R E V I E W S

Probing bacterial gene expression

within host ceils
Raphael H. Valdivia and Stanley Falkow

any bacterial patho- The study of bacterial gene expression in regulation and (3) determine

M gens can enter and

replicate within mam-
malian cells. This ability allows
the host environment is critical to our
understanding of the disease process.
New research tools, such as luciferase
their significance to the ability
of the microorganism to survive
inside the host.
the invading microorganism to and green fluorescent protein, provide
breach the epithelial barrier the means to measure bacterial responses Current methodologies for
and gain access to new niches to the intracellular environment the study of intracellular gene
in deeper tissues that are un- with minimal perturbations and with expression
available to other microbial single-cell resolution. f3-galactosidase (LacZ) is one of
species. An interesting feature of the most widely used reporters
some of these pathogens, such as R.H. Valdivia * and S. Falkow are in the of bacterial gene expression.
Dept of Microbiology, and Immunology,
Mycobacteria spp., Salmonella Stanford University School of Medicine,
Early genetic analysis of bac-
spp., Legionella pneumophila, Stanford, CA 94305, USA. terial pathogens involved the
Coxiella burnetii and Francis- • tel: +1 415 723 2671, fax: +1 415 723 1837, use of lacZ fusions to study tran-
ella tularensis, is their ability e-mail: valdivia@cmgm.stanford.edu scriptional and translational
to survive and replicate in regulation in response to lab-
phagocytic cells, such as macrophages, that are usually oratory conditions believed to mimic the host environ-
part of the host antimicrobial defense system 1. ment. These same lacZ fusions, in conjunction with
Understanding how these pathogens avoid the kill- highly sensitive fluorogenic substrate derivatives, can
ing mechanisms of the phagocyte has been the object be used to measure bacterial gene expression within
of intense study. F. tularensis and C. burnetii actually host cells. For example, Garcia del Portillo et al. have
survive in the harsh phagolysosomal environment. used lacZ fusions to genes known to be induced by
Other pathogens selectively redirect the intracellular specific stimuli to probe the microenvironment of the
trafficking of the host cell to prevent fusion with lyso- S. typhimurium-containing vacuole (STV) (Ref. 3).
somes while, at the same time, retaining the capacity This work predicted that the STV was acidic and de-
to fuse with other host cellular compartments. For ex- ficient in Fe2÷ and Mg 2÷. Some of these conclusions
ample, Mycobacterium avium blocks lysosome fusion were confirmed later when it was established that low
by preventing the prerequisite acidification of its vacu- Mg 2÷concentration is the signal that leads to the acti-
ole, whereas other species seem to reside in an endo- vation of a key regulator of intracellularly induced
somal-Golgi 'limbo' (e.g. Salmonella typhimurium) or genes (PhoP/PhoQ) (Ref. 4). These genes appear to be
an endoplasmic reticulum-like compartment (e.g.L. important for survival inside host cells, as phoP strains
pneumophila)2. Regardless of the mechanism of selec- of S. typhimurium are highly sensitive to antimicrobial
tive lysosomal avoidance, studies suggest that active bac- peptides and macrophage killing s.
terial protein metabolism is necessary for this process 1.2. Luciferases are an increasingly popular tool for the
How does the bacterial pathogen reroute endosomal analysis of intracellular gene expression. Luciferases
trafficking? We envision a microbial intruder that is emit photons during the oxidation of fatty aldehydes 6,
constantly 'sensing' its surroundings and determining and the substrate for this reaction can be added exogen-
(by parallel processing of diverse stimuli) whether it is ously or endogenously by supplying the fatty aldehyde
within a host cell. These stimuli would lead to the tran- biosynthetic genes in trans. Contag et al. have used the
scription of a wide spectrum of genes necessary for the luciferase genes (lux) from Photorhabdus luminescens
metabolic adaptation to new nutritional requirements, to demonstrate that S. typhimurium expressing the lux
protection from chemical and biological insults, and the genes can be tracked in infected animals and tissue cul-
direction of phagosome fusion with non-degradative ture cells 7. In principle, the activity of any gene fusion
compartments of the host cell. Many of these gene prod- to Iux can be followed in live animal tissues. In prac-
ucts are likely to be important in protecting the patho- tice, however, luciferase activity is highly sensitive to
genic microorganism from host defenses: some will act the concentration of molecular oxygen 7. This hinders
synergistically to offer full protection, whereas others the ability to draw strong correlations between photon
will have overlapping or even redundant functions. emission from the luciferase reaction and the level of
To begin to decipher the genetic basis of adaptations gene transcription, especially in oxygen-deficient tis-
to intracellular life, we need to be able to (1) identify sues. Nonetheless, if the substrates for the luciferase
the genes that are induced in the intracellular environ- reaction are not limiting, gene fusions to lux provide
ment, (2) measure their activity, kinetics and mode of reliable measurements of gene expression in a bacterial
Copyright © 1997 Elsevier Science Ltd. All rights reserved. 0966 842X/97/$17.00 PII: S0966-842X(97)01111-6

TRENDS IN MICROBIOLOGY 360 voc. 5 No. 9 SEPTEMBER 1997


population. Lux reporters are particularly useful in that
they permit monitoring of gene expression over time
within a sample, and the short half-life of luciferases
ensures that photon emission is representative of gene
activity occurring during the observation time. In ad-
dition, the availability of sensitive photon acquisition
cameras allows for the visualization of lux expression
with single-cell resolution. For example, by using
Yersinia pseudotuberculosis bearing a yopE-lux fu-
sion, