2N NH2

Amikacin OH
OH Inhibits protein synthesis rrs 3 133
HO O HO OH
O
H2N HO OH NH2
HO H O O
N NH2

H2N O

enzyme that is crucial for bacterial persistence during library that was used to discover SQ109 was screened
infection34,35, were considerably less successful. Again, this using both growth-inhibition and cell-wall-biosynthesis
might be explained structurally, as the active site of this assays37. Of nearly 5,000 compounds screened, 25 small
enzyme is shallow and highly charged. Small, hydrophilic molecules were active in both screens, but only one,
molecules, such as those that are predicted to bind to SQ775, showed significant activity in a mouse model
isocitrate lyase, are under-represented in most screening of infection.
libraries, as they are less likely to have desirable pharma-
cological properties. Finally, a high-throughput screen for Structural biology and virtual screening. Describing
inhibitors of pantothenate synthetase, an enzyme that is the proteome of M. tuberculosis has been the focus
crucial for the biosynthesis of the essential cofactors acyl of much research in the past few years. Primarily
carrier protein and coenzyme A, was recently reported owing to the efforts of the TB structural-genomics
Pharmacophore
by White and colleagues36. Of approximately 4,000 com- consortium, more than 260 X‑ray crystal structures
The chemical functional group
(or groups) that is present on a pounds assayed, one lead compound was identified and a of interesting proteins, a large percentage of which
molecule and that enables its preliminary structure characterized in complex with the were selected on the basis of their being potential
biological activity. target enzyme. This successful outcome suggests a path drug targets, have been completed, and many are
forward for the structure-based design of more potent currently being used to facilitate medicinal‑chemistry
Chemotype
A chemical functional group or
analogues that inhibit the enzyme and M. tuberculosis. efforts to rationally design new antibiotics38 (FIG. 1).
classification of a specific array Combining whole-cell and target-based screens The availability of these structures provides the
of functional groups. might avoid the drawbacks of each. For example, the opportunity to carry out virtual screening. This

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REVIEWS

O O
CH3 O O
N CH F
OH OH F
3
Br OH
H
N N
N N
N O O H
H3C HN O N
CH3 H3C N
NH H H
TMC207 Moxifloxacin Gatifloxacin SQ109

O HO NO2
O HO
N N
O N
OH O N
OH O O OH
OH O SQ775
NH OH OH
O NH Metronidazole
H3C O O
O N
H3C N
N O
O
O OH N N CF3
O HN N
O N
HO N N
N O
Rifalazil Rifapentine Sudoterb

F3C
O2N N O N O
O O2N O
CF3 O
N N O O N N
O H
N N
O F
CH3 O O
PA-824 OPC-67683 O Linezolid
N H3C OH
H3C N O
N N

N S
N Cl CH3 N CF3
H O O NH
O O
OH OH HN
S S O Sulbactam O
N S N N
Chlorpromazine CH3 Trifluoperazine S
O OH
S H HO H H
Thioridazine Clavulanic acid Imipenem

Figure 1 | Chemical structures of non-approved antituberculars.


Nature Reviews | Microbiology

powerful technique has had a beneficial impact on had a minimum inhibitory concentration (MIC) of
numerous drug‑discovery efforts39–41. Screening using 25 µg per ml. Using a different mathematical model,
computational methods can be complementary to a García-García et al. tested 5,000 commercially available
biochemical high-throughput screen because of the compounds and found 18 virtual hits43; 5 of these had an
potential for screening a larger chemical space quickly MIC90 of less than 50 µM.
and inexpensively. Virtual screening can be used in Other groups have instead focused on specific
two ways: to identify compounds that are consist- targets that have known structures. Agrawal and co-
ent with a pharmacophore model irrespectively of the workers45 performed a virtual screen for inhibitors of
Lipinski’s rules
A set of delimited
identity and structure of the pertinent protein target M. tuberculosis chorismate mutase, which is part
physiochemical properties or targets; or to develop inhibitors of a protein based of the essential shikimate pathway in M. tuberculosis46.
described by C. A. Lipinski that on its known three-dimensional structure. They started with known inhibitors of the homologous
best fit a studied subset of Recent examples of virtual screening that were based Saccharomyces cerevisiae enzyme47 and conducted a
drugs. In general, compounds
on known chemotypes include work from Manetti and three-dimensional pharmacophore search of a database
that adhere to these guidelines
are said to be drug-like. colleagues42 and García-García and colleagues43. Manetti that has more than 15,000 members. Of the 15 mol-
et al. used a training set of 471 small molecules that had ecules that scored highest, 4 demonstrated inhibition
Shikimate pathway a range of in vitro activities against M. tuberculosis to in an enzyme assay. Lin and colleagues48 focused on the
A series of biochemical construct a model that could be searched using a vir- elucidation of new small-molecule inhibitors of AccD5,
reactions in plants and
microorganisms that are
tual library of compounds that was filtered to follow an acyl-CoA carboxylase essential enzyme that cataly-
involved in the biosynthesis of Lipinski’s rules44. The virtual hits were assayed against ses the transformation of acetyl-CoA and propionyl­
aromatic amino acids. M. tuberculosis, and the two most potent compounds CoA to the corresponding malonyl-thioester and

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yet to yield a clinical candidate, X‑ray crystallography


and structure-based designs have had crucial roles in the
discovery of new inhibitors of this validated TB drug
target32,50–53. Sullivan and co-workers54 have translated
these insights into the discovery of potent triclosan-
based antituberculars. FIG. 2 depicts a select subset of
X‑ray structures that have had a prominent role in this
developing story.

New TB drugs in clinical trials


A chemical entity that has been discovered as an antitu-
bercular by the application of one or more of the methods
discussed above and has cleared the considerable hurdles
in pre-clinical development can enter clinical trials if it has
the approval of the pertinent governmental body (in the
United States, the Food and Drug Administration (FDA)).
The scientific community is hopeful that the drug candi-
dates described below (the structures of which are shown
in FIG. 1) will eventually be used in new treatment regimens
that meet the goals discussed earlier. It is clearly a testa-
Figure 2 | Overlay of small-molecule inhibitors of InhA. A cross-section through ment to the financial commitment of the involved funding
Nature
the surface of the active site of Mycobacterium tuberculosis Reviews
fatty-acid | Microbiology
enoyl-acyl organizations that these candidates are being supported
carrier protein reductase (InhA), coloured by atom type (carbon, grey; nitrogen, blue; through a process that is incredibly costly.
oxygen, red; and sulphur, yellow). Superimposed are the bound conformations
of NADH50 (carbon, yellow; nitrogen, blue; oxygen, red; and phosphorous, aqua), Fluoroquinolones. Fluoroquinolones, which have
C16 fatty acyl substrate analogue trans‑2-hexadecenoyl-(N-acetylcysteamine) been used for the treatment of TB since the 1980s55,
thioester32, shown in its bent conformation (carbon, yellow; sulphur, orange;
currently constitute the second line of defence against
nitrogen, blue; and oxygen, red) and 12 inhibitors (carbon, grey; nitrogen, blue;
M. tuberculosis and play a key part in the treatment of
oxygen, red; sulphur, yellow; and fluorine, purple) that have half-maximal inhibitory
concentration values ranging from 50 nM to 5 µM. Unlike isoniazid and MDR disease. Resistance to fluoroquinolones has been
ethionamide, these are non-covalent reversible inhibitors that form ternary attributed to mutations in the genes that encode gyrase
complexes with enzymes and NAD+. This figure was created using the molecular (gyrA and gyrB)56,57. As quinolone efflux pumps may also
modelling system Chimera128. have a role in resistance, it is noteworthy that the annota-
tion of putative pumps in the M. tuberculosis genome58
and the studies of Jacobs and co-workers59 on a probable
methylmalonyl-thioester, respectively. They screened over efflux pump, lfrA.
4 million compounds for binding to either the predicted Two approved fluoroquinolones, moxifloxacin
biotin or propionyl-CoA binding pockets and found that and gatifloxacin, have shown promising results both
1 of the 9 compounds that scored highest had an enzyme for treating resistant disease and, possibly, shortening
half-maximal inhibitory concentration (IC50) of 10 µM. the course of therapy. Phase II and III clinical trials
Approaches such as these underscore the usefulness that are currently underway are testing the efficacy of
and potential of virtual screening to enable new hits for moxifloxacin and gatifloxacin as replacements for either
drug-discovery efforts. INH or ethambutol in first-line therapy60,61. These com-
Each of the discovery technologies discussed in this pounds are particularly attractive, as they have already
section has potential as a starting point for the discov- been approved for use in various other infections and are
ery of a new antitubercular that, on successful passage known to be safe. As fluoroquinolones are prescribed for
through the pre-clinical drug-development pathway, can numerous respiratory infections, many cases of TB could
produce a clinical candidate. Undoubtedly, the most be treated with this monotherapy before they are diag-
efficient and expeditious way to seed drug discovery lies nosed. Theoretically, this could lead to drug resistance,
in the cooperative union of these methodologies in mov- although, as yet, widespread fluoroquinolone-resistance
ing from the validated drug target to a clinical candidate. mutations have not been observed.
Lead optimization This has been highlighted by examining retrospectively a
The process by which a GlaxoSmithKline antibacterial high-throughput screen- Rifampicin analogues. Rifapentine was approved by
promising small-molecule ing campaign, which, over a wide range of targets, pro- the FDA for the treatment of M. tuberculosis infection
entity is structurally modified
vided few compounds for follow up49. Additionally, lead in June 1998. The piperazinyl hydrazone functional-
to obtain drug-like
pharmacokinetic, optimization is often a labour- and time-intensive process ized rifampicin analogue, initially named DL 473,
pharmacodynamic and safety that many programmes do not endure. was first reported in 1975 (Ref. 62) and subsequently
profiles. A detailed understanding of the drug target (or has been shown to exhibit in vitro antimycobacterial
targets) and how the binding of a drug inhibits target efficacy that is, in most cases, superior to that of its
Efflux pump
An active transport system for
function helps immensely in developing a lead. Whereas parent63–65. A noteworthy advantage that rifapentine
the removal of toxic molecules, efforts to develop new InhA inhibitors, which, in the has compared with rifampicin is its longer serum half-
such as antibiotics, from cells. future, could complement and/or supplant INH, have life66, which has encouraged its examination in clinical

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settings to determine its potential for positively altering M. smegmatis. It is intriguing to note that the first mem-
the frequency of the antitubercular treatment regimen. ber of this DARQ class was isolated as a side product in
An extension of a Phase III trial that is currently evaluat- chemical experimentation that was designed to prepare
ing rifapentine and INH in the treatment of latent TB compounds for other early discovery programmes, thus
involves recruiting children (C. Dukes Hamilton, per- highlighting the importance of screening (targeted
sonal communication) to assess the efficacy, pharma- compounds and side products) across projects.
cokinetic and safety profiles of the drug combination. TMC207 achieves impressive in vitro and in vivo effi-
cacy against drug-sensitive and drug-resistant strains of
Rifalazil. Rifalazil, formerly known as KRM‑1648, has M. tuberculosis. Resistance studies identified the biologi-
a benzoxazinorifamycin structure67. This heterocyclic cal target as the F0 subunit of ATP synthase that is encoded
modification of ansamycin68 is much more potent against by atpE10,78,79. This finding, together with the lack of cross-
M. tuberculosis clinical isolates than rifampicin69 and is resistance of TMC207 with existing antitubercular drugs,
more efficacious in a mouse model70. Rifalazil presum- highlights the potential that undirected screens have for
ably has a similar mechanism of action to rifampicin68, as the discovery of compounds that have new mechanisms
mutants are cross-resistant69. Rifalazil has a longer half- of action. A Phase II trial is currently underway to
life than rifampicin in healthy human volunteers71 and, investigate the efficacy of TMC207-containing regimens
unlike rifampicin and rifabutin, is neither metabolized versus standard antitubercular therapy in patients who
by, nor an inducer of, rat and dog hepatic cytochrome have MDR TB. Given the previously reported animal
P450 enzymes72. These metabolic observations suggest studies80, TMC207 could be added to a second-line treat-
that rifalazil can be co-administered with drugs that are ment regimen or could replace a first-line drug to shorten
sensitive to oxidative metabolism, thereby reducing the the length of treatment.
potential for adverse drug–drug interactions. This is a
particularly important consideration, as co-infection Sudoterb. The tetra-substituted pyrrole Sudoterb was
with HIV-1 and TB is common and rifampicin consid- discovered by building on the previously described
erably lowers the serum levels of antiretroviral protease antimycobacterial SAR of pyrroles81. The N‑substituent
inhibitors73. Rifalazil, however, is not currently registered is similar to INH, as both contain an isonicotinoyl
with the FDA for a clinical trial of TB, probably owing hydrazide moiety. Because the only publicly available
to the toxicity that was observed during a Phase II trial information about this compound comes from the pat-
in Brazil74. ActivBiotics is currently investigating signifi- ent application82, we know little about its mechanism of
cantly lower once-weekly doses of rifalazil in Phase II/ action. However, the absence of cross-resistance with
III studies to ascertain the effect on patients that have existing therapies and the unique chemical structure sug-
carotid atherosclerotic disease and have been infected gests that it could be novel. Sudoterb has been reported
Structure–activity with Chlamydia pneumoniae (A. Sternlicht, personal to be more potent than INH as a monotherapy in a
relationship communication). It remains to be seen whether these mouse model for TB and displays an acceptable phar-
(SAR). The relationship lower doses, if safe, would be efficacious for TB. macological profile in mice and dogs83. The compound
between the chemical structure
is reportedly in Phase I clinical trials83.
of a compound and its
biological or pharmacological SQ109. SQ109 was discovered using a high-throughput
activity. This type of screen of ethambutol analogues that had the dual goals Nitroimidazoles. Metronidazole has frequently been
relationship can be assessed of inhibiting mycobacterial cell-wall synthesis and cell used to treat infections of microaerophilic or anaerobic
by considering a series of growth in general26. Barry and colleagues26 used the bacteria84. Owing to the proposed relevance of limiting
molecules, each with a slightly
structure–activity relationship (SAR) study results reported oxygen conditions to the latent phase of TB85, metroni-
different structure, and then
noting the effect on the by Lederle Laboratories75,76 to prepare more than 63,000 dazole has also been investigated as an antitubercular.
biological activity that is compounds. The compounds were assayed for the inhibi- Metronidazole kills only dormant M. tuberculosis and
associated with each structural tion of M. tuberculosis growth and cell-wall biosynthesis. not actively growing cultures86. The drug had no effect
variation.
They found 119 hits in the cell-wall-biosynthesis assay on the growth of M. tuberculosis-infected mouse macro-
Fast-track status and 60 hits in the growth assay that were at least as potent phages, but a measurable, although small, efficacy in a
The FDA status that is reserved as ethambutol. SQ109, the optimal hit, shares some struc- mouse model of chronic-stage M. tuberculosis87. Despite
for products that demonstrate tural similarity with ethambutol, but has superior in vitro these mixed results, a Phase II clinical trial in South
the potential to treat a serious and in vivo activity77. In fact, DNA microarray analysis Korea is currently recruiting patients to examine the
or life-threatening condition.
suggests that this compound has a different mode of effect of adding metronidazole to a standard second-line
F0 subunit of atp synthase action from ethambutol9. SQ109 has achieved fast-track therapeutic regimen (see ClinicalTrials.gov in Further
The transmembrane portion   status and is in Phase I clinical trials. information for details of an ongoing clinical trial).
of the enzyme complex that is Attempts to discover other nitroimidazole-based
involved in the biosynthesis of
TMC207. Originally named R207910 by Johnson and antituberculars led researchers at Ciba-Geigy to discover
ATP, which has a role in the
passage of protons through   Johnson Pharmaceutical Research and Development10, CG‑17341 — a nitroimidazooxazole that displays potent
the membrane. TMC207 is a new antitubercular agent from the dia- activity both in vitro and in vivo against drug-sensitive
rylquinoline (DARQ) class. The drug was found by a and drug-resistant strains88,89. A toxicity issue noted
Ames mutagenicity test high-throughput screen of a library of approximately in an Ames mutagenicity test hindered this compound
A sensitive biological method
for measuring the mutagenic
10,000 compounds (K. Andries, personal communi- from being developed further. Stover and colleagues90
potency of chemical cation), during a search for those that inhibited the removed this concern, however, with the discovery of the
substances. growth of the rapidly growing environmental bacterium potent antitubercular PA‑824 of the nitroimidazopyran

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class. In vivo studies demonstrated comparable activity for its early bactericidal efficacy in Phase II studies in
between PA‑824 and INH91. Most importantly, PA‑824 combination with the standard front-line regimen.
displays no cross-resistance with existing TB drugs and The clinical candidates outlined in this section hold
is efficacious against non-growing M. tuberculosis under promise as next-generation antituberculars, but each
hypoxic conditions. Intriguingly, this activity against must now pass through the phases of clinical trials to
persistent mycobacteria is not shared with the original receive approval. This process is not without considerable
lead compound CG‑17341. However, in vivo tests using uncertainty, as demonstrated by the high attrition rates
a mouse model have failed to demonstrate an advan- for clinical compounds in the United States.
tage in including PA‑824 with first-line antituberculars
in terms of shortening the duration of treatment 92. Approved non-TB drugs as antituberculars
Mechanistically, the mycobacterial target (or targets) A strategy to reduce the risk that is associated with
of PA‑824 is unknown. Biochemical evidence suggests failure in clinical trials owing to an inadequate human-
that both protein and lipid synthesis are inhibited by a safety profile lies in the use of already-approved drugs
compound (or compounds) that is generated from the as antituberculars. In this section, we will discuss the
bioreduction of the imidazole nitro group84,90,93. Fatty- potential for three classes of non-TB therapeutics in
acid analysis of treated mycobacteria suggested the inhi- the fight against TB. The main hurdle has become the
bition of hydroxymycolate oxidation to ketomycolate90. demonstration of sufficient efficacy at a dosage level that
Further mechanistic work is required to understand the was previously deemed to be safe.
mechanism of action of PA‑824. Overall, the likelihood
of a novel mechanism of action and activity against per- Linezolid. Linezolid received FDA approval for MDR
sistent mycobacteria bodes well for the positive impact Gram-positive bacterial infections in 2000. This
of PA‑824 on antitubercular chemotherapy. This com- 3‑aryl‑2-oxazolidinone antibiotic, and its analogues, has
pound is in Phase I clinical trials that are supported by displayed promising in vitro and in vivo efficacy against
the TB Alliance. drug-sensitive and drug-resistant M. tuberculosis96,97. In
Staphylococcus aureus, and presumably other bacteria
OPC‑67683. Building on the work of Ciba-Geigy in such as M. tuberculosis, linezolid inhibits protein syn-
nitroimidazoles and the discovery of PA‑824, researchers thesis by binding to the 23S ribosome to prevent transla-
at the Otsuka Pharmaceutical Company in Japan tion98. Clinical examination of linezolid as a potential
examined the SAR around PA‑824 and proposed that antitubercular, although demonstrating promising
further variation of the furan portion of the hetero- efficacy against MDR and XDR TB, has detected sig-
cycle could yield potent antituberculars that are free nificant toxicity99–102. The rate of incidence and severity
of mutagenicity93,94. Whereas PA‑824 features a pen- of these adverse events might be reduced by decreasing
dant 4‑trifluoromethoxybenzyloxy group, the Otsuka the dosage amount of linezolid. A clinical trial that is
researchers introduced a 4‑piperidine moiety in place expected to be completed in late 2007 is currently being
of the trifluoromethoxy group to improve oral bioavail- conducted in Brazil to determine the efficacy of lower
ability. OPC‑67683 was eventually prepared in an effort drug doses (J. Johnson, personal communication). In
to decorate the piperidine 4‑position with hydrophobic addition, further optimization of the oxazolidinone
groups such as 4‑trifluoromethoxyphenyl. series for M. tuberculosis activity is underway103.
OPC‑67683 is free of the mutagenicity of its nitroimi- The potential for using already approved drugs
dazole progenitor, is orally bioavailable in mice and as antitubercular agents holds considerable promise.
provides efficacy that is equivalent to rifampicin against Given that marketed drugs have well-documented
M. tuberculosis Kurono at less than one-fifteenth of and acceptable safety profiles, a major hurdle has been
the dose93. OPC‑67683 was equally efficacious against removed in terms of finding new therapies for TB.
cultures of drug-sensitive and drug-resistant strains However, as most antibiotics have little activity against
of M. tuberculosis and superior to INH, ethambutol, M. tuberculosis, the crucial step in using these drugs is
rifampicin, streptomycin, CGI‑17341 and PA‑824, and to demonstrate good efficacy.
also produced rapid eradication of infection in mice.
OPC‑67683 was not metabolized by a panel of human β-lactam. Although the β‑lactam class of antibiotics
microsomes and did not positively or negatively interfere has been used in clinics for over 60 years, none of its
with their catalytic activities, thereby lending hope to the representatives has been used for the treatment of TB.
idea that this drug could be used in combination with β‑lactams act by inhibiting bacterial-cell-wall biosynthe-
HIV‑1 therapies. Mechanistically, OPC‑67683 seems to sis and would be a welcome addition to the antitubercular
inhibit mycolic-acid biosynthesis and, more specifically, arsenal. Unfortunately, M. tuberculosis produces only a
methoxy- and keto-mycolic-acid biosynthesis93, although single β‑lactamase that has broad specificity104–106. Other
it probably requires biotransformation for its activity95. genes may also have a role in resistance by affecting cell-
Given the limited amount of target information that is wall permeability and/or binding affinity for the perti-
currently available, it is unclear if OPC‑67683 and PA‑824 nent penicillin-binding proteins107. Although β‑lactams
have different mechanisms of action. OPC‑67683 suc- can penetrate the cell and inhibit their targets108, their
cessfully completed Phase I clinical trials, demonstrating potency is primarily limited by β‑lactamase-mediated
satisfactory safety and pharmacokinetic profiles in healthy degradation. Two strategies could avoid this problem.
individuals. The small molecule is now being evaluated First, in some cases, β‑lactamase inhibitors, such as

nature reviews | microbiology volume 6 | january 2008 | 49


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REVIEWS

clavulanic acid and sulbactam, allowed growth inhibi- an attractive target for other compounds. One way to
tion below the µg per ml level if used in combination achieve this would be to start with the known active
with β‑lactams. Second, the carbapenem imipenem is phenothiazines and construct analogues that have more
resistant to cleavage. Unfortunately, both imipenem108–110 selectivity, a goal that is currently being pursued123,124.
and amoxicillin in combination with clavulanic acid111
have produced mixed results in early bactericidal- Conclusions
efficacy assessments in humans. However, this remains M. tuberculosis would seem to be a vulnerable organism,
a promising area for investigation and recent structural given that it has no noteworthy animal or environmental
work using M. tuberculosis and Mycobacterium fortuitum reservoir and limited genetic diversity. However, despite
β‑lactamases106,112 might prompt the further design the availability of several effective antibiotics, TB con-
of β‑lactamase inhibitors and/or β‑lactams that have tinues to be a widespread and devastating disease. The
reduced β‑lactamase susceptibility. need for new fast-acting drugs is clear. Fortunately,
several chemical entities are currently in clinical trials,
Phenothiazines. Phenothiazines have a rich and diverse and numerous promising compounds are in the earlier
history of medicinal uses113 and antitubercular activ- stages of drug development. It is likely that new drugs will
ity against drug-sensitive and drug-resistant strains become available in the near future that can cope with
of M. tuberculosis114,115. The major examples are chlo- the resistance problem as it currently exists. However,
rpromazine115, thioridazine116 and trifluoperazine117,118. resistance continually evolves. Persistence is currently
These drugs have substantial, and sometimes even an even more formidable enemy, requiring a much more
disabling, side effects that limit their use at effective thorough understanding of its basic biological underpin-
plasma concentrations. However, they might be more nings and the small molecules that can modulate its role
effective in vivo than in vitro as they are concentrated in in the disease state. Therefore, little hope exists in the
macrophages119,120. short term for a drastic reduction in the time for treat-
Phenothiazines appear to target the type‑2 nico- ment, given the targets and drugs that are under clinical
tinamide adenine dinucleotide (NADH):menaqui- investigation. Despite the considerable challenges that are
none dehydrogenase (NDH‑2) 121. NDH‑2 is crucial posed by these ‘bugs’, the recent improvement in the scale
to the mycobacterial electron-transport chain and is of efforts to find new drugs lends hope that science will
the only such enzyme in M. tuberculosis that is absent deliver on the promise of eradicating ‘The Great White
in humans. Notably, this target seems to be important in Plague’ that was celebrated prematurely by some owing
starved cultures122, suggesting that NDH‑2 could be to Selman Waksman’s Nobel Prize.

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104. Fisher, J. F., Meroueh, S. O. & Mobashery, S. Bacterial pulmonary tuberculosis. Am. Rev. Respir. Dis. 81, 128. Pettersen, E. F. et al. UCSF chimera — a visualization
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Microbiology 151, 521–532 (2005). 117 Ratnakar, P. & Murthy, P. S. Antitubercular activity of pyrazinamide, and ethambutol pharmacokinetics in a
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treatment of tuberculosis in mice and humans. antitubercular drugs. Proc. Natl Acad. Sci. USA 102, The authors thank M. Spigelman, R. Goldman, J. Garcia,
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(2005). 122. Xie, Z., Siddiqi, N. & Rubin, E. J. Differential antibiotic A. Sternlicht for insightful conversations and, in some cases,
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Hopewell, P. C. Activity of amoxicillin/clavulanate in quaternized promazine and promethazine derivatives. DATABASES
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Chlorpromazine in nonpsychotic patients with Sci. 60, 218–220 (2005).

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Salmonellae interplay with host cells


Andrea Haraga*, Maikke B. Ohlson‡ and Samuel I. Miller*‡§
Abstract | Salmonellae are important causes of enteric diseases in all vertebrates.
Characterization of the molecular mechanisms that underpin the interactions of
salmonellae with their animal hosts has advanced greatly over the past decade, mainly
through the study of Salmonella enterica serovar Typhimurium in tissue culture and animal
models of infection. Knowledge of these bacterial processes and host responses has
painted a dynamic and complex picture of the interaction between salmonellae and
animal cells. This Review focuses on the molecular mechanisms of these host–pathogen
interactions, in terms of their context, significance and future perspectives.

Pinocytosis Salmonellae are Gram-negative bacterial pathogens multiple mechanisms for crossing the intestinal barrier
A nonspecific process by which that are capable of infecting a wide range of animals, indicates the importance of this strategy to the lifestyle
small volumes of extracellular which can result in several manifestations of disease1. of salmonellae.
fluid are taken up by certain The host specificities and the disease symptoms that are M-cell-mediated uptake also allows salmonellae
eukaryotic cells owing to the
engulfment of fluid in small
caused by some of the thousands of Salmonella serovars to interact with, and possibly enter, intestinal epithe-
membrane vesicles. are listed in TABLE 1. Salmonellae are typically acquired
  lial cells through their basolateral surface. Epithelial
by the oral ingestion of contaminated food or water; turnover and shedding then, in turn, could return
Tight junction however, exposure to pet reptiles and amphibians, which the bacteria to the lumen of the intestine, simply
The connection between two
are often carriers of the bacteria, can also pose a risk for requiring that they survive and, perhaps, replicate
adjacent cells in a monolayer
that is formed by extracellular-
salmonellosis in humans (FIG. 1). Although conditions that
  intracellularly. Such mechanisms are possibly impor-
matrix and protein complexes; increase the gastric pH reduce the infectious dose of the tant for intestinal colonization and even for acute
impermeable to water and bacterium, salmonellae have an adaptive acid-tolerance disease by salmonellae, as a great deal of data, from
other molecules. response that might promote their survival in the low both animal and cell culture models, indicate that the
Macropinocytosis
pH milieu of the stomach2. After entering the small specialized ability to invade and survive in epithe-
Used to refer to the intestine, salmonellae traverse the intestinal mucous lial cells is essential for their pathogenesis 13–17. By
endo­cytosis of large volumes   layer and evade being killed by digestive enzymes, bile contrast, the importance of phagocytosis and traf-
of extracellular fluid and salts, secretory IgA, antimicrobial peptides and other ficking through CD18-positive cells is unknown and
particles by membrane ruffles.
innate immune defences in order to gain access to the might represent another mechanism, redundant to
underlying epithelium3–5. Salmonellae have the ability to M-cell-mediated entrance, for salmonellae to cross
invade the non-phagocytic enterocytes of the intestinal the intestinal barrier without actively interacting with the
epithelium by bacterial-mediated endocytosis6. After epithelial surface. It is interesting to speculate that this
adherence to the apical surface of the cell, using vari- method may be important for typhoid fever, in which so
ous fimbrial adhesins, the bacteria disrupt the epithelial few organisms can cause disease but induce only mini-
brush border and induce membrane ruffles that engulf mal intestinal disturbance in the majority of cases. This
the organisms7,8. However, salmonellae preferentially could allow Salmonella enterica serovar Typhi (S.  typhi)
enter microfold (M) cells, which are specialized epithelial to disseminate systemically in a relatively silent fashion,
*Department of Genome cells that sample intestinal antigens through pinocytosis as opposed to mechanisms that involve bacterial-medi-
Sciences, ‡Department of and transport them to lymphoid cells in the underlying ated endocytosis, which are associated with intestinal
Microbiology, §Department of
Peyer’s patches9,10. Alternatively, they can translocate inflammation and diarrhoea13,14.
Medicine, University of
Washington, Seattle, through the intestinal epithelia after uptake by CD18- Once the epithelial barrier has been breached,
Washington 98195, USA. expressing phagocytes11. Moreover, in vitro, salmonellae Salmonella serotypes that are associated with systemic
Correspondence to S.I.M. are able to disrupt tight junctions, which seal the epithelial illness can enter intestinal macrophages by inducing
e‑mail: millersi@u.washington. cell layer and restrict the paracellular passage of ions, macropinocytosis , sensing the phagosomal environ-
edu
doi:10.1038/nrmicro1788
water and immune cells12. This, in addition to intes- ment and activating various virulence mechanisms
Published online   tinal inflammatory responses, probably contributes in order to survive in the microbicidal environment
19 November 2007 to the induction of diarrhoea. The fact that there are of the macrophage 18–25 . This promotes bacterial

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REVIEWS

Table 1 | Examples of Salmonella enterica serovars; their hosts and diseases and a hollow needle29,30. Another set of proteins comprise
the translocon, which is thought to be involved in the
Salmonella Host Disease and symptoms translocation of effectors by forming a translocation pore
enterica serovar specificity
in the host cellular membrane31. The effector proteins
Typhoid have been shown to contain specific targeting signals
Typhi; Human- Enteric fever: fever; abdominal pain; transient located in their amino termini that route them to the
Paratyphi restricted diarrhoea or constipation; and a salmon- T3SS32–35. In addition to the short secretion signal, many
coloured maculopapular rash on the trunk effectors have a binding site for a specific chaperone,
Non-typhoid the function of which is thought to be to stabilize and
Typhimurium; Broad-range Gastroenteritis: abdominal pain; vomiting; and target its cognate substrate to the translocation appara-
Enteriditis inflammatory diarrhoea tus34,36,37. Most chaperones are specific for a single effec-
tor, but some can facilitate the secretion of more than one
effector protein38–41. An ATPase that is located in the base
replication and subsequent dissemination throughout of the NC not only drives the export process but also
the reticulo­endothelial system (RES). Infection with facilitates the release of effectors from chaperones before
non‑typhoidal strains in healthy human adults, however, transport42. Although much of the T3SS is similar among
is usually limited to the intestine, where the bacteria Gram-negative bacteria, the set of effector proteins is
induce an early inflammatory response that results in the unique to each species. For a complete list of Salmonella
infiltration of polymorphonuclear leukocytes (PMNs) spp. effectors, their host-cell functions and binding
into the intestinal lumen1,26. Production of the potent partners, see Table 2.
PMN chemokine interleukin (IL)‑8, and, perhaps, other Salmonellae encode two distinct virulence­­‑associated
similar molecules, by the infected epithelia is thought T3SSs within Salmonella pathogenicity islands 1 and 2
to be required for this process27. The release of cyto- (SPI1 and SPI2) that function at different times during
toxic granules by PMNs, as well as the effects of various infection28. Whereas the SPI1-encoded T3SS is active
bacterial molecules in stimulating innate immune on contact with the host cell and translocates bacterial
inflammatory responses and manipulating host- proteins across the plasma membrane, the SPI2 T3SS is
cell processes, may then result in the destruction or expressed within the phagosome and translocates effectors
turnover of the intestinal mucosa, which contributes across the vacuolar membrane. The SPI1 system has been
to inflammatory diarrhoea. shown to be required for invasion of non-phagocytic
Recent progress has been made in our understanding cells, induction of intestinal inflammatory responses
of the molecular mechanisms by which salmonel- and diarrhoea, as well as colonization of the intestine.
lae interact with host cells and manipulate various host The SPI2 T3SS, by contrast, has an important role in
responses that result in the induction of intestinal bacterial survival in macrophages and establishment of
inflammatory responses and systemic illness. Specifically, systemic disease. Interestingly, Brown and colleagues43
the identification of bacterial and host proteins that are have recently shown by RIVET (recombination-based
involved in such processes as the invasion of epithelial in vivo expression technology) that expression of the
cells, stimulation and repression of signalling cascades, SPI2 T3SS in mice might begin in the early stages of
sensing of the intracellular environment and establishment Salmonella enterica serovar Typhimurium (S. typhimu-
of a niche for intracellular replication have contributed to rium) infection, before intestinal penetration. As there
insights into the complex pathogenesis of this bacterium, is currently no evidence that this T3SS is also involved
and will be discussed here in detail. in intestinal colonization, the authors speculated that its
early induction in the intestinal lumen might prepare
Reticuloendothelial system The two virulence-associated T3SSs the bacterium for the inhospitable intracellular environ-
(RES). The meshwork of A specialized apparatus, named the type III secretion ment of the macrophage and ease the transition to the
connective tissue that contains
system (T3SS), is essential to salmonellae pathogen- later systemic phase of the disease. However, it is also
immune cells, such as
macrophages, and surrounds esis and the colonization of host tissues. The T3SS possible that early expression of this T3SS is required for
tissues that are associated mediates the transfer of bacterial virulence proteins, the optimal function of the invasion-associated T3SS, as
with the immune system, such known as effectors, from the bacterial cell into the mutations in the SPI2 apparatus can cause a significant
as the spleen and lymph host-cell cytoplasm28. Once inside the eukaryotic cell, reduction in the expression of several SPI1 T3SS genes
nodes. Immune cells in the RES
provide surveillance of antigens
these effectors can alter host cellular functions, such as and impair the ability of the bacterium to invade epi-
that the body encounters and cytoskeletal architecture, membrane trafficking, signal trans- thelial cells44,45. Conversely, there is mounting evidence
can be quickly recruited to duction and cytokine gene expression, in order to promote that some of the effectors of the SPI1 T3SS are expressed,
sites of infection. bacterial survival and colonization. The T3SS is or persist within, host cells long after bacterial inter-
evolutionarily related to the flagellar export system and nalization and contribute to events that were previously
Pathogenicity island
A large region of genomic DNA is present in multiple Gram-negative animal and plant attributed exclusively to effectors of the SPI2 T3SS46–51.
that encodes genes that are pathogens. It comprises more than 20 proteins, some of Although it is unknown whether mutations in the SPI1
associated with virulence. A which form a supramolecular structure that is known as T3SS could cause pleiotropic effects, these findings indi-
pathogenicity island is typically the needle complex (NC) (FIG. 2). Electron micrographs of cate that the two Salmonella T3SSs do not operate in an
transferred horizontally
between bacterial strains and
purified T3SSs revealed that the NC consists of a multi- independent manner, as previously thought, and pos-
is often inserted into tRNA ring base that spans the inner and outer membranes of sibly cooperate to facilitate the intracellular lifestyle of
genes within the genome. the bacterial envelope, an inner rod that joins the rings the bacteria. This is particularly intriguing, because the

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© 2008 Nature Publishing Group
REVIEWS

older data that had suggested a role for the SPI1 T3SS
during systemic disease17,63. This, combined with the
Salmonella spp. previously mentioned putative function of the SPI2 T3SS
prior to intestinal penetration, further demonstrates that
M cell the dogmatic compartmentalization of the roles of the two
Epithelial cell T3SSs is simplistic. In addition, the recent resurrection of
the streptomycin-treated mouse model of acute intestinal
infection with inflammation and diarrhoea might enable
dissection of the roles of the host and bacteria in acute
intestinal disease64. It is likely that, in the near future,
models of salmonellae-induced chronic intestinal disease
Macrophage at the intestinal mucosal surface will be further devel-
T cell B cell oped and studied. In fact, our group has demonstrated
Gastroenteritis: that the A/J mouse strain develops this type of disease
PMN influx (L. Peiser, K. Smith and S.I.M., unpublished observa-
tions). These animal models should be useful in under-
standing the contributions of mucosal immune defences,
as well as bacterial factors such as T3SS effectors, to
Enteric fever: dissemination chronic intestinal disease. However, these experimental
to lymph nodes, liver and spleen systems also have limitations, as specific disease outcomes
may be different in different hosts for broad host-range
Figure 1 | Biology of salmonellae infection. Orally ingested salmonellae
Nature Reviews |survive at the
Microbiology salmonellae. A future challenge in understanding these
low pH of the stomach and evade the multiple defences of the small intestine in order to animal models will be to determine the effects of specific
gain access to the epithelium. Salmonellae preferentially enter M cells, which transport effectors in both the model and natural hosts. In addi-
them to the lymphoid cells (T and B) in the underlying Peyer’s patches. Once across the
tion, the diversity and evolution of the effector content of
epithelium, Salmonella serotypes that are associated with systemic illness enter intestinal
macrophages and disseminate throughout the reticuloendothelial system. By contrast, different Salmonella strains isolated from natural sources
non-typhoidal Salmonella strains induce an early local inflammatory response, which might also reveal different functions or roles that cannot
results in the infiltration of PMNs (polymorphonuclear leukocytes) into the intestinal be explored by these model systems. In this regard, many
lumen and diarrhoea. S. typhimurium effectors are present in only a percentage
of strains, and in some serotypes, such as Typhi, are only
present as pseudogenes. Selection for the presence or
sequence information and genetic organization of the loss of effectors in specific animal populations or human
two Salmonella T3SSs, as well as their phylogenetic disease settings might, ultimately, be more informative of
distribution among Salmonella species, indicate that the actual natural disease or colonization than the phe-
they were acquired independently, at different periods notypes of deletion mutants in model systems. Therefore,
and from different sources44,52–57. Therefore, it is likely the acceptance of the limitations of these animal mod-
that selection pressure from animal environments led els is important to prevent dogmatic viewpoints about
to the cooperation between the two T3SSs, to opti- the roles of such mechanisms or specific T3SS effector
mize colonization, invasion and intracellular survival proteins. This is particularly relevant to the study of effec-
of salmonellae in animals. tor proteins with redundant functions, as the necessity
The most extensively used animal model for investi- for their similar tasks during pathogenesis might not be
gating the contributions of the two T3SSs to salmonellae apparent in model systems. A complete picture will prob-
virulence is the highly sensitive natural-resistance- ably require the integration of animal model information
associated macrophage protein 1 (Nramp1)‑null mouse, with real world information about effector content and
which is susceptible to mortal infection if inoculated with disease course in nature.
as few as ten bacteria58. Nramp1 is a macrophage-specific
ion exporter that removes ions from the Salmonella- Bacterial-mediated endocytosis
containing vacuole (SCV) and, thus, restricts bacterial The concerted function of at least five SPI1 T3SS effec-
replication59. The Nramp1-null mouse model system tors is required for efficient invasion of cultured epithe-
has been used successfully to identify many important lial cells, although optimal invasion in animal tissues
genes that are required for T3SS-associated virulence60,61. might be more complex and diverse (FIG. 3). SopE, SopE2
However, it is too sensitive to detect phenotypes for and SopB (also known as SigD) activate the host Rho
T3SS effectors that contribute to long-term persist- GTPases Cdc42, Rac1 and RhoG, which leads to actin
ence, because these mice do not survive long enough cytoskeletal reorganization, membrane ruffling and bac-
to study the proteins that are necessary for systemic terial internalization by macropinocytosis65–70. However,
colonization. The resurgence of interest in the resistant recent evidence suggests that only Rac1 and RhoG are
Nramp1-positive mouse model has enabled studies to indispensable for the actin remodelling events that are
be performed that are more physiologically relevant generated by Salmonella spp. during host-cell entry70.
to long-term systemic infection49,62. These reports SopE, SopE2 and SopB are all essential for the invasion of
confirmed that SPI2 T3SS effectors are important for epithelial cells, as an S. typhimurium mutant that is
persistent infection and colonization, but also validated defective in all three effectors cannot induce actin

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a modulating actin dynamics directly. SipA (also


called SspA) helps to initiate actin polymerization
at the site of S. typhimurium entry by decreasing the
critical concentration and increasing the stability

Needle
of actin filaments, whereas SipC (also called SspC),
an effector that also functions as a translocon and is
inserted in the host cell’s plasma membrane with a
cytoplasmic domain that may bind to intermediate
filaments, can nucleate and bundle actin 75–77. In
addition, SipA can enhance the activity of SipC

Base
independently of any host cellular protein, which
indicates that there might be a unique collaboration
between the two effectors78. Although SipA and SipC
might act in concert with SopE, SopE2 and SopB to
mediate the cytoskeletal changes that are required
b
Base
for the formation of membrane ruffles, they can-
not induce membrane ruffling and invasion by
themselves 69,76,77,79. Instead, they seem to facilitate
efficient bacterial uptake by directing the spatial
localization of actin foci beneath the invading bac-
teria and, perhaps, preventing disassembly of the
S. typhimurium-induced actin structures.

Needle Intestinal inflammatory responses


The stimulation of Cdc42 by SopE, SopE2 and SopB
also triggers several mitogen-activated protein kinase
(MAPK) pathways, including the Erk, Jnk and p38 path-
ways, which results in the activation of the transcription
factors activator protein‑1 (AP‑1) and nuclear factor-κB
(NF-κB)16,70,80 (FIG. 3). These transcription factors then
direct the production of pro-inflammatory cytokines,
Figure 2 | Structure of the Salmonella type III
Nature Reviews | Microbiology such as IL‑8, which stimulate PMN transmigration and
secretion system (T3SS). a | Electron micrographs of a
the inflammatory response leading to diarrhoea. Boyle
purified Salmonella enterica serovar Typhimurium
(S. typhimurium) needle complex (NC). b | Electron
and colleagues81 have recently reported that disrup-
cryomicroscopy and surface images of the structures of tion of tight junction structure and function — one
the base and NC of the S. typhimurium T3SS. Part a aspect of S. typhimurium-induced enteritis that is
reproduced, with permission, from Ref. 31 (2002) likely to be important — by the effectors SopB, SopE,
Elsevier Science. Part b reproduced, with permission, SopE2 and SipA also occurs by activation of Rho fam-
from Ref. 185  (2004) American Association for the ily GTPases. In addition, SopB also promotes intestinal
Advancement of Science. disease by increasing the intracellular concentration of
d‑myo-inositol 1,4,5,6-tetrakisphosphate, a compound
rearrangements and, therefore, become intracellular69. that stimulates cellular chloride secretion and fluid
Whereas SopE and SopE2 are potent guanine nucleotide flux69,82. SipB (also called SspB), an effector protein that
exchange factors (GEFs) for all three GTPases, SopB is also part of the SPI1 T3SS translocation machinery,
only stimulates Cdc42 and RhoG indirectly through its adds to the inflammatory response by increasing pro-
phosphoinositide phosphatase activity65,67–70. Although duction of the pro-inflammatory cytokines IL‑1β and
the mechanism of action of SopB is not yet understood, IL‑18 through binding and activating caspase‑1 (Ref. 83).
Patel and colleagues70 have recently shown that activa- Caspase‑1 activation can also occur by the recognition of
tion of RhoG by SopB occurs by a cellular exchange flagellin by the Ipaf cytoplasmic signalling pathway84,85.
factor called SGEF. As SGEF has a phosphoinositide- It is plausible that this is the result of the accidental
binding pleckstrin homology domain, it is plausible translocation of flagellin into the host cell’s cytoplasm
that it is activated by the phosphoinositide fluxes that are by the SPI1 T3SS owing to the conserved evolution of
generated by the enzymatic action of SopB71. Activation the flagellar and the SPI1-encoded secretion systems.
of Rho GTPases then results in the activation of the The observation that caspase‑1-deficient mice are more
Wiskott–Aldrich Syndrome protein (WASP) family susceptible to orally administered S. typhimurium than
members N‑WASP and WAVE2, which leads to recruit- wild-type mice indicates that SPI1 T3SS-mediated cas-
ment of the actin-related protein‑2/3 (Arp2/3) complex pase‑1 activation could be essential to diarrhoeal disease
to sites of membrane ruffles and stimulation of actin and intestinal survival86,87. SopA and SopD also contrib-
polymerization67,72–74. ute to enteritis in calves13,88,89. Recent evidence indicates
There are two SPI1 T3SS effectors that promote that SopA has a HECT (homologous to E6-AP carboxyl
bacterial internalization by binding to actin and terminus)-like E3 ubiquitin ligase activity, which is

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Table 2 | Effectors of the Salmonella SPI1- and SPI2-encoded type III secretion systems (T3SSs)
Effector Cellular function Host-cell target References
SPI1 T3SS
AvrA Inhibits nuclear factor (NF)‑κB signalling and interleukin (IL)‑8 Unknown 99,186
production; also prevents ubiquitination of β‑catenin
SipA or SspA Decreases the critical concentration of G‑actin and increases the F-actin; T‑plastin 76, 81,187,188
stability of F‑actin; also induces PMN transepithelial migration
and disrupts tight junctions
SipB or SspB* Binds and activates caspase‑1 and induces autophagy in Caspase‑1; cholesterol 83,189,190
macrophages
SipC or SspC* Nucleates and bundles actin F-actin; cytokeratin‑8 and 75,77,191
cytokeratin-18
SopA Stimulates PMN transmigration by HECT-like E3 ubiquitin ligase Unknown 89,90
activity
SopB or SigD Activates Cdc42, RhoG, AktA and chloride secretion through its Unknown 69,70,81,82,192
inositol phosphatase activity and disrupts tight junctions
SopD Stimulates fluid accumulation in bovine ligated ileal loops and Unknown 13,82,91
contributes to diarrhoea in calves and systemic disease in mice
SopE Activates Cdc42, Rac1 and RhoG by its GEF activity and disrupts Cdc42, Rac1 and Rab5 65,81,193
tight junctions
SopE2 Activates Cdc42, Rac1 and RhoG by its GEF activity and disrupts Cdc42 and Rac1 67,68,81
tight junctions
SptP Inhibits Cdc42 and Rac1 by its GAP activity and MAPK signalling Rac1 92,94,95
and IL‑8 secretion through its tyrosine phosphatase activity
SPI2 T3SS
GogB Unknown Unknown 194
PipB Unknown Unknown 157
PipB2 Contributes to Sif formation Kinesin‑1 157,165,170
SifA Induces Sif formation, maintains integrity of the SCV and SKIP and Rab7 154,164,166, 181,195
downregulates kinesin recruitment to the SCV
SifB Unknown Unknown 156
SopD2 Contributes to Sif formation Unknown 91
SpiC* Interferes with endosomal trafficking Hook3 150,151–153
SpvB‡ Actin-specific ADP-ribosyltransferase and downregulates Sif Actin 160, 175,176,196
formation
SseF Contributes to Sif formation and microtubule bundling Unknown 162,169
SseG Contributes to Sif formation and microtubule bundling Unknown 162,169
SseI or SrfH Contributes to host-cell dissemination Filamin and TRIP6 160,197
SseJ Maintains integrity of the SCV and has deacylase activity Unknown 155,180
SseK1 Unknown Unknown 198
SseK2 Unknown Unknown 198
SseL Deubiquitinase Ubiquitin 199
SspH2 Inhibits the rate of actin polymerization and contributes to Filamin and profilin 96,160
virulence in calves
SteA Unknown Unknown 200
SteB Unknown Unknown 200
SteC Unknown Unknown 200
SPI1 and SPI2 T3SS
SlrP Contributes to virulence in calves Unknown 201
SspH1 Inhibits NF‑κB signalling and IL‑8 secretion, contributes to PKN1 95–98
virulence in calves and has E3 ubiquitin ligase activity
*Also a component of the secretion apparatus. ‡Has not been definitively shown to be an SPI2 T3SS effector. GAP, GTPase-activating protein; GEF, guanine
nucleotide exchange factor; HECT, homologous to E6-AP carboxyl terminus; MAPK, mitogen-activated protein kinase; PMN, polymorphonuclear leukocyte; SCV,
Salmonella-containing vacuole; Sif, Salmonella-induced filament; SPI, Salmonella pathogenicity island.

nature reviews | microbiology volume 6 | january 2008 | 57


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a multifunctional protein that is also involved in the


Membrane reversal of the pro-inflammatory signalling cascade
Tight
ruffle
junction that is associated with invasion94. It has tyrosine phos-
Salmonella phatase activity, which has a role in downregulation
spp.
of the S. typhimurium-induced activation of the MAPK
Actin
Erk. In addition, S. typhimurium can downregulate IL‑8
production after invasion of intestinal epithelial cells,
SopE SipA SopA and SptP and SspH1 participate in this process95. SspH1
Epithelial
cell
SopE2 SipC SptP is a leucine-rich-repeat protein that is localized to the
SopB
SopB Rho GTPases mammalian nucleus and inhibits NF‑κB-dependent
gene expression95,96. It has also been shown to have
Rho GTPases MAPK CI– MAPK SspH1
AvrA ubiquitin ligase activity97. As it interacts with a host
NF-κB
CI– NF-κB serine/threonine protein kinase called PKN1, which if
AP-1 activated also inhibits NF‑κB-dependent gene expres-
sion, SspH1 could interfere with inflammatory signal-
ling by binding to and activating PKN1 (Ref. 98). In fact,
IL-1β
IL-8
IL-18 PKN1 is activated in epithelial cells during infection
with S. typhimurium. However, as this also occurs in
Macrophage the absence of SspH1, it is possible that SspH1 is not
SipB required for this process or that there are additional
PMN
effectors or bacterial mechanisms that are capable of
activating PKN1. Another SPI1 T3SS effector, AvrA,
Figure 3 | SPI1 T3SS-induced changes in host cells. On contact with the epithelial has been reported to inhibit NF‑κB activity and pro-
Nature Reviews | Microbiology
cell, salmonellae assemble the Salmonella pathogenicity island 1(SPI1)-encoded type III inflammatory cytokine secretion99. However, unlike
secretion system (T3SS) and translocate effectors (yellow spheres) into the eukaryotic its Yersinia spp. homologue, YopJ, which binds and
cytoplasm. Effectors, such as SopE, SopE2 and SopB, then activate host Rho GTPases, acetylates MAPK kinases and the inhibitor of NF-κB
which results in the rearrangement of the actin cytoskeleton into membrane ruffles, kinase (IKK), thereby preventing their phosphorylation
induction of mitogen-activated protein kinase (MAPK) pathways and destabilization of
and activation, AvrA has not been shown to interact
tight junctions. Changes in the actin cytoskeleton, which are further modulated by the
actin-binding proteins SipA and SipC, lead to bacterial uptake. MAPK signalling with or affect the activity of any host proteins that are
activates the transcription factors activator protein‑1 (AP‑1) and nuclear factor-κB involved in the NF‑κB pathway100,101.
(NF‑κB), which turn on production of the pro-inflammatory polymorphonuclear The fact that salmonellae use multiple effectors to
leukocyte (PMN) chemokine interleukin (IL)‑8. SipB induces caspase‑1 activation in dampen the host’s inflammatory response indicates
macrophages, with the release of IL‑1β and IL‑18, so augmenting the inflammatory that this function is important for their pathogenesis,
response. In addition, SopB stimulates Cl– secretion by its inositol phosphatase activity. perhaps by contributing to colonization of the intestinal
The destabilization of tight junctions allows the transmigration of PMNs from the tract over prolonged periods of time. Furthermore, it is
basolateral to the apical surface, paracellular fluid leakage and access of bacteria to the interesting to speculate that the evolutionary pressure
basolateral surface. However, the transmigration of PMNs also occurs in the absence of on the host–pathogen interaction for non-typhoidal
tight-junction disruption and is further promoted by SopA. The actin cytoskeleton is
salmonellae is for asymptomatic intestinal colonization,
restored and MAPK signalling is turned off by the enzymatic activities of SptP. This also
results in the down-modulation of inflammatory responses, to which SspH1 and AvrA whereas for typhoidal strains, which have poor intesti-
also contribute by inhibiting activation of NF‑κB. Figure reproduced from an original nal colonization, the selective pressure is for replication
kindly provided by A. Haraga, University of Washington, USA. within professional phagocytes, a condition that is also
less inflammatory than extracellular growth. The bacte-
ria might induce a mild state of inflammatory response
involved in the S. typhimurium-induced transepithelial and their goal might be to evolve towards parasitism or
migration of PMNs90. However, how SopD, an effector that commensalism. In fact, the long period of intestinal colo-
is also expressed under SPI2 T3SS-inducing conditions nization greatly exceeds that of the acute disease phase for
and continues to contribute to later stages of infection, non-typhoidal strains102. Also, typhoidal disease in the
exerts its effects remains unknown33,91. In summary, all absence of antibiotic therapy is often characterized by
of these mechanisms strongly implicate the SPI1 T3SS in periods of few or no symptoms, followed by a relapse that
the induction of gastroenteritis and intestinal disease. is more of a chronic, prolonged febrile illness, in which
intracellular replication is the predominant bacterial
Reversal of cytoskeletal and signalling responses lifestyle1. Thus, it is likely that a dynamic induction of
Interestingly, shortly after bacterial invasion the actin inflammatory responses, followed by their dampening, is
Transepithelial migration
cytoskeleton regains its normal architecture7. SptP has important to the diseases caused by salmonellae.
The movement of cells, such as
neutrophils and invading been shown to participate in this reversal process by
bacteria, from the basolateral its GTPase-activating protein activity on Cdc42 and The SCV as a niche for replication
(bottom) to the apical (top) Rac1 (Ref. 92) (FIG. 3). The opposing activities of SopE Salmonellae are capable of infecting and replicating in
surface, or the reverse, of an or SopE2 and SptP are mediated by the temporal regula- many cell types, but are thought to primarily replicate
epithelial cell layer. Migration
can also occur between two
tion of these proteins93. SopE has a shorter half-life in in macrophages, as they are found in the lymphatic tis-
adjacent cells through tight eukaryotic cells than SptP owing to its ubiquitination sues and organs of the RES during systemic infection1,103.
junctions. and rapid proteasome-dependent degradation. SptP is Salmonella strains that are defective for macrophage

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Salmonella spp.

intracellularly from hours to days, making it a unique


phagosome with respect to the normal progression of
phagolysosomal maturation and recycling. Although
there has been controversy within the field, several
SspH2 SifA reports have shown that salmonellae can survive within
SpvB PipB2 s macrophages in which the lysosomal compartments
Spacious Ssel tor
phagosome le mo have fused with the SCV107–110. Consequently, the avoid-
u
tub ance of phagolysosomal fusion is unlikely to be a major
M icro Sif
SCV pathogenic strategy of salmonellae. Studies in various
cell types have also demonstrated that the vacuole acidi-
Actin SseJ fies; however, depending on the mechanism of host‑cell
Epithelial cell entry, vacuolar acidification may be delayed in both
macrophages and epithelial cells19,110–112. The SCV has
Microtubules
been shown to interact transiently with the early endo-
cytic pathway and quickly gain and lose early endocytic
markers, such as EEA1 (early endosomal antigen 1), TfR
Nucleus
(transferrin receptor) and the early endocytic traffick-
ing GTPases Rab5 and Rab11 (Refs 113,114). Several late
SseF
SseG endosomal markers are commonly associated with the
SCV at later time points, including the GTPase Rab7,
Golgi Secretory vesicles LAMP1 (lysosomal associated membrane protein 1),
LAMP2, LAMP3 and the vacuolar ATPase110,113,115,116.
There is conflicting data concerning the occurrence of
Figure 4 | Formation of the SCV and induction of the SPI2 Nature
T3SSReviews Microbiology
within| the host cell. M6PR (mannose‑6-phosphate receptor), LBPA (lysobi-
Shortly after internalization by macropinocytosis, salmonellae are enclosed in a sphosphatidic acid) and the lysosomal hydrolase cathep­
spacious phagosome that is formed by membrane ruffles. Later, the phagosome fuses sin D on the SCV115,117–120. Furthermore, cholesterol has
with lysosomes, acidifies and shrinks to become adherent around the bacterium. This is
been reported to accumulate on the SCV118,121. The pres-
called the Salmonella-containing vacuole (SCV), which contains the endocytic marker
lysosomal associated membrane protein 1 (LAMP‑1; purple). The Salmonella
ence or absence of different markers on the persistent
pathogenicity island 2 (SPI2) T3SS (type III secretion system) is induced within the SCV SCV may simply indicate that they are variably detected
and translocates effector proteins (yellow spheres) across the phagosomal membrane rather than reflect whether or not the SCV has matured
several hours after phagocytosis. The SPI2 T3SS effectors SifA and PipB2 contribute to through a normal endocytic pathway. As it has been
Salmonella-induced filament (Sif) formation along microtubules (green) and regulate shown that the SCV can fuse with lysosomes and acidify,
microtubule-motor (yellow star shape) accumulation on the Sif and the SCV. SseJ is a the ability of salmonellae to survive exposure to lyso-
deacylase that is active on the phagosome membrane. SseF and SseG cause somal contents reveals that resistance to antimicrobial
microtubule bundling adjacent to the SCV and direct Golgi-derived vesicle traffic peptides, nitric oxide and oxidative killing are important
toward the SCV. Actin accumulates around the SCV in a SPI2 T3SS-dependent manner, to its survival within macrophages and to virulence122–129.
in which SspH2, SpvB and SseI are thought to have a role.
This is supported by the observations that Salmonella spp.
mutants that are sensitive to these chemicals are attenu-
ated for mouse virulence, whereas mice that are deficient
replication are avirulent in mouse models of infection, for the production of these compounds have increased
which underscores the importance of bacterial survival susceptibility to Salmonella spp.23,104,129–131
and replication in macrophages to disease104. In addi-
tion, the observation that many attenuated strains of Sensing and response to the vacuole
salmonellae are auxotrophic, particularly for purines, Salmonellae sense the acidic environment of the SCV,
pyrimidines, amino acids and other metabolites that resulting in the induction of various regulatory systems
are required for their replication but that are not readily that promote intracellular survival, for example, by
available within mammalian cells, supports the concept surface remodelling of the protein, carbohydrate and
that intracellular replication is essential to salmonellae membrane components of the bacterial envelope19. Such
virulence105. Unlike non-phagocytic cells, salmonellae regulatory systems include OmpR/EnvZ, PhoP/PhoQ,
can enter macrophages by several endocytic processes, RpoS/RpoE, PmrA/PmrB, Cya/Cyp and cyclic diGMP,
including SPI1 and non-SPI1 T3SS-induced macropino- all of which confer resistance to antimicrobial peptides
cytosis20. Following SPI1 T3SS-induced macropinocyto- and oxidative stress18,124,127,129,132–135. The phagosomal
sis, a few SPI1 T3SS effectors, such as SipA, SopB, SopD environment is acidic, with a pH range of <5 to 5.5, has
Auxotrophic
An organism that cannot
and SopE2, persist within the cell and have recently been a concentration of magnesium and calcium in the 1 mM
synthesize certain organic implicated in contributing to the intracellular stages of range and contains antimicrobial peptides and oxygen
compounds, such as amino the infection process47,48,50,51,91,106. Once intracellular, and nitrogen radicals that can damage the bacterial
acids, that are necessary for its salmonellae remain inside a vacuolar compartment, cell19,111,112. Various studies indicate that both pH and
metabolism. For growth,
which has been named the spacious phagosome (SP)20 antimicrobial peptides are important signatures of the
auxotrophic organisms must
be able to take up the lacking (FIG. 4). The SP shrinks over minutes to hours to form phagosomal environment and such conditions activate
compound from the an adherent membrane around one or more bacteria, many of the regulators that are implicated in salmonellae
surrounding environment. which is then referred to as the SCV. The SCV can persist virulence24,25,125,136. It is likely that several sensory systems

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REVIEWS

respond to the phagosome environment and cooperate endosome–endosome fusion in vitro and binds to pro-
to orchestrate the complex cascade of events that are nec- teins that are implicated in vesicular trafficking, its role
essary to alter the bacterial surface and promote intracel- as a T3SS effector protein remains controversial150,151. As
lular survival137. The best characterized of these is the it has not been universally observed to be translocated
PhoQ sensor, which promotes resistance to antimicro- into eukaryotic cells and it is required for the transloca-
bial peptides18,25. PhoQ contains a novel acidic domain tion of several, if not all, SPI2 T3SS effectors, as well as
that is bridged to the inner membrane by interactions the surface expression of translocon proteins, it is more
with metal ions and binds to and initiates responses to likely that SpiC is part of the SPI2 secretion apparatus
antimicrobial peptides138,139. It also responds to pH by and is not an effector152,153. The most important trans-
structural changes that are determined by regions of the located effectors, by virtue of causing virulence defects
protein separate from the metal ion bridges involved in in mice when mutated, are SifA, SseJ, SseF, SseG, SopD2
antimicrobial peptide sensing136. and PipB2 (Refs 91,154–158) . The observation that
After phagocytosis, salmonellae undergo extensive S. typhimurium that lacks any single SPI2 T3SS effector
bacterial surface remodelling, as has been shown for the protein cannot cause the same virulence attenuation in
lipid A component of lipopolysaccharide (LPS) during mice as a mutant strain that lacks the entire SPI2 T3SS
growth within macrophages137,140. Bacterial molecules suggests that many effectors function cooperatively to
that the host can recognize as indicators of infection, exert their effects on the host cell. Furthermore, the
such as the SPI1 T3SS and flagellin, are repressed and deletion or mutation of many effector genes has no
the LPS structure is altered85,141. Some of the specific sur- virulence phenotype, which implies that their functions
face modifications include: decreasing the length of the might be redundant. Early studies of SPI2 T3SS effectors,
O antigen, which is the repeating carbohydrate polymer which primarily focused on determining their subcel-
of LPS; alterations to the number of acyl chains in the lular localization in mammalian cells, revealed that
structure of the lipid A component of LPS; and changes they might have specific targeting sequences that direct
in the protein content of the outer membrane, the inner localization to endosomal compartments, the Golgi
membrane and the peptidoglycan layer142–145. Synthesis apparatus, the actin cytoskeleton and the microtubule
of enzymes that allow the bacteria to cope with oxida- network33,155–157,159–162. This indicates that components
tive and nitrogenous radicals also occurs146. Microarray of these host-cell structures might be the intracellular
studies have shown that up to 919 S. typhimurium targets of the SPI2 T3SS.
genes are differentially regulated in response to the
phagosomal environment, demonstrating that dra- Salmonella-induced tubular endosomes
matic transcriptional and post-translational changes In epithelial cells and interferon‑γ-primed macrophages,
probably occur when salmonellae make the transition the SPI2 T3SS induces the formation of long filamen-
from a nutrient-rich extracellular environment to the tous membrane structures — Salmonella-induced fila-
intracellular environment147. ments (Sifs) — that originate from the SCV and extend
throughout the cell157,163 (FIG. 4). These structures are
Protein delivery across the vacuolar membrane LAMP1-positive and have a similar membrane composi-
As a result of sensing the phagosomal environment, tion to the SCV118. Sif formation requires microtubules,
the expression and assembly of the SPI2-encoded T3SS but not the actin cytoskeleton, although these filaments
is induced134,148. Although the function of this T3SS in can be decorated with actin159. Possible mechanisms that
pathogenesis remains poorly defined, it has been shown contribute to Sif formation include repetitive initiation
to be essential for virulence in the mouse model of infec- of vesicular budding from the SCV, in which fission
tion22,149. As mutants of the SPI2 T3SS cannot replicate events are incomplete, or continuous fusion of endocytic
in tissue-culture cells and animal models, it is likely vesicles with the SCV, which would result in endosomal
that the role of this T3SS during disease is to promote tubulation or elongation of the SCV. Regardless of the
intracell­ular replication within the intestine during the mechanism of Sif formation, the filaments may func-
acute phase of the infection and in other organs during tion to increase the size of the SCV to accommodate
the chronic state. A reasonable hypothesis for the main bacterial replication during systemic infection. Sif
function of the SPI2 T3SS is that it promotes intracellular formation is dependent on the SPI2 T3SS effector SifA,
replication by altering host vesicular trafficking, so that and to a lesser extent on SseF, SseG, SopD2 and PipB2
useful metabolic molecules, such as amino acids and lip- (Refs 91,164,165). However, it is a dynamic phenotype
ids, are routed to the SCV and the vesicular compartment that may also be modulated by other effectors. SifA was
membrane is expanded. To date, at least 20 Salmonella recently shown to bind to the host cell protein SKIP (SifA
spp. effector proteins are known to be translocated by and kinesin interacting protein)166. The same authors
the SPI2 T3SS across the phagosomal membrane into the also found that, if SKIP was depleted by RNA interfer-
eukaryotic-cell cytoplasm; however, their specific roles ence in S. typhimurium-infected cells, the cells failed to
in promoting intracellular replication or modifying form Sifs, suggesting that Sif formation requires SKIP.
vesicular movement are not yet understood (for a list In addition, Alto and colleagues167 identified SifA as a
of SPI2 T3SS effectors, their functions and binding part- member of the WxxxE (tryptophan (W)-variable (x)-x-
ners, see TABLE 2). In addition, no individual translocated x-glutamate) family of bacterial effectors that function as
effector has definitively been shown to alter vesicular mimics of GTPases. As SifA contains a carboxy-terminal
trafficking. Although it has been reported that SpiC alters Caax (cysteine (C)-aliphatic residue (a)-a-x) motif that

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is prenylated and S‑acylated by host-cell enzymes, similar membrane, which has led us to propose that its most
to GTPases, it is possible that SifA uses a GTPase-type likely function during S. typhimurium infection is to
mechanism for membrane localization and, perhaps, Sif cleave phospholipids from the SCV membrane (Megha,
formation168. Furthermore, the region of SKIP that inter- M.B.O. and S.I.M., unpublished observations). The
acts with SifA is a pleckstrin homology domain, which cleavage of acyl chains from phospholipids could then
is commonly found in proteins that bind to signalling promote curvature of the membrane or produce discrete
lipids and other regulatory molecules, including those lipid environments that serve as platforms for promot-
that modulate Rho GTPases166. ing vesicle fusion and binding scaffolding proteins and
The roles of PipB2, SopD2, SseF and SseG in Sif for- such activity could modulate Sif formation. However,
mation are not entirely clear. S. typhimurium strains that how these functions, and Sif formation in general, are
lack any of these effectors do not induce Sifs as efficiently important for Salmonella-induced disease is currently
as wild-type S. typhimurium91,165,169. Instead, infection of not understood. It is plausible that Sifs increase the size
cultured cells with these mutants tends to lead to the for- of the SCV in a specific and directional fashion that
mation of ‘ pseudo-Sifs’, which extend from the SCV and promotes bacterial replication and/or redirect nutri-
co-localize with effectors, but do not contain LAMP1. ent-rich organelles to the SCV. In this regard, the SPI2
This suggests that in the absence of these proteins the T3SS could help salmonellae to replicate inside the
ability to form Sifs — which is defined as being entirely phagosome and gain important nutrients for growth,
LAMP1-positive — is impaired and that induction of but yet avoid some of the host-defence mechanisms and
Sifs involves multiple steps that are orchestrated by dif- inflammatory responses that would result from their
ferent effectors. Transient expression of PipB2 in mam- release into the cytoplasm.
malian cells induces the movement of LAMP1-positive
compartments to the cell periphery, which is probably Microtubule motors and movement of the SCV
the result of its interaction with the plus-end-directed As Sif formation requires microtubules — which have
microtubule motor kinesin165,170. This activity might been observed to accumulate and bundle around the
contribute to the outward extension of the SCV, which SCV — there has been an increased interest in under-
would promote Sif formation. SopD2, which has homo­ standing how microtubules and microtubule motors
logy to the SPI1 T3SS translocated effector SopD, has contribute to the intracellular life cycle of salmonellae
also been shown to cooperate with SifA to induce Sifs, and whether targeting vesicular trafficking along micro-
but by an as yet unidentified mechanism33,91. SseF and tubule networks in infected cells is indeed one of their
SseG promote the aggregation of endosomal vesicles pathogenic strategies162 (FIG. 4). SifA has been reported to
and recruit Golgi-derived exocytic vesicles to the SCV, downregulate the recruitment of the plus-end directed
which suggests that salmonellae are able to usurp both microtubule motor kinesin to the SCV, which is medi-
endocytic and exocytic cellular transport processes171–174. ated by PipB2 (Refs 166,170). It does so by interacting
However, it is not known how these activities directly with SKIP, which, if in a complex with SifA, displaces
influence Sif formation. kinesin from the SCV166. This interference is thought to
The deletion of SseJ and SpvB can cause an increase be crucial for maintaining the integrity of the SCV. The
in Sif formation at later time points during the infection inhibitory activity of SifA on kinesin seems to be domi-
of cultured cells, which suggests that these proteins have nant over the kinesin-recruiting activity of PipB2, which
Sif downregulatory functions175. Furthermore, these is suggestive of a potential coordinate or temporal regu-
effectors have virulence defects in the Nramp1-null ani- lation between the two effectors170. In addition, SseF and
mal model, which indicates that their activities contrib- SseG have been found to co-localize with microtubules
ute to systemic infection156,176. Although SpvB has not and cause microtubule bundling in S. typhimurium-
been formally demonstrated to be an SPI2 T3SS effector, infected cells. In particular, SseF has been implicated
some studies suggest that the SPI2 T3SS is required for in recruiting the minus-end directed motor dynein to
its activity in infected cells177,178. SpvB is an actin-specific the SCV162,173. Therefore, it would appear that the SCV
ADP-ribosyltransferase that promotes actin depolym- accumulates dynein, but not kinesin. However, as SifA
erization176. SseJ has homology to glycerophospholipid inhibits the interaction between Rab7 and its effector
cholesterol acyl transferase enzymes, which can remove RILP, a protein that is associated with the dynein motor
acyl chains from phospholipids and transfer them to complex, it is plausible that SifA also prevents dynein
cholesterol in a two-step deacylase-acyltranferase accumulation on the SCV181,182. Although these results
reaction32,179. In fact, purified SseJ has deacylase activ- paint a paradoxical picture of the need to promote or
ity in vitro, which has been shown to be required for inhibit microtubule-motor accumulation on the SCV,
its virulence in mice180. It has been proposed that SseJ salmonellae might employ a carefully choreographed
Prenylated
and SifA have complementary roles in maintaining the combination of both the recruitment and inhibition
The post-translational addition
of lipid chains, such as farnesyl integrity of the SCV membrane, because sifA-mutant of both types of microtubule motors. For example, at
or geranylgeranyl, to cysteine S. typhimurium tends to lose the SCV membrane but specific times during infection, the recruitment of
residues in proteins that does not do so if SseJ is also lacking. This suggests that, dynein could promote minus-end-directed movement
contain a prenylation motif in the absence of SifA, SseJ could cause damage to the of the SCV to stabilize it near the nucleus or the Golgi
called a CaaX box. This
process facilitates membrane
SCV by its enzymatic activity. In addition, our labora- apparatus, where the SCV would have increased access
localization and/or protein– tory has obtained evidence that SseJ has phospholipase to trafficking vesicles that contain nutrients and mem-
protein interactions. activity in vivo that might be localized to the endosomal brane, whereas at other times the recruitment of kinesin

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REVIEWS

to the SCV could induce plus-end-directed movement of VAP, presumably by ADP-ribosylating actin and
towards the cell periphery to promote the enlargement promoting actin depolymerization160,176. SspH2 has also
of the SCV and/or elongation of Sifs. been reported to inhibit the rate of actin polymerization
Recent studies have also shown that the SCV in vitro160. In addition, SspH2 can interact with newly
in infected cultured epithelial cells is localized in a polymerized actin, probably by binding to the actin-
perinuclear position and that effectors from both the binding proteins filamin and profilin, through its amino
SPI1- and the SPI2-encoded T3SSs are involved in this and carboxy termini, respectively. Another effector, SseI
process50,161,166,170,173,181. In particular, SifA, SseF, SseG (also called SrfH), shares a high degree of identity with
and SipA seem to control this phenomenon, because the amino terminus of SspH2 and has been shown to
the SCV containing S. typhimurium strains lacking any bind to polymerized actin, probably through filamin.
of these effectors does not localize near the nucleus. As Although transiently expressed SspH2 and SseI are
the disruption of perinuclear localization of the SCV is associated with VAP during S. typhimurium infection,
correlated with decreased bacterial replication, it has they are not required for the formation of this structure.
been proposed that salmonellae require juxtanuclear Thus, unlike the clearly defined and temporally control-
positioning for optimal growth. In fact, Salcedo and led roles of the SPI1 T3SS effectors in manipulating the
Holden161 have shown that SCVs in close proximity actin cytoskeleton during invasion of epithelial cells, the
to the nucleus contain more bacteria on average than biological significance of the SPI2 T3SS-mediated actin
SCVs further away from the nucleus. However, it recruitment to the SCV, and the interaction of the effec-
has not been directly demonstrated that non-nuclear tors of this T3SS with the actin network, remains to be
localization of the SCV, per se, is inhibitory to bacterial understood. It might be important for translocon stabil-
replication. Rather, it seems more likely that the lack ity and translocation, thereby allowing the full effector
of these effectors or disruption of vesicle trafficking complement to be delivered consistently, and it could
along the microtubule network correlates with defective explain the importance of this process to intracellular
replication and that non-nuclear SCV positioning is a replication183. Subsequently, as T3SS effectors seem to
by-product of this more direct effect. Furthermore, the decrease or remodel actin polymerization, there might
positioning of the SCV might be different in polarized be a period in which actin removal is important, for
epithelial cells, in which movement of the vacuole in a example, to decrease inflammatory responses or allow
directional fashion, such as during transcytosis, could fusion with, or remodelling of, the endosomal mem-
be more physiologically relevant. As discussed above, it brane. Alternatively, actin removal could be essential for
is possible that positioning the SCV in close proximity the effectors to interact with the membrane surface, as
to the Golgi, or other organelles, has some advantages certain protein and phospholipid domains that are cov-
for salmonellae in terms of acquiring nutrients, inhibit- ered in polymerized actin may inhibit their binding to
ing immune responses, prolonging the maturation of important regions within the phagosome membrane.
intestinal epithelial cells and allowing more intracellular
time before epithelial shedding into the intestinal lumen. Future perspectives
All of these effects could then contribute to intestinal The study of the molecular basis of Salmonella spp.
colonization or the intracellular growth of salmonellae. pathogenesis in mammals has advanced greatly over
the past 15 years owing to the identification of many
Actin polymerization around the SCV of the key molecules and mechanisms in mammalian
SPI2 T3SS-dependent actin condensation around the model systems and the discovery of important innate
SCV has been observed, which suggests that this T3SS immune receptors and responses to the bacteria. For
is also involved in cytoskeletal modifications160,183 (FIG. 4). example, important findings have been made in our
In addition, the treatment of S. typhimurium-infected understanding of the mechanisms of entrance into non-
cells with inhibitors of actin polymerization results in phagocytic cells, the way bacteria sense the intra­cellular
decreased bacterial replication, which indicates that the environment and remodel their surfaces, and the inflam-
manipulation of the actin cytoskeleton is important for matory responses to salmonellae. Concurrently, the
bacterial replication183. Although the effectors that are identification of innate immune receptors for bacterial
responsible for the recruitment of actin to the SCV have products and characterization of ligand binding by these
not been identified, several have been shown to interact receptors has progressed greatly. We anticipate a fur-
with actin-binding proteins or to directly manipulate ther explosion of information in the next decade about
actin polymerization. However, it is also possible that the details of the intracellular lifestyle of salmonellae,
SCV-associated actin polymerization is not mediated the function of effectors that are translocated across the
by effectors, but is due to the insertion of the T3SS phagosome membrane and the response of the host to
translocon into the SCV membrane, which has also the activity of these bacterial proteins. Studying how
been observed with the similar Yersinia spp. T3SSs184. salmonellae manipulate endosomal trafficking through
Indeed, most SPI2 T3SS effectors that have been shown to the endocytic pathway should lead to important obser-
interact with components of the actin cytoskeleton have vations about unknown mechanisms of endocytic traf-
actin-inhibitory activities, which suggests that the most ficking. In addition, how effectors function to bring
likely goal of salmonellae is to reduce or reverse vacuole- together mammalian protein components that may
associated actin polymerization (VAP). For example, not normally be together could offer important find-
SpvB has been shown to be involved in the disassembly ings with relevance for non-infectious diseases. The

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© 2008 Nature Publishing Group
REVIEWS

study of salmonellae in more-resistant mouse models, model systems. In addition, the availability of human and
coupled with sophisticated imaging, should also lead to animal genotyping will lead to an increase in data about
a greater understanding of the pathophysiological role of the diversity of human and animal responses to infec-
identified virulence mechanisms and the development tion with various salmonellae. Model systems in various
of chronic disease. This may have relevance for other dis- animals that have diverse innate immune modifications
eases at mucosal surfaces, such as inflammatory bowel will also need to be developed to further understand the
disease. The future decade may also take the field of natural world in the context of our available tools and
salmonellae pathogenesis and host responses beyond to, ultimately, reach the stage in which the evolution of
model systems to the diversity of bacteria and hosts in microorganisms and the emergence of infectious diseases
nature. Emerging technologies, such as whole-genome can be observed in real time. Therefore, the study of sal-
sequencing, will allow us to investigate the diversity monellae pathogenesis should yield a rich treasure trove
of effectors that are used by the many pathogenic of information for those who are interested in microbial
Salmonella species, contributing to our view of host pathogenesis, innate immunity, cell biology and genom-
specificity. Specific hypotheses will be generated by sta- ics, and should remain an important model system of
tistical association, which will then need to be tested in host–pathogen interactions for many years to come.

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An integrated model of the recognition


of Candida albicans by the innate
immune system
Mihai G. Netea*, Gordon D. Brown‡, Bart Jan Kullberg* and Neil A. R. Gow §
Abstract | The innate immune response was once considered to be a limited set of responses
that aimed to contain an infection by primitive ‘ingest and kill’ mechanisms, giving the host
time to mount a specific humoral and cellular immune response. In the mid‑1990s, however,
the discovery of Toll-like receptors heralded a revolution in our understanding of how
microorganisms are recognized by the innate immune system, and how this system is
activated. Several major classes of pathogen-recognition receptors have now been
described, each with specific abilities to recognize conserved bacterial structures. The
challenge ahead is to understand the level of complexity that underlies the response
that is triggered by pathogen recognition. In this Review, we use the fungal pathogen
Candida albicans as a model for the complex interaction that exists between the host
pattern-recognition systems and invading microbial pathogens.

The clinical spectrum of Candida spp. infections ranges unlikely that antifungal agents will have an impact on the
Innate immune system
The suite of host responses to from benign colonization of the skin and mucosal sur- grim mortality statistics for systemic fungal infections
microbial invaders that results faces to mucocutaneous forms of candidiasis and sys- without the aid of new clinical approaches, such as com-
in rapid defence without temic infections (candidaemia and deep-seated organ bining chemotherapy with immunotherapy5. However,
requiring prior stimulation. candidiasis). Despite the fact that fungal infections are augmenting the ability of the immune system to elimi-
typically self-limiting, the number of life-threatening nate a pathogen requires a sophisticated understanding
systemic fungal infections has risen steadily over the of the molecular mechanisms that are involved in patho-
past three decades1, although this increase might now gen recognition and in the host immune response. In
be reaching a plateau2. The increased prevalence of fungi this Review we describe the recent progress that has been
*Department of Medicine as agents of disseminated infection has been restricted to made in research into the mechanisms that are involved
and Nijmegen University
patients in whom surgical or chemotherapeutic interven- in the recognition of fungal pathogens, and present
Centre for Infectious
Diseases, Radboud University tions and/or underlying immunological deficiencies have how this progress has changed our understanding of
Nijmegen Medical Centre, allowed fungi to overwhelm the protective host defence the pathogenesis of infections in general, and invasive
Nijmegen, The Netherlands. mechanisms2. In healthy and immunologically normal candidiasis in particular.

Institute of Infectious individuals, the innate immune system is an efficient
Diseases and Molecular
Medicine, Division of
sentinel that provides protection from the thousands of Innate immunity and host defence
Immunology, CLS, University fungal species that humans regularly encounter. Until recently, little was known about the ways in which
of Cape Town, Rondebosch Candida albicans is a ubiquitous fungal organism that neutrophils and macrophages, the major players in
7925, South Africa. often colonizes the skin and the mucosal surfaces of nor- innate immunity, recognized C. albicans as a pathogenic
§
School of Medical Sciences,
mal individuals, without causing disease. However, when microorganism, or how the fungal–leukocyte interaction
Institute of Medical Sciences,
University of Aberdeen, the normal host defence mechanisms are impaired (for triggers an inflammatory response. The dogma that had
Foresterhill, Aberdeen AB25 example, in patients who are undergoing chemotherapy been blindly accepted over the past 50 years was that
2ZD, UK. for malignancies, receiving immunosuppressants after although effective, innate immunity was non-specific
Correspondence to M.G.N. & an organ transplant, or patients with AIDS), C. albicans and “rather primitive and dumb” (Ref. 6). However, this
N.A.R.G.
e-mails: m.netea@aig.umcn.nl;
can become a pathogen. Candidaemia has an incidence simplistic model, in which innate immunity performs
n.gow@abdn.ac.uk of between 1.1 and 24 cases per 100,000 individuals, and only simple ‘ingest and destroy’ tasks, could not explain
doi:10.1038/nrmicro1815 an associated mortality of more than 30%3,4. It seems how innate immune cells recognize microbial pathogens

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Box 1 | The recognition of microorganisms by pattern-recognition receptors


The innate recognition of pathogens is achieved through germ-line-encoded receptors, the pattern-recognition
receptors (PRRs), which sense conserved chemical signatures called pathogen-associated molecular patterns (PAMPs).
Four major classes of PRRs have been identified.
• Toll-like receptors (TLRs) are cell-membrane-associated (TLR1, TLR2, TLR4, TLR5 and TLR6) or intracellular (TLR3, TLR7,
TLR8 and TLR9) receptors. Several TLRs have been implicated in the recognition of fungal components: TLR2 recognizes
phospholipomannan, TLR4 recognizes O‑linked mannans, TLR6 is involved in the recognition of zymosan and TLR9
detects fungal DNA.
• C-type lectin receptors (CLRs) are mainly membrane-bound receptors that recognize polysaccharide structures from
Candida albicans: dectin 1 recognizes β-glucans, whereas the macrophage mannose receptor (MR) and DC‑SIGN
recognize N‑linked mannans.
• Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and retinoic-acid-inducible gene I (RIGI)
receptors are intracellular receptors for bacterial peptidoglycans and viral nucleic acids. So far, no studies have
documented the involvement of NLRs or RIGI receptors in the recognition of fungi.
• The blueprint for the structure of PRRs is represented by the extracellular pathogen-recognition domain (the Leu-rich
repeat (LRR) domain in TLRs and the C‑type lectin domain (CLD) in CLRs). The intracellular signalling domain (the TLR-
interleukin 1 receptor, TIR domain) of TLRs and the immunoreceptor Tyr-based activation-like motif (ITAM) from some
CLRs, such as dectin 1, ultimately induce the intracellular signals responsible for the functional activity of the receptor.
The four major classes of PRRs discussed here are restricted to families of mammalian molecules with a PRR
function at a cellular level that have a proven signalling pathway that leads to activation of the innate host response.
There are other classes of molecules with less well established functions as PRRs. (For example, peptidoglycan-
recognition proteins (PGRPs), which recognize peptidoglycans in insect and mammalian cells. Although PGRPs
induce defensins in Drosophila melanogaster, their function in mammalian cells has not yet been established.) Some
circulating proteins that bind bacterial structures (for example, pentraxins and mannose-binding lectin (MBL)), are
also considered by some to be PRRs.

as ‘non-self ’, or why different responses are triggered by chains to form a tough three-dimensional network of
different classes of microorganisms. Only in the past microfibrils. Most models suggest that the skeletal com-
decade has it become clear that the innate immune ponents of the cell wall are found close to the cell mem-
system not only specifically recognizes various classes brane in an inner layer, although some chitin and glucan
of microorganisms, it also initiates and modulates the can be present throughout the thickness of the wall. In
subsequent adaptive responses that are delivered by T- budding yeast cells, a scar is left on the mother cell after
cells and B cells through their interactions with antigen- separation of the bud, and at this site the components
presenting cells (especially dendritic cells (DCs))7. The of the inner layers of the cell wall, such as chitin and
tasks of recognizing an invading pathogen and acti- β-(1,3)-glucan, can become exposed at the surface11.
vating the host response are accomplished by pattern- In addition to the glucan and chitin skeleton, the
recognition receptors (PRRs), which recognize conserved C. albicans cell wall contains a matrix that mainly com-
microbial chemical signatures called pathogen-associated prises glycosylated proteins. In C. albicans, the major
molecular patterns (PAMPs) (BOX 1). class of cell wall proteins are glycosylphosphatidylinositol
(GPI)-anchor-dependent cell wall proteins (GPI-CWPs),
The fungal cell wall which are attached through a GPI remnant to β-(1,3)-
The fungal cell wall is an essential organelle that main- glucan or chitin by a highly branched β-(1,6)-glucan
tains the viability of fungal cells. The regulation of fungal linker. The CWPs are normally highly glycosylated with
cell wall biosynthesis and glycosylation has been exten- mannose-containing polysaccharides (sometimes called
sively reviewed elsewhere8–10, but a basic outline of the mannan), and carbohydrates can account for up to 90%
components of the cell wall is necessary to understand of their molecular mass. Many CWPs have a lollipop
how it is recognized by the host immune system. To be structure with a globular domain that is presented to
strong, yet plastic, fungal cell walls combine skeletal and the outside of the cell and a Ser/Thr-rich polypeptide
matrix components in a way that resembles the engineer- stem-like domain that is stabilized in the cell wall by
ing principles that are involved in constructing elaborate O‑linked mannan side chains 8. These ether-linked
structures, such as reinforced concrete buildings, that O‑mannans are relatively short, linear polysaccharides
are made of mesh and mortar. The cells of the innate that, in C. albicans, consist of one to five mannose (Man)
Dendritic cells
immune system recognize elements of both the skeletal sugars that are almost exclusively α-(1,2)-linked (FIG. 1).
‘Professional’ antigen-
presenting cells that are found and matrix components of the C. albicans cell wall. N‑mannan consists of a core Man8GlcNAc2 trianten-
in the T-cell areas of lymphoid The skeletal component of the cell wall of the major- nary complex to which a highly branched structure is
tissues and as minor cellular ity of fungal pathogens, including C. albicans, is based attached, comprising up to 150 mannose sugars arranged
components in most tissues. on a core structure of β-(1,3)-glucan covalently linked as an α-(1,6)-linked backbone with side chains of α-
They have a branched or
dendritic morphology and are
to β-(1,6)-glucan and chitin (a β-(1,4)-linked polymer of (1,2)-, α-(1,3)-mannose and phosphomannan9 (FIG. 1).
important stimulators of T-cell N‑acetylglucosamine (GlcNAc)) (FIG.1). These polymers The mannan structures define many of the serotypes
responses. form hydrogen bonds between adjacent polysaccharide of Candida spp.12 For example, in serotype B strains

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Mannan N-acetylglucosamine
Cell wall protein α-(1,3)-mannose
β-(1,6)-glucan α-(1,2)-mannose
β-(1,3)-glucan α-(1,6)-mannose
Chitin β-(1,2)-mannose
β-(1,4)-mannose P
β-(1,6)-glucose
β-(1,3)-glucose n

NH O
Asn X Ser/Thr Ser/Thr

Pir-CWP

CWP

Figure 1 | The structure of the Candida albicans cell wall. The schematic shows the major components of the cell wall
and their distributions. β-(1,3)-glucan and chitin (poly-β-(1,4)-N‑acetylglucosamine) are the main structural
Nature Reviewscomponents,
| Microbiology
and these are located towards the inside of the cell wall. The outer layer is enriched with cell wall proteins (CWP) that are
attached to this skeleton mainly via glycosylphosphatidylinositol remnants to β-(1,6)-glucan or, for mannoproteins with
internal repeat domains (Pir-CWP), via alkali-sensitive linkages to β-(1,3)-glucan. The insets show the structure of the
glucan and mannan components.

β-(1,2)-mannose is found exclusively in the phospho- types ultimately determines the type of response that is
mannan, whereas in serotype A strains β-(1,2)-mannose initiated following recognition of C. albicans.
also occurs in the acid-stable side-chain fraction9.
Mannans and mannoproteins. Both mannans and man-
Fungal recognition noproteins from the C. albicans cell wall have important
Cytokines Owing to the localization of mannoproteins and man- immunostimulatory activities, ranging from stimulation
Biologically active molecules
nans in the outermost part of the cell wall, mannan of cytokine production13,14 to induction of DC matura-
that are released by cells and
that affect the function of other detection would be expected to be one of the first steps in tion15 and T-cell immunity16,17. Mannoproteins induce
cells. the recognition of C. albicans by the host innate immune mainly T helper 1 (TH1)-type cytokine profiles, which
system. However, the presence of β‑glucans and chitin, have protective effects against disseminated C. albicans
Fcg receptor especially at the level of the bud scar, is also likely to infection18.
A surface molecule on various influence the recognition of C. albicans by leukocytes. The first receptor on the surface of macrophages to
cells that binds to the Fc
regions of immunoglobulins,
So how is the recognition of the C. albicans cell surface be described as a mannan receptor was the C‑type-lectin
thereby initiating effector achieved by the immune system? mannose receptor (MR)19,20. The MR recognizes oligosac-
functions. The main cells of the host innate immune response charides that terminate in mannose, fucose and GlcNAc21,
that recognize invading pathogens are monocytes and and this binding is mediated by carbohydrate-recognition
T helper 1
neutrophils in the circulation, together with macro- domains (CRDs) 4 to 8 in the extracellular region of
An immune response that is
characterized by a subset of phages in infected tissues. Monocytes express high levels the receptor22. In vitro studies have shown that the MR
helper T-cells that secrete a of Toll-like receptors (TLRs) on their cell membranes, as preferentially recognizes α-linked oligomannoses with
particular set of cytokines, well as moderate levels of lectin receptors (LRs). During branched, rather than linear, structures23, and this was
including interleukin 2, differentiation into macrophages, they retain expression supported by data that demonstrated that in mono-
interferon-γ and TNFα, the
main function of which is to
of TLRs while strongly upregulating their expression of cytes and macrophages, the MR recognizes branched
stimulate phagocytosis- LRs. Important differences in the expression of several N‑bound mannans from C. albicans24. By contrast,
mediated defences against receptors have also been reported between resident and recognition of the shorter linear structures of O‑bound
intracellular pathogens. inflammatory macrophages, as the expression of PRRs mannan is performed by TLR4 (Ref. 24), and results in
can be modified by cytokines or microbial products. DCs, cytokine production25. Interestingly, TLR4 stimulation
C-type lectin
which are crucial for antigen processing and presenta- is lost during the germination of yeast into hyphae,
C-type lectins are largely
calcium-dependent animal tion, also express most of the PRRs that are important which leads to a loss of interferon-γ (IFNγ) production
lectins that are carbohydrate- for the recognition of fungal pathogens. Neutrophils capacity26. On DCs, however, the binding of C. albicans
binding proteins. The binding show moderate expression of TLRs and strongly express mannans is mediated through the MR and DC‑SIGN
activity of C-type lectins is phagocytic receptors such as complement receptor 3 (DC‑SIGN is a receptor that is specifically expressed on
based on the structure of the
carbohydrate-recognition
(CR3) and Fcγ receptors (FcγRs), but the expression of the cell membrane of DCs)27.
domain, which is highly PRRs on T cells is much more restricted (FIG. 2). The The α-linked mannose structures on the surface
conserved in this family. mosaic of PRRs that is expressed by each of these cell of C. albicans are recognized by the MR, TLR4 and

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Monocyte suggests that β-glucans are exposed on the cell surface35,


TLR4 although possibly restricted to specific regions, such as
Dectin 1
bud scars11. β‑glucans can stimulate leukocytes in vitro,
TLR2 which induces cytotoxic and antimicrobial activities as
MR
TLR6 well as the production of pro-inflammatory mediators,
Neutrophil Macrophage cytokines and chemokines36. β‑glucans are released into
TLR9
TLR2 TLR6 the circulation during systemic fungal infections37.
TLR2 TLR4 The recognition of β‑glucans has primarily been
Dectin 2 Dectin 1
ascribed to two receptors, CR3 and, more recently,
dectin 1. Although other β‑glucan receptors have been
CR3 Galectin 3 TLR9 described, including lactosylceramide and scavenger
MR
receptors, the physiological role of these receptors is
FcγR Dectin 1 TLR4 still unclear. CR3 is a widely expressed β2-integrin that
CR3
FcγR recognizes several endogenous and exogenous ligands,
Dendritic cell
CD4
pathogens that have been opsonized by iC3b (the inac-
TLR2 Dectin 2 tivated form of complement component C3b) and carbo-
TLR2
hydrates, including β‑glucans. Carbohydrate recognition
DC- Dectin 1
is mediated by a lectin domain38,39, which is distinct from
SIGN
TLR9 TLR9 the normal ligand-binding site (the I domain) of CR3
MR
TLR4 (Ref. 39). The lectin domain mediates recognition of both
TLR4
the yeast and hyphal forms of C. albicans40,41, as well as
FcγR
CR3 several other fungi. Recognition by CR3 does not trigger
Figure 2 | Cell populations and pattern-recognition receptors involved in
protective host responses, such as the respiratory burst42,
Candida albicans recognition. The main populations involved Nature Reviews
in the | Microbiology
recognition and can repress pro-inflammatory signals43,44.
of C. albicans during the innate immune response are the monocytes, neutrophils and Dectin 1 is a myeloid-expressed transmembrane
macrophages. Dendritic cells are crucial for processing of, and antigen presentation to, receptor and possesses a single extracellular, non-
T cells, and thus to activation of specific immunity. The differential expression of pattern- classical C‑type-lectin-like domain that specifically rec-
recognition receptors by these cell types is shown. CR3, complement receptor 3; FcγR, ognizes β-(1,3)-glucans. Dectin 1 can recognize several
Fcγ receptor; MR, mannose receptor; TLR, Toll-like receptors. fungi, including C. albicans yeast, although it does not
appear to recognize C. albicans hyphae11,45. The cyto-
plasmic tail of dectin 1 contains an immunoreceptor
DC‑SIGN. By contrast, β-(1,2)-mannosides, which are tyrosine-based activation-like motif (ITAM), which can
present in the acid-stable and acid-labile components mediate various protective responses through spleen
of mannoproteins and in phospholipomannan (PLM), tyrosine kinase and caspase recruitment domain pro-
are recognized by other mechanisms. On the one hand, tein 9 (Syk–CARD9)-dependent pathways, such as the
PLM reportedly stimulates cytokine production through stimulation of interleukin 2 (IL-2) and IL‑10 (Ref. 46),
TLR2 (Ref. 28). On the other hand, it has been suggested IL‑6 (Ref. 47), and IL‑17 production48. Although Syk-
that recognition of the acid-labile β-(1,2)-mannosides is dependent signalling from dectin 1 is sufficient for
redundant in human monocytes24, although it might play a these responses, stimulation of the mitogen-activated
role in tissue macrophages, particularly in the gut mucosa. protein kinase (MAPK) and nuclear factor (NF)-κB
In this respect, a recent study has shown that galectin 3 pathways, with subsequent production of pro-inflam-
on the surface of murine macrophages can discriminate matory cytokines, such as tumour necrosis factor (TNF),
between pathogenic C. albicans and non-pathogenic requires collaborative signalling with the TLR2 recep-
Saccharomyces cerevisiae, and that an association between tor49,50. The precise nature of the TLR2 ligand from C.
galectin 3 and TLR2 is involved in this process29. albicans has not yet been completely elucidated, although
Another lectin family member, dectin 2, has also PLM might be recognized by TLR2 and TLR6 (Ref. 28).
been described to function as a receptor for C. albicans It has recently been suggested that phagocytosis of
mannans30, although this interaction is less well charac- C. albicans by neutrophils can be mediated by the minor
terized. Dectin 2 is mainly expressed on myeloid cells cell wall component β-(1,6)-glucan51. Beads coated with
and maturing inflammatory monocytes31. Owing to its β-(1,6)-glucan are ingested by neutrophils, and the
short intracytoplasmic tail, dectin 2 must interact with phagocytosis of yeast cells treated with β-(1,6)-glucanase
the FcγR to induce intracellular signals and, interest- is reduced. This recognition appears to be mediated by
Chemokines
ingly, seems to be mainly involved in the recognition of CR3 following opsonization by C3d fragments that bind
Small, inducibly secreted
proteins that induce the C. albicans hyphae32. Dectin 2 is encoded by a different β-(1,6)-glucan.
activation and migration of gene cluster to the β-glucan receptor dectin 1 (see below)
lymphocytes. and does not recognize β‑glucans32; rather, dectin 2 has Other C. albicans components. In addition to manno­
binding specificity for mannose33. proteins, mannans and β‑glucans, other structures of
Complement C. albicans can also be recognized as fungal PAMPs.
A part of the innate immune
system that comprises serum
β-glucans. The C. albicans cell wall consists of approxi- Chitin, a less studied polysaccharide component of the
proteins that can protect mately 60% β-glucan34. Although initially thought to be C. albicans cell wall, is a β-(1,4)-linked homopolymer of
against infection. buried beneath the mannoprotein layer, recent evidence GlcNAc that forms antiparallel hydrogen-bonded chains

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called microfibrils52. Recently, it has been demonstrated fungi, an activity that can be enhanced by TLR signal-
that chitin induces recruitment of immune cells that ling50,64. The respiratory burst is an essential antifungal
mainly release IL‑4 and IL‑13 (Ref. 53). Little is known effector mechanism that results in the production of
about the recognition pathways of chitin and its role dur- toxic oxidants65,66 and in the activation of granule pro-
ing C. albicans infections, and no recognition receptors teases that can kill C. albicans65–67. Killing of C. albicans
for chitin have yet been described. also occurs extracellularly, through the as-yet-undefined
Bacterial and fungal DNA are poorly methylated, in actions of PRRs such as galectin 3 (Ref. 68).
contrast to mammalian DNA. This difference has been
proposed to be instrumental in the recognition of non- Cytokine induction. More is known about the receptors
self DNA by TLR9 (Ref. 54). Although the recognition of that are involved in the induction of cytokine production
fungal DNA has not been formally demonstrated, the by C. albicans. At least four TLRs (TLR2, TLR4, TLR6
involvement of TLR9 in the recognition of C. albicans is and TLR9) are involved in triggering these responses.
supported by the observation that cytokine production Upon recognition of microbial structures, TLRs activate
from CD4+ T-cells from TLR9–/– mice is skewed (higher either the NF‑κB or MAPK pathway, which lead to the
IL‑4, lower IFNγ) compared to cytokine production stimulation of pro-inflammatory cytokine produc-
from CD4+ T cells from wild-type mice, upon challenge tion69. The balance between the signals that are induced
with C. albicans yeast55. As yet, no studies have investi- by TLR2 and TLR4 seems to have a crucial role in the
gated the possible role of RNA recognition systems (that regulation of the immune response. TLR4 can strongly
is, TLR3, and TLR7 and TLR8) in the host response to stimulate pro-inflammatory cytokines through two path-
C. albicans infection. ways: one involves the myeloid differentiation primary
response gene 88 (MyD88)–Mal-mediated induction
Activation of host defence by PRRs of the NF‑κB-dependent release of pro-inflammatory
At first glance, ligation of the various host immune cytokines and chemokines; the other involves the inter-
receptors by C. albicans PAMPs appears to lead to a feron regulatory factor 3 (IRF3)-dependent release of
set of standard, and possibly redundant, pathways that type I interferons70, which induce the secondary production
stimulate cytokine production, phagocytosis and fun- of TH1-type cytokines such as IFNγ 26,55,71.
gal killing. However, this early model of a ‘standard’ By contrast, several studies have demonstrated that
PRR response has been refined over the past few years. although TLR2 ligation can induce pro-inflammatory
Various PRRs enable the innate immune system not cytokines, this effect is weaker than that mediated by
only to recognize specific PAMPs, but also to specifi- TLR4 ligation72. Moreover, TLR2 ligands fail to induce
cally modulate the response that follows. In addition, by the release of IL‑12 and TH1-type IFNγ, thus promoting
inducing specific cytokine profiles, PRRs bring a certain conditions that are favourable for TH2- or regulatory T cell
degree of specificity to the innate response. (TReg)-type responses73. Stabilization of the transcription
Type I interferons factor c‑Fos, which is a suppressor of IL‑12 expression,
Interferons that are rapidly C. albicans uptake. Phagocytosis of C. albicans is medi- is an important step in this process74. In support of
induced by virus replication, as ated by the concerted action of several opsonic and non- this, a recent in vitro study has reported that zymosan
well as by some bacterial and opsonic receptors. Complement binding and activation induces the development of a tolerigenic DC population
fungal infections.
is mediated by the alternative pathway, and complement through TLR2 and dectin 1 (Ref. 75). The induction of
TH2
activation is mainly important for the chemotaxis and this tolerigenic cytokine profile depends on extracel-
A type of activated T helper opsonization of C. albicans, but not for C. albicans lysis, lular signal related kinase (ERK)/MAPK phosphoryla-
cell that participates in which is prevented by the thick and complex cell wall56. tion, a mechanism that is distinct from the p38/Jun
phagocytosis-independent Furthermore, although mannose-binding lectin does N‑terminal kinase (JNK) pathway that is induced by
responses and that
bind and recognize C. albicans, the lectin pathway of TLR4 stimulation75,76. Also, in vivo studies have shown
downregulates pro-
inflammatory responses that complement activation probably plays only a minor role that knockout mice that lack TLR2 are more resistant
are induced by TH1 cells. TH2 in C. albicans uptake57. to disseminated candidiasis, and this is accompanied
cells secrete interleukin 4 Several membrane-bound receptors contribute to by a TH1 bias55,77. C. albicans induces immunosuppres-
(IL-4), IL-5, IL-6 and IL-10. the phagocytosis of C. albicans, among which dectin 1 sion through TLR2-mediated IL‑10 release, and this
(Ref. 58), the MR59,60 and DC‑SIGN27 have been directly leads to the generation of CD4+CD25+ TReg cells with
Regulatory T-cell
A small population of CD4+
shown to mediate uptake of fungal particles in trans- immunosuppressive potential77,78. Similar data have
T-cells that expresses the fected cell systems, although the ability of the MR to been reported in models of Schistosoma mansoni and
transcription factor forkhead mediate phagocytosis has recently been questioned61. Borrelia burgdorferi infection79,80.
box P3 and that has TLRs do not mediate fungal uptake, but they might be The role of TLR6 and TLR9 in the induction of
suppressor activity towards
involved in directing the subsequent maturation of the cytokines is less prominent. Although TLR2–TLR6
other T-cells either by cell–cell
contact or cytokine release. phagosome62 and the presentation of antigens, although heterodimers are involved in the recognition of
this is still controversial62. zymosan81, cytokine production is only moderately
Zymosan reduced in TLR6–/– macrophages that are stimulated
Particulate preparation of C. albicans killing. Following uptake, killing of with C. albicans, and this does not result in increased
S. cerevisiae cell walls that is C. albicans occurs through both oxidative and non- susceptibility to disseminated candidiasis82. Similar to
rich in β-glucans and mannan
and that can be used to
oxidative mechanisms63. Although the receptors that are TLR2, the absence of TLR9 also results in a slight shift
activate the innate immune involved in triggering these events are largely unknown, of the cytokine production that is induced by C. albicans
system. dectin 1 induces the respiratory burst in response to towards a more anti-inflammatory profile55.

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Of the non-TLR receptors, dectin 1, dectin 2 and surface of a polycarbonate support membrane. Immune
the MR have been implicated in initiating responses to cells such as polymorphonuclear leukocytes (PMNs)
C. albicans. Not much is known about dectin 2, but this can be added to such ex vivo reconstituted human epi-
receptor does preferentially bind C. albicans hyphae thelial (RHE) tissue cultures along with an inoculum of
and induces the production of TNFα and IL-1 receptor fungal cells. The expression of TLRs in RHE models is
antagonist (IL-1Ra)32. Dectin 1 induces the production similar to the TLR expression profile in vivo96. Using such
of numerous cytokines and chemokines in response approaches, PMNs have been shown to be active in the
to fungi, including TNFα, macrophage inflammatory innate immune response at these primary barriers to sys-
protein 2 (MIP2), macrophage inflammatory protein 1α temic invasion97, and differences have been noted between
(MIP1α), granulocyte–macrophage colony-stimulating the local immunity of the oral and vaginal mucosae97,98. For
factor (GM-CSF), granulocyte colony-stimulating fac- example, PMNs enhance an inflammatory TH1 response
tor (G-CSF), IL‑10, IL‑2, IL‑1α, IL‑1β, IL‑23 and IL‑6 in the oral epithelia, which results in the induction of pro-
(Refs 46,50,83–86). Whereas the production of IL‑10, inflammatory cytokines, such as IFNγ and TNFα, effec-
IL‑6 and IL‑2 can be mediated by dectin 1 directly87, the tively mimicking the situation that is observed in vivo97.
induction of pro-inflammatory cytokines and chem- Vaginal epithelial cells express TLR2 and TLR4 in vivo,
okines requires collaborative signalling with TLR2 and the presence of heat-inactivated C. albicans cells and
(Refs 50,83), although evidence suggests that dectin 1 is zymosan induces these cells to secrete inflammatory
sufficient for these responses in certain cell types, such cytokines, chemokines and to produce β-defensin 2 — an
as alveolar macrophages85. By contrast, the interaction of antimicrobial compound that also induces PMN chemo-
dectin 1 with the tetraspanin CD37 seems to inhibit the taxis99. Work to characterize fungal PAMPs and PRRs that
function of dectin 1 and stimulation of IL‑6 (Ref. 88). induce local innate responses is ongoing. For example, it
The intracellular pathways that are activated by dec- has recently been shown that human epithelial TLR4 is
tin 1 for the induction of cytokines involve the recruit- directly involved in the PMN-dependent protection of
ment of Syk46 and downstream signalling via CARD9 oral mucosa96.
(Ref. 89). The dectin 1–Syk–CARD9 pathway induces
DC maturation and directs TH17 responses that are inde- In vivo models in TLR- and LR‑deficient mice
pendent of the interaction with TLRs48, and this effect In vitro stimulation models that use either cell lines or
has been particularly linked with hyphal stimulation of primary cells have suggested specific roles for the various
DCs90. The role of IL‑17 in the host response to dissemi- PRRs in the recognition of C. albicans, as detailed above.
nated candidiasis has been suggested to be protective, by However, whether these pathways are indeed important
inducing neutrophil recruitment91, whereas it exerts a for the antifungal host response in vivo can only be
predominantly inflammatory pathological effect in gas- demonstrated in experimental models of infections in
tric C. albicans infections92. In addition to its mediation genetically modified mice that lack particular receptors,
of the dectin 1–Syk pathway, CARD9 is also a central or in patients with specific immune defects.
adaptor molecule that mediates the pro-inflammatory Since the first observation that TLRs recognize
signals that are induced by other classes of receptors, C. albicans and activate the innate immune response74,
such as the nucleotide-binding oligomerization domain additional studies have confirmed the important role
(NOD)-like receptors93, ITAM-associated receptors and that TLRs have in the recognition of C. albicans and the
possibly the TLRs94. Recently it has also been suggested anti-candidal host response. A global role for TLRs in
that TRIF-dependent pathways modulate the balance defence against disseminated candidiasis was demon-
between deleterious TH17 responses and protective TReg strated by the increased susceptibility of MyD88–/– mice
cells in mice with gastric candidiasis95. to C. albicans infection55,100,101. A subsequent study
The receptors and intracellular pathways that are reported increased susceptibility of TLR2–/– mice to
involved in the recognition of C. albicans by leukocytes disseminated candidiasis, and this effect was attributed
are depicted in FIG. 3. Although all of these pathways have to decreased production of TNF and chemokines102.
Tetraspanin been demonstrated to be involved in the recognition of However, the increased susceptibility of TLR2–/– mice
A family of transmembrane fungal components, it has not been verified that identical to infection with C. albicans was not confirmed in two
proteins that have four mechanisms are responsible for the recognition of intact, other studies, which found reduced mortality and a
transmembrane domains and
two extracellular domains of
live C. albicans cells. For example, zymosan has been used decreased fungal burden in TLR2–/– mice, accompanied
different sizes. The function of extensively for immune stimulation, but the relationship by decreased production of IL‑10 and increased pro-
tetraspanins is unclear, but between the response to zymosan and the response to duction of IL‑12 and IFNγ 55,77. An additional in vitro
they seem to interact with native fungal cells has not yet been clarified. The spe- study also confirmed the increased ability of macro-
many other transmembrane
cificity of the antifungal response is evident in differences phages from TLR2–/– mice to contain C. albicans103.
proteins and form large
multimeric protein networks. between the kinetics of the various immunological effects These latter studies55,77,103 suggest that TLR2, by induc-
and in the magnitude of each of these responses. ing mainly an anti-inflammatory response, inhibits
TH17 response Epithelial and endothelial surfaces also have PRRs the innate immune response to C. albicans infections
The TH17 subpopulation are that recognize surface PAMPs of invading and colonizing in certain circumstances.
T-cells that release mainly microorganisms such as C. albicans. Studies of the role of It has also been suggested that TLR4 is involved in
IL-17, which has both
neutrophil chemotactic activity
epithelial cell immunity have been significantly enhanced the recognition of C. albicans24,25,71, and TLR4-defective
and the potential to promote by the availability of model epithelial and endothelial tis- C3H/HeJ mice have been reported to be more suscepti-
autoimmunity. sues in which a cell line of keratinocytes is grown on the ble to disseminated candidiasis71. However, other studies

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Monocytes/macrophages Dendritic cells


Mannan β-Glucans β-Glucans Mannan Mannan Mannan β-Glucans Mannan Mannan
(O-linked) PLM? β-Mannosides (N-linked) Chitin (N-linked) (O-linked) PLM? β-Glucans (N-linked) (N-linked)

TLR4 TLR6 TLR2 + Galectin 3 Dectin 1 MR Chitin R? Dectin 2 FcγR TLR4 TLR2 Dectin 1 MR DC-
SIGN

Mal Mal Syk TRIF Mal Mal Syk


MyD88

MyD88

MyD88
+

MyD88
TRAM
+

IκB ERK IκB CARD9 IκB p38/JNK ERK CARD9


NF-κB NF-κB NF-κB

NF-κB
NF-κB NF-κB NF-AT IRF3 NF-κB c-Fos

IL-8 TGFβ TNF IL-10 TNF IL-4 TNFα Type I TNF IL-12 TGFβ IL-23 TNF IL-10
TNF IL-10 IL-2 IL-1β IFN IFNγ IL-10 IL-6 IL-1β

TReg TH2 TH1 TH1 TReg TH17

Figure 3 | Recognition of Candida albicans at the membrane level. Recognition of C. albicans at the level of the cell
membrane is mediated by TLRs and LRs. TLR4 induces mainly pro-inflammatory signals in monocytic cell types
(monocytes, macrophages and DCs) through the MyD88–Mal-mediated NF‑κB and MAPK pathways, Nature Reviews | Microbiology
while stimulating TH1
responses through IRF3-dependent mechanisms mainly in plasmacytoid DCs. TLR2 stimulates the production of moderate
amounts of pro-inflammatory cytokines and strong IL‑10 and TGFβ responses. On the one hand, this leads to the induction
of a tolerant phenotype in DCs, through an ERK/MAPK-dependent mechanism121. On the other hand, in monocytes and
macrophages it induces TGFb and IL-10, and subsequent proliferation of TReg cells and immunosuppression78. The pro-
inflammatory effects of TLR2 can be amplified by dectin 1 and galectin 3 — the latter especially in macrophages. In
addition to amplifying the effects of TLR2, the non-classical lectin-like receptor dectin 1 induces IL‑2, IL‑10 and TH17
responses through a Syk–CARD9 cascade, independently of its interaction with TLR2. The classical lectin-like receptor, the
MR, induces pro-inflammatory effects in monocytes and macrophages, whereas chitin-dependent stimulation of these
cells induces mainly TH2 responses122, although this effect has yet to be demonstrated for C. albicans, and the identity of
the chitin receptor is unknown. Other less well characterized pathways include stimulation of TNF and IL‑1Ra by dectin 2,
while engagement of DC-SIGN in DCs induces production of the immunosuppressive cytokine IL‑10 . CARD9, caspase
recruitment domain protein 9; DC, dendritic cell; ERK, extracellular signal related kinase; FcγR, Fcγ receptor; IL,
interleukin; IL‑1Ra, interleukin‑1 receptor antagonist; IFNγ, interferon-γ; IRF3, interferon regulatory factor 3; JNK, Jun
N‑terminal kinase; LR, lectin receptor; MAPK, mitogen-activated protein kinase; MR, mannose receptor; MyD88, myeloid
differentiation primary response gene 88; NF‑κB, nuclear factor κB; PLM, phospholipomannan; Syk, spleen tyrosine kinase;
TGFβ, transforming growth factor-β; TH, T helper; TLR, Toll-like receptor; TNF, tumour necrosis factor; TReg, regulatory T-cell.

have observed variable results, with TLR4–/– mice being a deleterious outcome of infection, probably owing to
more susceptible in models of intragastric infection or compensatory pathways that are mediated either by
intravenous re-infection 55, not different from wild- other TLRs or by LR‑dependent mechanisms.
type animals in models of intravenous infection with The study of the role of LRs in the in vivo response
C. albicans yeast104, or even surviving longer in a model to C. albicans infections is still in its infancy. Recently, it
of intravenous infection with C. albicans hyphae55. The has been shown that dectin 1–/– mice are more susceptible
differences between the experimental models and/or to C. albicans infection, and infection results in increased
the C. albicans strains used are thought to be responsi- fungal growth in the organs and accelerated death105.
ble for these differences. This is consistent with the increased susceptibility of
Although no increased susceptibility of TLR9–/– mice CARD9–/– mice to disseminated candidiasis89. Notably,
to disseminated candidiasis has been observed, the all of the major components of antifungal defence
fungal burden in the organs of TLR9-deficient animals — cytokine release, phagocyte recruitment, phagocytosis
tends to be lower than in control mice55. Although and microbial killing — are impaired in the knockout
pro-inflammatory cytokine production was slightly mice, which suggests an important role for dectin-1-
lower in TLR9–/– macrophages that had been stimulated mediated recognition of β-glucans for anti-candidal
with C. albicans, this mild difference did not result in defence in vivo105. However, these results are at odds with

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REVIEWS

a b c
C. albicans C. albicans C. albicans
yeast hyphae yeast

TLR2 TLR4 Dectin 1 CR3 FcγR TLR4

Mal Mal Mal

MyD88

MyD88

MyD88
Syk

β-Glucans shielded from


dectin 1 recognition
ERK NF-κB NF-κB

c-Fos

Anti-inflammatory Pro-inflammatory Pro-inflammatory


signals signals signals

Figure 4 | Candida albicans mechanisms to escape the innate response using pattern-recognition receptors.
Nature Reviews
| Microbiology
a | The balance between the signals that are induced by Toll-like receptor 2 (TLR2) and TLR4 seems to have a crucial role in
the regulation of the immune response. TLR2-mediated signals are mainly anti-inflammatory and can be deleterious when
activated prematurely or excessively. TLR4-mediated signals are mainly pro-inflammatory. b | Shielding of the β‑glucans by
mannans in hyphae prevents activation of the dectin 1 signalling pathways. c | Inhibitory signals from complement
receptor 3 (CR3) and Fcγ receptor II (FcγRII)/FcγRIII have inhibitory effects on the activation of the immune system via
TLR4. ERK, extracellular signal related kinase; MyD88, myeloid differentiation primary response gene 88; NF‑κB, nuclear
factor κB; Syk, spleen tyrosine kinase, TLR, Toll-like receptor.

another study in dectin 1–/– mice, which failed to discern still be done to fully understand the role of PRRs in the
increased susceptibility to candidiasis106. Differences in various types of C. albicans infections in vivo. Studies of
the experimental host or between different C. albicans disseminated candidiasis are more advanced, but little
strains might account for these discrepancies, and addi- work has been done in other forms of C. albicans infec-
tional studies are needed to pinpoint the origin of these tion, such as oropharyngeal or vaginal candidiasis, and
differences. little attention has been paid to the mechanisms that lead
Studies on the in vivo roles of other LRs in the to colonization rather than infection.
response to C. albicans defences are also scarce. In one
study, intraperitoneal administration of C. albicans Escape mechanisms based on PRRs
resulted in an increased fungal burden in the organs As evidence supporting the important role of PRRs
of MR–/– mice, compared to controls, although survival in the recognition of C. albicans for the induction
was not affected107. However, intraperitoneal injection of an immune response accumulates, data have also
of C. albicans is not an established experimental model emerged to show that certain fungal pathogens have
of invasive candidiasis, as it mainly induces peritonitis evolved strategies to curtail recognition by these recep-
rather than a disseminated infection108. A recent study tors, or to use it to their own advantage to evade the
has demonstrated an important role for galectin 3 in host response. For example, it was recently shown in
the recognition of C. albicans in the gut29, with β-(1,2)- Drosophila melanogaster that Gram-negative bind-
mannosides able to block colonization and invasion of ing protein 3 (GNBP3) acts as a dectin-1-like lectin-
C. albicans in the intestines9. Finally, like mice defective recognition mechanism for fungal glucans, including
in TLR2, mice defective in CR3 or FcγR are more resistant that of C. albicans109. Certain fungal insect pathogens
to disseminated candidiasis, and display more vigorous secrete proteases that degrade GNBP3 to avoid detec-
cytokine release and antifungal killing, in line with the tion, but the D. melanogaster persophone protease is
immunosuppressive effect of these receptor pathways on activated by the fungal protease virulence factor, which
the inflammatory response60. results in the bypass of GNBP3 and the activation of the
It has become clear that PRRs are important for the Toll pathway downstream of the PRR. It remains to be
host response to C. albicans, with various TLRs and LRs seen whether there is a resonance of this co-evolution
having distinctive roles in innate immunity: some recep- between recognition mechanisms and virulence factors
tors have a more pro-inflammatory role (for example, in human–fungal interactions.
TLR4, dectin 1 and the MR), whereas others exert immu- In the mammalian host, inappropriate or premature
nosuppressive effects (for example, TLR2, CR3 and FcγR) activation of immunomodulatory receptors can be used
(FIGS 3,4). In addition, the choice of experimental model as an escape route by C. albicans, as detailed above for
and the C. albicans strain used might have an important the outcome of systemic candidiasis in TLR2–/– mice
impact on the outcome of infection. More work must (FIG. 4a). Modulation of DC function can also be a target

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for escape mechanisms, with the MR and DC‑SIGN pos- the β-glucan–dectin 1 interactions? The answer to this
sibly inducing anti-inflammatory cytokine production paradox might reside in the complex interaction between
and immunosuppression110,111. Thus it seems that the TLRs and LRs. Although both mannans and glucans can
MR might have a dual role, either pro-inflammatory (in induce pro-inflammatory signals, concomitant stimula-
monocytes and macrophages) or immunosuppressive tion of the mannan–TLR and β-glucan–dectin 1 path-
(in DCs), although this assumption is still controver- ways has a synergistic effect on the amplification of the
sial. In addition, the recognition of β‑glucans by CR3 immune response. Amplification mechanisms between
does not trigger protective host responses, such as the dectin 1 and TLR2 (Refs 49,50), and between dectin 1
respiratory burst42, and can repress pro-inflammatory and TLR4 (K. M. Dennehy and colleagues, unpublished
signals44 (FIG. 4). In line with these immunosuppressive observations) have recently been documented. If the
effects in vitro, CR3-knockout mice are more resistant interaction of β-glucans with dectin 1 is shielded by
to disseminated candidiasis60, and are also more resist- mannans, this synergism may be lost, with inefficient
ant to Blastomyces dermatitidis infection43. Some studies activation of host defence, despite recognition of man-
have suggested that the differential activation of stimula- nans. It is therefore mainly the loss of synergistic effects,
tory and inhibitory receptors is caused by the dimorphic rather than the inhibition of an individual recognition
nature of C. albicans yeast and hyphae, which induce dif- pathway, that may lead to defective cytokine release.
ferent DC activation profiles. The recognition of yeasts
by DCs is mediated mainly by the MR, dectin 1 (Ref. 60) An integrated model of innate recognition
and DC‑SIGN27,112, and hyphae are recognized mainly Progress in our understanding of the mechanisms respon-
through dectin 2, CR3, FcγRII and FcγRIII60. sible for C. albicans recognition, as described above,
In addition to inducing direct anti-inflamma- allows us to suggest an integrated view of how C. albicans
tory effects, C. albicans has developed strategies to is recognized and of how the host innate immune
either block or avoid recognition by stimulatory PRRs response is activated during candidiasis. Although we
(FIG. 4b) . The morphogenetic change of C. albicans are still in the early stages of understanding the com-
from a yeast to a hyphal form, after adhesion of yeast plexity of fungal recognition, and there is still much
cells to the intravascular endothelium and invasion of to learn, there are several principles that characterize
the surrounding tissue, might represent a mechanism recognition of C. albicans.
whereby the pathogen can avoid recognition of β‑glucans • First, recognition depends on ‘tasting’ several
by the immune system113. Hyphal filaments lack surface- PAMPs in the fungal cell wall. Specific receptor sys-
exposed β‑glucan and have a range of other cell-surface tems have evolved for the recognition of the major
modifications that could influence immune detection11. polysaccharide cell wall components, such as the
In addition, the induction of the pro-inflammatory MR and DC‑SIGN for the recognition of branched
response through dectin 1 can be prevented, because the N‑linked mannan, TLR4 for linear O‑linked mannan,
mannoprotein layer covers the β‑glucan layer of the cell galectin 3 for β-mannosides, dectin 1 and TLR2 for
wall, thus blocking the β‑glucan–dectin 1 interaction11. β-glucan and PLM, and CR3 for β-(1,6)-glucan.
Accordingly, exposure of β‑glucan through treatment • Second, despite overlapping and sometimes redun-
with the antifungal caspofungin, by heat treatment of dant functions, each ligand–receptor system activates
cells or by mutation of glycosylation genes that result specific intracellular signalling pathways, and this has
in depletion of surface mannan all led to an enhanced distinct consequences for the activation of the various
dectin-1-mediated pro-inflammatory cytokine signal. components of the host immune response.
This suggests that the mannoprotein layer is a mask that • Third, differential expression of the various PRRs is
downregulates the pro-inflammatory dectin-1-mediated an important mechanism for the cell-type-specific
response114. As the exposed β‑glucan of intact C. albicans response to fungal pathogens (FIG. 2).
yeast cells is thought to be presented mainly at the sur- • Fourth, the fully integrated response to a specific
face of the wall as bud scars, the attenuation of dectin‑1- pathogen depends on the mosaic of PRRs and recep-
mediated recognition during germ tube evagination tor complexes that are engaged. Co-stimulation via
could be due to the expansion of the hyphal cell surface multiple PAMP–PRR interactions can increase both
that lacks any bud scars. In line with this, Rappleye et al. the sensitivity and the specificity of the immune rec-
showed that the β‑glucan–dectin 1 interaction between ognition process.
Histoplasma capsulatum and macrophages can be These four principles can be extrapolated to immune
shielded by a different cell wall component — α-(1,3)- recognition of any microorganism. For example, the
glucan115. The presence of α-(1,3)-glucan in yeast cells final response upon stimulation of monocytes by
prevents recognition through dectin 1, and mutation of C. albicans depends on integrating the signals that are
AGS1 (which encodes α-(1,3)-glucan synthase) results received from TLR2, dectin 1, TLR4 and the MR. This
in greatly enhanced TNFα production115. Therefore, response differs from the stimulation that is induced
both mannans and α‑glycans can act as shields that mask by the Gram­-negative bacterium Escherichia coli, which
dectin-1-mediated immune responses. can be ascribed to recognition of lipopolysaccharide by
It is important to note that mannans are in them- TLR4, membrane lipopeptides by TLR2 and flagellin by
selves immunostimulatory 15,24,116–118. How can the TLR5. By contrast, Gram-positive bacteria are mainly rec-
immuno­stimulatory properties of mannans be recon- ognized by TLR2 (which recognizes lipoteichoic acid) and
ciled with the apparent inhibitory effect of masking NOD2 (which recognizes peptidoglycans). The different

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REVIEWS

intracellular events that are stimulated by these pathways in which human primary cells are exposed to C. albicans.
are ultimately responsible for the tailored innate response However, valuable information can also be gathered from
to infection by different classes of microorganisms. immunogenetic studies. For example, TLR4 polymor-
From this perspective, although described here for phisms have recently been identified as a susceptibility
C. albicans, these general principles can be considered trait for systemic candidiasis, which supports a role for
as a blueprint for pattern recognition of all pathogenic this receptor in the host immune response to C. albicans119.
microorganisms by the innate immune response. Studies of additional functional polymorphisms in larger
cohorts of patients are needed to confirm the role of the
Future directions various pathways in the pathogenesis of candidiasis
In parallel with the general renaissance in the field of The ultimate ambition of research into the biology of
innate immunity, our understanding of C. albicans rec- host–fungus interactions is to be able to translate findings at
ognition by the innate immune system has improved the molecular level of the interactions into new treatments
considerably over the past decade. Despite this progress, for patients who are vulnerable to lethal fungal infections.
more challenges lie ahead. Although we have gained a Although direct blockade or stimulation of PRRs might
much better understanding of the basal (polysaccharide) be problematic as a viable therapeutic approach, vaccine
structures that are recognized by the PRRs, more work biology has become a domain in which our newly acquired
must be done to understand the details of the interac- knowledge of PRRs is already demonstrating potential120.
tions of these PAMPs with their receptors on the host The understanding of the modulation of DC function by
cell membrane. Recent evidence, for example, demon- PRRs has led to the design of novel and specific vaccine
strates that dectin 1 associates with tetraspanins, which adjuvants, and this work is likely to have a major impact on
suggests that dectin 1 forms receptor complexes on the the development of future antifungal vaccines.
cell surface that might be involved in regulating immune In conclusion, in this Review we have presented
responses88. In addition, little is known about the recog- an integrated model of innate pattern recognition
nition of the other components of the C. albicans cell of an important human pathogen, the fungus C. albicans,
wall, such as the proteins themselves, or the possible including a description of the PAMPs of C. albicans that
sensing of products that are secreted by C. albicans. are recognized by the host, to the receptor pathways
The differential recognition of the yeast and hyphal that are stimulated by specific fungal molecular struc-
forms also promises to be a fertile area for future tures. We have also discussed and speculated on how these
research. signals are integrated to bring about efficient activation
An additional challenge is to provide evidence that the of the host innate response. This model, which focuses
pattern-recognition pathways identified in experimental on the interaction of C. albicans with host defences,
models are also applicable during C. albicans infections in could be conceptually pertinent to the modelling
humans. This challenge can be partly met by experiments of various other host–pathogen interactions.

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Kiss and spit: the dual roles of


Toxoplasma rhoptries
John C. Boothroyd* and Jean-Francois Dubremetz‡
Abstract | Toxoplasma gondii is a single-celled, eukaryotic parasite that can only
reproduce inside a host cell. Upon entry, this Apicomplexan parasite co-opts host
functions for its own purposes. An unusual set of apical organelles, named rhoptries,
contain some of the machinery that is used by T. gondii both for invasion and to
commandeer host functions. Of particular interest are a group of injected protein
kinases that are among the most variable of all the T. gondii proteins. At least one of
these kinases has a major effect on host-gene expression, including the modulation
of key regulators of the immune response. Here, we discuss these recent findings and
use them to propose a model in which an expansion of host range is a major force that
drives rhoptry-protein evolution.

Apicomplexa The phylum Apicomplexa includes a large number of Rhoptry ultrastructure and content
A phylum of unicellular obligate intracellular parasites. Among these are some The size, electron density and number of rhoptries var-
eukaryotes that are obligate notorious human and animal pathogens from genera ies among Apicomplexan species and between the dif-
parasites and defined by a such as Plasmodium (the causative agent of malaria), ferent developmental stages of a single species. In this
collection of apical organelles
that are involved in invasion of
Toxoplasma (an important cause of congenital disease Review, we will mainly focus on the rapidly dividing,
a host cell. and infection in immuno-compromised patients1; see asexual form of T. gondii that is known as the tachyzoite3.
BOX 1 for a brief description of the Toxoplasma life cycle Tachyzoites predominate during the acute stages of
Microneme and the clinical spectrum of human toxoplasmosis), infection in an intermediate host, which can be virtually
A small, cylindrical organelle
Cryptosporidium (a cause of serious gastrointestinal any warm-blooded animal (BOX 1). This breadth of host
that is found at the periphery
of the anterior end of
disease) and Eimeria (a major problem in the poultry range also extends to the cellular level, as tachyzoites
Apicomplexan parasites that industry and cause of chicken coccidiosis). The phy- can invade and replicate in almost any host cell that they
secretes its contents onto the lum is defined by the presence of an apical complex encounter, at least in vitro. In vivo, the cellular tropism
surface of a gliding or invading that comprises a microtubule anchoring ring through of tachyzoites has not been well characterized as, until
parasite.
which dedicated secretory organelles release their con- recently4,5, methods to survey parasite growth in the
tents2. There are two types of apical secretory organelle entire animal have been lacking. Most in vitro studies use
— the small, rod-shaped micronemes and the much fibroblasts as the host cell because of their availability,
larger, bulb-shaped rhoptries (Greek for club). FIG. 1a the fact that they are a primary cell line (making studies
provides an electron micrograph of one of the asexual on host-gene expression more relevant than in a trans-
*Department of Microbiology
and Immunology, Stanford
forms of Toxoplasma gondii, which clearly shows an formed cell line) and the ease with which they can be
University School of Medicine, apical cytoskeleton and a substantial complement of examined by light microscopy (as they have full contact
Stanford, California micronemes and rhoptries. inhibition and grow as a uniform, flat monolayer).
94305‑5124, USA. Given their conservation across much of the phy- Tachyzoites typically have ~12 rhoptries, each of

UMR 5539 CNRS, Université
lum, it has long been suspected that rhoptries have a which is ~2 to 3 micrometres in length6 (FIG. 1). Using
de Montpellier 2, CP 107,
Place Eugène Bataillon, key role in the intracellular lifestyle of these pathogens. light microscopy, the rhoptries can be visualized as an
Montpellier 34090, France. Recently, exciting results have shed light on at least two elongated cluster that is present at the anterior end of
Correspondence to J.C.B. functions of the rhoptries. In this Review, we will discuss the parasite and is frequently located on one side. They
e‑mail: john.boothroyd@ the ultrastructure and content of rhoptries, their role in are visible using differential interference contrast micro-
stanford.edu
doi:10.1038/nrmicro1800
invasion and function in subverting host-cell processes, scopy, but are more easily observed by immunofluores-
Published online as well as the evolutionary pressures that might underlie cence using a range of antibodies that are specific for their
3 December 2007 the extreme variability of rhoptry proteins. protein contents. Interestingly, most of these antibodies

nature reviews | microbiology volume 6 | january 2008 | 79


© 2008 Nature Publishing Group
REVIEWS

Box 1 | Toxoplasma gondii biology pathway that involves rough endoplasmic reticulum and
the Golgi apparatus, but the process of rhoptry forma-
Asexual Sexual tion and the signals that ultimately target proteins to this
Warm-blooded intermediate Feline organelle have yet to be precisely identified10–14. There is
some evidence that rhoptries are related to exosomes15,
which are membrane-limited bodies that are extruded
by some cells. This has interesting implications for the
final topology of a rhoptry protein during release, as
exosome formation might involve an invagination of
the rhoptry membrane and, therefore, place an integral
membrane protein in an inverted orientation relative to
their position by alternative models16.
There are 29 proven rhoptry proteins, of which 24 are
present in the rhoptry bulb (most of these are therefore
4 termed ROP proteins) and 5 are present in the rhoptry
Bradyzoite Gamete
neck (termed RON proteins) (TABLE 1). Many of these
3 1 2 5 6
were first discovered by proteomic analyses of purified
Tachyzoite Oocyst rhoptries9, which identified an additional 28 proteins
7 that have yet to be verified as being truly rhoptry in
T. gondii is an extremely common protozoan parasite of warm-blooded animals that has origin. Given the high percentage of proteins that were
a host range that extends from birds to humans. Its life cycle comprises two, potentially verified as being rhoptry proteins from the first group
independent cycles, one asexual and one sexual, although Nature Reviewsbetween
movement | Microbiology
the two that was analysed, most of these 28 proteins are prob-
is almost certainly key to efficient transmission (see the figure). ably also from this compartment. Further evidence of a
The asexual cycle (see the figure, left-hand panel) can occur in virtually any warm- rhoptry origin comes from the fact that many of these 28
blooded animal and ~100 mammalian and avian species have been documented as
proteins are paralogues of known rhoptry proteins, as are
being infected with T. gondii in nature. In these ‘intermediate’ hosts, the parasite
population initially expands by rapid proliferation of the tachyzoite form (‘tachy’ means
the predicted products of several additional genes that
fast in Greek and in this context refers to speedy replication). Once an immune response have yet to be analysed in detail (TABLE 2).
is elicited, the parasite differentiates to the more slowly growing bradyzoite form The largest rhoptry gene family shows clear homol-
(‘brady’ means slow in Greek) (arrow 1). The bradyzoite form can persist for the life of the ogy to protein kinases17. The canonical member of this
host in cyst-like structures that are present deep in the brain and other tissues. During family is ROP2 and, in at least one instance, a member
immunosuppression (for example, in patients with AIDS) the parasite can resume rapid of this family (ROP18) has been confirmed to have
replication in the form of tachyzoites (arrow 2). Transmission between intermediate kinase activity17,18, although the vast majority seem to
hosts is by the ingestion of raw or under-cooked meat and other organs that contain the have lost the key catalytic residues and, hence, the ability
infectious, encysted bradyzoites (for example, a mouse eaten by a hawk or an under- to phosphorylate proteins19 (TABLE 1). Apart from their
cooked lamb chop eaten by a human) (arrow 3). The sexual cycle (see the figure, right-
kinase domains, these proteins seem to be unique to the
hand panel), which begins when bradyzoite-bearing tissue from an intermediate host is
ingested by a feline (arrow 4), involves gametogenesis and fertilization (arrow 5) in the
Toxoplasma genus and its close relatives (for example,
gut epithelium of felines. The cat family is the only group of animals that is known to species of Neospora); no bona fide homologues have been
serve as a definitive host for this parasite; that is, it is the only host in which sexual described in the distantly related Plasmodium spp. This
reproduction occurs, although felines can also support asexual reproduction. The could reflect an incomplete knowledge of the rhoptry
sexual cycle culminates in the shedding of up to 100,000,000 highly stable oocysts in the proteome in species of Plasmodium and/or a difference
faeces of an infected cat (these oocysts are initially shed in an immature state and in the intracellular niches that they occupy. Other ROPs
require approximately 2 days to mature in the environment and gain full infectivity). are homologous to phosphatases20 and proteases21,22, but
Mature oocysts are highly infectious and can either infect another cat (arrow 6) or, more several ROPs, including ROP1, ROP6 and ROP9, seem to
probably, a grazing intermediate host (arrow 7) that is foraging in a farm. be unique to the Toxoplasma genus and are of unknown
T. gondii causes serious disease in humans, but this disease is primarily confined to
function23,24.
the developing foetus of a woman who acquires her first infection during pregnancy
and individuals who are immunocompromised as a result of HIV-1 infection,
In contrast to the extensive ROP2 family, each of the
lymphoma or other immune-suppressive syndromes. Disease pathologies range from RON proteins is encoded either by a unique gene that has
asymptomatic to severe and, sometimes, fatal55. Occasionally, otherwise healthy no similarity to any other gene in the T. gondii genome
adults can also experience acute symptoms, especially in the eye54. These different (for example, RON1 and RON5) or by a gene that has
disease outcomes may be related to which of the three most common strains of one or two paralogues elsewhere in the T. gondii genome
T. gondii (Type I, II or III) is responsible for the infection46,47,55. (for example, RON2, RON3 and RON4) (TABLE 2). The
initial discovery of the sequence of the RON proteins
gave no clue as to their biological function9. Several
stain either the bulbous base7,8 or the tapering neck of the T. gondii RONs have clear orthologues in related genera,
Rhoptry rhoptry9, but not both. It seems, therefore, that the rhop- including Plasmodium, which suggested their involve-
A club-shaped secretory try contents are not a random mixture but are sorted ment in processes that are common to the Apicomplexa
organelle that is found at the into discrete subcompartments. No internal membranes phylum. Although their exact function has not been deter-
anterior end of Apicomplexan
seem to separate these domains and the mechanism by mined, the unusual secretion of RON4 and its subsequent
parasites that releases its
contents during invasion; which proteins are sorted to a particular region is not yet migration down the length of the parasite during invasion
subdivided into a bulbous known. After translation, at least initially, rhoptry pro- has led to clear models about the overall role of RON4 and
base and tapering-neck. teins move through a conventional eukaryotic secretory other RONs, as discussed below.

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Finally, in addition to their protein complement, a


rhoptries also contain lipids25 and this lipid comple-
ment has a high ratio of cholesterol to phospholipids. AC
Interestingly, these lipids are sometimes present as RON
membrane whorls inside the rhoptry organelle, which
can be visualized using electron microscopy. This might
explain the unusual topology of ROP proteins that are PVM
M
released into the host cell during invasion; as discussed
below, some ROPs seem to enter the host-cell cytosol in ROP
A
a freely soluble form whereas other ROPs are associated
with unusual vesicle-like bodies that seem to fuse with G
the nascent parasitophorous vacuole.

Role of rhoptries in cell invasion N


The only circumstance in which rhoptries are known to
secrete their contents is during the process of invasion
into a host cell (FIGS 2,3; see Supplementary information
S1 (movie)). The trigger for release is unknown, but it
evidently depends on a direct recognition between the
apical surface of the parasite and the receptor molecule
(or molecules) on the host cell. The identities of the mol-
ecules on either side of this interaction are also unknown
and no stimulus has yet been identified that will induce
rhoptry secretion in the absence of host-cell contact. The
mechanics of the fusion event that allows rhoptry pro-
tein release are a mystery. It could be as simple as fusion b
of the rhoptry to the parasite’s plasma membrane or,
perhaps, an intermediate, anterior compartment. Whatever
the process, a distinct opening at the anterior-most tip of PV
the tachyzoite is clearly observed during host-cell inva-
RON
sion26 and this is presumed to be the opening through HC
which the rhoptry contents ultimately flow. HPM
Once released, rhoptry proteins have various destina-
tions (FIG. 3). The RON proteins, RON2, 4 and 5, form PVM ROP
a complex with the micronemal protein AMA1 and
this multimeric complex colocalizes with the so-called
moving junction (MJ)27,28. The MJ is a ring-like structure
that represents the circular point of contact between the
parasite surface and host plasma membrane29. During
invasion, the MJ migrates down the length of the parasite Mito
Paralogue
(FIGS 2,3; see Supplementary information S1 (movie)).
A gene that shares a common Figure 1 | Toxoplasma gondii Nature
ultrastructure. a | An
Reviews | Microbiology
evolutionary origin and has AMA1 seems to be necessary for the MJ to form, because intracellular T. gondii tachyzoite inside a parasitophorous
evolved in parallel with another parasites in which AMA1 expression has been reduced vacuole membrane (PVM), showing the apical cytoskeleton
gene that is located in the to ~1% of the wild type (using parasites that harbour a (AC) and neighbouring micronemes (M), rhoptry bulbs
same genome or organism,
tetracycline-regulated copy of the AMA1 gene) release (ROP) and rhoptry necks (RON). Other components of the
typically to serve different but
related functions. the RONs, but the MJ fails to form and the parasites do parasite, such as the nucleus (N), the Golgi apparatus (G)
not invade host cells28. and the plastid that is specific to the Apicomplexa phylum,
Orthologue The MJ might represent the mechanism by which the ‘Apicoplast’ (A), are shown. The scale bar represents
A gene in one species that the parasite makes contact with the host cytoskeleton. 0.5 µm. b | A lower magnification of a rosette of T. gondii in
shares a common evolutionary the parasitophorous vacuole (PV) inside an infected cell
origin with a related gene in a
The most widely accepted model is one in which the
(HC). The host plasma membrane (HPM), as well as host
different species and that parasite anchors itself on integral proteins of the host
mitochondria (Mito) in close apposition to the PVM, can
serves essentially the same plasma membrane and drags them backward (rela- also be seen. The scale bar represents 1 µm. Image in part a
function. tive to the parasite surface), effectively converting the reproduced, with permission, from REF. 56  Elsevier
host plasma membrane into parasitophorous vacuole Science. Image in part b reproduced, with permission, from
Parasitophorous vacuole
The vacuole that harbours the membrane that surrounds the intracellular portion of REF. 57  Elsevier Science.
parasite. the parasite (FIG. 2). This wrapping of the parasite in
parasitophorous vacuole membrane is simultaneous
Moving junction with a clear forward motion of the parasite into the grip on something that is connected to fixed anchors
(MJ). A migrating ring of
contact between a host-cell
host cell, relative to the host-cell’s normal perim- within the host cell; that is, the host plasma membrane
plasma membrane and the eter (Supplementary information S1 (movie)), which integral proteins must be connected to the host-cell
surface of an invading parasite. strongly argues that the parasite must have a firm cytoskeleton. Finally, the model predicts that the

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Table 1 | Known rhoptry proteins


Protein Gene identification Final Predicted Biological Comments Plasmodium Refs||
name number*; GenBank destination‡ coding function falciparum§
accession number function
ROP1 583.m00003; M71274 PV Unknown Unknown Knock-out in a type I strain is still No 23
virulent
ROP2A¶ 33.m01398; Z36906 PVM Protein kinase Mitochondria More adjacent genes probably exist No 37
recruitment but are missing from ToxoDB4.2
ROP2B¶ 63.m00146 PVM Protein kinase Unknown Should be a tandem, identical copy of No 37,58
ROP2A but appears as a fragment in
the ME49 sequence in ToxoDB4.2
ROP4¶ 83.m02145; Z71787 or PVM Non-catalytic Unknown Phosphorylated; mis-annotated in No 19,37
AY662677 kinase ToxoDB4.2 as one gene (fusion with
ROP7)
ROP5¶ 551.m00238; EF466101 PVM Non-catalytic Unknown Approximately 3 or 4 more tandem No 9,35,59
or DQ116423 kinase copies exist that are not present in
the ME49 sequence in ToxoDB4.2
ROP7¶ 83.m02145; AM056071 PVM Non-catalytic Unknown Mis-annotated in ToxoDB4.2 as one No 36
kinase gene (fusion with ROP4)
ROP8¶ 33.m00005; AF011377 PVM Non-catalytic Unknown None No 58
kinase
ROP9 49.m00048; AJ401616 Unknown Unknown Unknown Distinct from another protein No 24
named ROP9 (Ref. 60), for which the
sequence and gene are unidentified
ROP10 583.m05686; DQ124368 Unknown Unknown Unknown None No 9
ROP11¶ 42.m03584; AAZ29607 Unknown Protein kinase Unknown None No 9
or DQ077905
ROP12 20.m08222; DQ096559 Unknown Unknown Unknown None No 9
ROP13 583.m09115; DQ096560 Unknown Unknown Unknown Mis-annotated in ToxoDB4.2; No 9
encoded on the opposite strand
ROP14 583.m00692; DQ096565 Unknown Transmembrane Unknown None Yes 9
ROP15 27.m00091; DQ096561 Unknown Unknown Unknown None No 9
ROP16¶ 55.m08219; DQ116422 Host nucleus Protein kinase Host subversion Extremely different in T. gondii type I, No 9
and virulence II and III strains
ROP17¶ 55.m08191; AM075203 Unknown Protein kinase Unknown None No 17
ROP18¶ 20.m03896; AM075204 PVM Protein kinase Virulence Extremely different in T. gondii type I, No 17
or EF092842 II and III strains
TgSUB2 583.m00011; AF420596 Unknown Serine protease ROP protein None Yes 22
processing
Toxopain1 50.m00008; AY071839 Unknown Cathepsin B ROP protein None No 21
protease processing
TgPP2C-hn 74.m00766 + 74m.00767; Host nucleus Protein Unknown Injected into host cell where it ends No 20
EF450457 phosphatase up in the nucleus; knock-out has a
2C slight growth defect in vitro
TgNHE2 129.m00252; AY735393 Unknown Na+ and H+ Ionic Knock-out in type I strain is still Yes 61
exchanger homeostasis virulent
Toxofilin 33.m02185; AJ132777 Unknown Actin-binding Unknown Possible interference with host actin No 62
BRP1 583.m09133 Unknown Unknown Bradyzoite- and Knock-out in type II strain is still No 63
merozoite- virulent
specific
TgRAB11 80.m00009 Unknown Small GTPase Protein None Yes 9
trafficking
RON1 583.m11443 + 583. Unknown Possibly GPI- Unknown None Yes 9
m00597; DQ096562 anchored
RON2 145.m00331; DQ096563 MJ Transmembrane Invasion Covalently attached to RON4 Yes 9
RON3 42.m00026; DQ096564 Unknown Transmembrane Unknown None Yes 9
RON4 44.m06355; DQ096566 MJ Unknown Invasion Covalently attached to RON2 Yes 9
RON5# 583.m09191 + 583. Possibly MJ Unknown Invasion Cleaved into three major fragments Yes None
m09192 + 583.m00636
*The ToxoDB4.2 (Toxoplasma gondii Genome resource; see Further information64) gene identifier is provided for the T. gondii ME49 strain, along with the GenBank
entry for (typically, but not always) the T. gondii RH strain, if such an entry exists. The GenBank entry should be a verified coding sequence and, therefore, encode the
definitive amino-acid sequence of the primary translation product. The gene identifiers in ToxoDB4.2 are tentative predictions that in most cases have yet to be
experimentally confirmed. Consequently, although they are approximately correct, the transcription start sites, splice junctions and predicted protein sequences
might be incorrect. This is why some genes have multiple identifiers — for example, the large RON1 gene is currently incorrectly annotated as two different genes
(indicated by the + symbol, which separates the gene identifiers listed). ‡ Final location of the protein after invasion. § Significant orthologue, with homology that
extends beyond the presence of a conserved enzymatic domain (for example, more than just a kinase or protease active site is conserved throughout evolution),
exists in P. falciparum. ||The reference that reports the definitive sequence and/or identification of this protein as a rhoptry protein. ¶Homologue of ROP2. No gene has
yet been linked to the ROP2 family member that has been dubbed ROP3. ROP3 could simply be a post-translational modification of one of the known ROP2 family
members or encoded by one of the ROP2-like genes for which the protein product has yet to be detected. #P. Bradley and M. Lebrun, unpublished observations.
GPI, glycophosphatidylinositol; MJ, moving junction; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane.

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Table 2 | Suspected rhoptry proteins


Protein Gene identification Final Predicted coding Comments Plasmodium Refs||
name number*; GenBank destination‡ function falciparum§
accession number
ROP6 55.m00092; AY792971 Possibly HPM Possible protease and None No 665
GPI-anchored
ROP2L3¶ 55.m08224 Unknown Protein kinase Mass spectrometry data for expression**; No 9,17
detected in rhoptry proteome‡‡
ROP2L4¶ 57.m01774 Unknown Protein kinase None No 17
ROP2L5¶ 49.m03275 Unknown Protein kinase None No 17
ROP2L6 ¶
80.m02343 Unknown Non-catalytic kinase Mass spectrometry data for expression**; No 9,17
detected in rhoptry proteome‡‡
ROP2L7¶ 49.m03159 Unknown Protein kinase None No None
ROP2L8 ¶
52.m01543 Unknown Protein kinase Mass spectrometry data for expression**; No 9
detected in rhoptry proteome‡‡
ROP2L9¶ 55.m04788 Unknown Protein kinase Mass spectrometry data for expression** No None
ROP2L10¶ 59.m06126 Unknown Protein kinase None No None
ROP2L11 ¶
86.m00398 Unknown Unknown Truncated pseudogene No None
ROP2L12¶ 86.m00844 Unknown Unknown Truncated pseudogene No None
ROP2L13 ¶
25.m01746 Unknown Non-catalytic kinase None No None
RON2L1# 83.m01266 Unknown Unknown None Yes 28
RON2L2# 57.m01722 Unknown Unknown None Yes 28
RON3L1 #
20.m03905 Unknown Unknown Mass spectrometry data for expression** Yes None
RON4L1# 52.m01582 Unknown Unknown Mass spectrometry data for expression** Yes 28
*The ToxoDB4.2 (Toxoplasma gondii Genome resource; see Further information64) gene identifier is provided for the T. gondii ME49 strain, along with the GenBank
entry for (typically, but not always) the T. gondii RH strain, if such an entry exists. The GenBank entry should be a verified coding sequence and, therefore, encode
the definitive amino-acid sequence of the primary translation product. The gene identifiers in ToxoDB4.2 are tentative predictions that in most cases have yet to be
experimentally confirmed. Consequently, although they are approximately correct, the transcription start sites, splice junctions and predicted protein sequences
might be incorrect in their detail. ‡Final location of the protein after invasion. § Significant orthologue, with homology that extends beyond the presence of a
conserved enzymatic domain (for example, more than just a kinase or protease active site is conserved throughout evolution), exists in P. falciparum. ||Reference
that originally reported this gene or protein. ¶ROP2-like (ROP2L) protein predicted in ToxoDB4.2 but not yet confirmed to be rhoptry localized and, except as
noted, not even confirmed to be expressed. #RON2-, RON3- or RON4-like protein (RON2L, RON3L and RON4L, respectively) predicted in ToxoDB4.2 but not yet
confirmed to be rhoptry localized and, except as noted, not even confirmed to be expressed. **Mass spectrometry has revealed that one or more peptides are
present in tachyzoites based on data presented in ToxoDB4.2. ‡‡Detected by mass spectrometry in rhoptry-enriched fraction but not yet confirmed to be rhoptry-
localized. The biological function of these proteins is unknown. GPI, glycophosphatidylinositol; HPM, host plasma membrane.

region of contact between the parasite and the host are embedded within. One exciting possibility is that
cell needs to be a circular band, as otherwise a gliding RON2, or a different MJ protein, is inserted into the
parasite would not be able to take a ‘dive’ into the host host plasma membrane during the first steps of inva-
cell — it would simply keep moving along the sur- sion and that it then contacts the host cytoskeleton to
face. AMA1, which was first described in Plasmodium provide the anchoring that is described above. This is
falciparum, might organize the MJ into a ring28. The analogous to a phenomenon that has been reported
association of AMA1 with RON4 is also observed in for enteropathogenic Escherichia coli, in which the
P. falciparum, which indicates that the overall collabo- bacterium inserts a protein (Tir) into the host cell
ration of rhoptry RONs with micronemal AMA1 is that then plays a part in attachment and subsequent
a conserved feature of most, if not all, species of the invasion31. An appealing feature of such a model is
Apicomplexa phylum30. that it would explain the ability of T. gondii to invade
How the RON proteins (RON2, 4 and 5) that almost any cell from a wide range of warm-blooded
form the MJ complex associate with each other is animals; if they provide their own anchor, they could
not known, although RON2 and RON4 seem to be make a home in almost any port, as long as they
linked by disulphide bonds 28. How these proteins are able to make contact with a highly conserved
further associate with a micronemal protein such as cytoskeletal protein. Alternatively, of course, the
AMA1 is also unclear; it is presumed, however, that MJ proteins could remain anchored in the parasite’s
they come into contact with one another at, or just plasma membrane and associate with host-plasma-
below, the apical surface of the parasite. Likewise, membrane structures that are, in turn, anchored to
the topology of the various components of the MJ the host-cell cytoskeleton. If so, a portion, or domain,
complex within the membrane (or membranes) has of that host‑plasma‑membrane molecule would need
not yet been determined. RON2 has at least 2, and to be structurally conserved among many species and
possibly 3, predicted transmembrane domains, but it cell types to explain the extraordinary range of hosts
is unclear which membrane (host or parasite) they and cells that T. gondii can productively infect.

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one parasite, the nearest parasitophorous vacuole is


usually that of the same parasite that released the ROP1
HC during invasion. Small vesicle-like structures referred
to as evacuoles have been observed that contain ROP1
but are devoid of parasites (hence they have also been
named ‘empty’-vacuoles or ‘e’-vacuoles32). Evacuoles
AC
* are observed in a small, but significant, percentage of
invasion events in tissue culture and their frequency
can be increased if invasion is blocked by drug treat-
MJ
ment. For example, treatment with cytochalasin D
prevents the actin and myosin motors of the parasite
from exerting their propulsive force, although attach-
PVM
ment is not affected (the host cell’s actin and myosin
MJ motors appear to be irrelevant to parasite invasion17,33).
The result is a ‘frustrated’ parasite that is stuck to the
outside of a host cell that contains many evacuoles but
lacks a developing parasitophorous vacuole membrane
for the ROP1-containing evacuoles to fuse to.
The ROP2 family of proteins generally migrates to
the second location for rhoptry proteins, the parasito-
phorous vacuole membrane19,34–37. It seems that several
members of this protein family are intimately associated
with the parasitophorous vacuole membrane, and are
possibly even integral membrane proteins. Early sug-
gestions that a hydrophobic alpha helix functions as a
transmembrane domain34 have been called into ques-
tion now that we know that this helix is a conserved
Figure 2 | Toxoplasma gondii invasion. A T. gondii feature of most protein kinases and its hydrophobicity
Nature Reviews | Microbiology
tachyzoite invading an HeLa cell (HC). An irregularly is a necessary feature of its being buried within the
shaped organelle that is derived from rhoptry exocytosis interior of the protein. Although no crystal structure
(asterisk) is found near the apical cytoskeleton (AC). The of a ROP2 family member has been reported, it seems
parasitophorous vacuole membrane (PVM) that is derived unlikely that a helix would be used to span a membrane,
from the host cell membrane is found around the portion especially given the clear conservation of sequence (and
of the parasite that has invaded the host cell. The invasion presumably structure) on either side of the hydrophobic
is thought to be driven by parasite motors acting at the portion. ROP2 has also been implicated in the recruit-
moving junction (MJ). The scale bar represents 0.5 µm. ment of host-cell mitochondria 38,39. This has been
Image reproduced, with permission, from REF. 56 
postulated to be through recognition of the processed
Elsevier Science.
amino terminus of ROP2, which resembles a mitochon-
drial-import signal (that is, an amphipathic helix). The
ROP proteins are injected into host cells proposal is that host mitochondria mistakenly attempt
ROPs seem to have completely different functions to import ROP2, which is somehow firmly tethered to
from RONs. Like the neck proteins, ROPs are released the parasitophorous vacuole membrane. The result is
during invasion but they do not form organized struc- that as the mitochondria attempt to ‘reel in’ the ROP2
tures and are not found at the MJ. Instead, following protein, they ratchet down onto the parasitophorous
release, they migrate to one of three general locations: vacuole membrane, which is where they are routinely
the lumen of the nascent parasitophorous vacuole; the observed by electron microscopy.
parasitophorous vacuole membrane; or the interior of Recent data on ROP18, a member of the ROP2
the host cell (FIG. 3). family, might indicate an interesting aspect of its asso-
ROP1 is an example of migration to the first loca- ciation with the parasitophorous vacuole membrane;
tion: it is released during invasion and accumulates when expressed inside an infected host cell, by direct
within the lumen of the nascent parasitophorous transfection of the ROP18 gene (minus the portion
vacuole23. Remarkably, based on studies that used a encoding a signal peptide), the protein is eventually
combination of ROP1 knock-out parasites and parasite found concentrated on the parasitophorous vacuole
lines that express epitope-tagged versions of ROP1, it membrane, presumably on the face that is exposed to
seems that ROP1 can be synthesized in one parasite the host cytosol18. This is also the location of a putative
but end up in the parasitophorous vacuole of another32. parasite-derived kinase that phosphorylates host iκB40.
This suggests that ROP1 is not simply ‘dumped’ into Whether ROP18 or another member of the ROP2 fam-
the nascent parasitophorous vacuole during inva- ily is responsible for iκB phosphorylation remains to be
sion, but instead is released into the host cell where it directly investigated and the mechanism by which these
then migrates to the nearest parasitophorous vacuole. proteins associate with the parasitophorous vacuole
Because in nature most cells will be infected by only membrane is likewise unknown.

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Nucleus proteins from concentrating in the nucleus41,43. There


is no evidence for the existence in T. gondii of genes or
proteins that are related to those used by bacteria for
introducing proteins into a host cell (for example, type
Rhoptry bulb
Moving
III or type IV secretion systems44), although there are
Microneme junction Host plasma many parallels between these processes.
Rhoptry neck membrane The function of the rhoptry PP2C-hn is unknown,
as a knock-out strain showed no changes in host-gene
expression (based on microarray analysis of infected
Toxofilin? cells) or virulence (based on infection studies in mice),
PVM
although the knock-out strain was partially compro-
mised in its ability to grow in fibroblasts in vitro43. Any
ROP2 family conclusion concerning the lack of a virulence phenotype
ROP1 must be qualified, however, because the knock-out of
PP2C-hn was in an RH strain in which virulence is so
high (LD100 of 1 parasite) that anything short of a dra-
matic reduction in virulence might still yield a parasite
that is capable of killing a mouse.
ROP16
PP2C-hn
Nucleus ROP16 and ROP18 — roles in virulence
Figure 3 | Schematic model for rhoptry contribution to invasion. Rhoptry bulbs It has long been known that different strains of T. gondii
(grey) and rhoptry necks (red) release their contents during the invasion
Nature process
Reviews in
| Microbiology produce radically different pathologies in mice and
concert with simultaneous release by micronemes (green). RON2, RON4 and RON5 maybe even in humans45. Using F1 progeny from crosses
collaborate with micronemal AMA1 to create the moving junction, which migrates between two strains that differ in their virulence, two
down the surface of the parasite, forming a ring of contact with the host plasma research groups have mapped the parasite loci that are
membrane. This effectively excludes many host plasma membrane integral proteins and responsible for the different pathogenicities46,47. The
results in the generation of a parasitophorous vacuole membrane (PVM), which results showed that virulence in a given host (in these
envelopes the parasite. ROP2 family members are injected during invasion, perhaps in
cases, mice) is influenced by which allele is present at
association with small vesicles, and ultimately end up on the host cytosolic side of the
PVM. ROP1 is also observed in association with the injected vesicles, but most of this
each of at least five loci. Two of the most important
protein ends up inside the parasitophorous vacuole lumen. ROP16 and PP2C-hn (hn is loci are ROP16 and ROP18; for example, depending on
the abbreviation for host nucleus) are not observed in the vesicles, but accumulate inside which allele of ROP18 a strain carries, its LD50 in mice
the host nucleus. Other soluble rhoptry proteins (for example, toxofilin) are also can vary by over 4 logs. The impact of the ROP16 locus
presumed to be injected, but in the absence of a concentrating mechanism they will be is less dramatic but still significant. The identities of the
present at too low a concentration to be detectable (only a few rhoptries secrete their other three virulence loci have yet to be determined.
contents during invasion and any proteins that they release will be diluted by up to a For ROP18, there is still much to be learned about
million-fold or more in the host cytosol). Figure adapted from Ref. 28. the exact mechanism by which the different alleles effect
such dramatic differences in disease outcome. It is known
that the allele that is associated with low virulence yields
The third known destination for ROPs is the interior a tiny fraction (~0.1%) of ROP18 mRNA compared
of the host cell or, more specifically, the host-cell nucleus. with the amount that is produced by the alleles found
So far, two rhoptry proteins have been observed in in more virulent strains46,47. This seems to be due to the
the nucleus, a protein phosphatase of the 2C class presence of a large insertion and a small deletion within
(PP2C-hn; hn is the abbreviation for host nucleus)20 the presumptive promoter region of the low virulence
and a putative protein kinase that is a highly diver- allele, which seems to render the promoter inactive. The
gent member of the ROP2 family (ROP16 (Ref. 41)). precise function of the ROP18 protein, however, is not
As for the other ROPs, how these proteins enter yet known, although it clearly does have potent kinase
the host cell is a mystery. The only clues come from activity, and a parasite substrate has been observed but
patch-clamp experiments, which show that there is a not yet identified18.
break in the continuity of the host plasma membrane
at the earliest times of invasion42, perhaps reflecting ROP16 subverts host gene expression
the moment when injection occurs (FIGS 2,3). The proc- Microarray experiments using infected host cells in vitro
ess is clearly an early one, as both ROP16 and PP2C-hn have shown that the infected host cell responds differ-
are detectable in the host nucleus within 15 minutes of ently depending on which strain of T. gondii is used and
allowing infection to commence, which is close to the that these differences also segregate as distinct pheno-
time that such proteins would need to reach the host types among the F1 progeny41. Importantly, ROP16 is
nucleus if they were introduced at time zero. It should among the main parasite loci that have been shown to
be noted that once ROP16 and PP2C-hn enter the be responsible for these differences. Testing of specifi-
host cytosol, conventional nuclear localization signals cally engineered strains showed that this putative protein
(NLSs) are used to traffic them to the nucleus. This has kinase somehow intersects the host signal transducer
been demonstrated by showing that ablation of a classic and activator of transcription (STAT) pathways41. These
NLS signature, which both proteins have, stops these pathways are central to the regulation of many host genes,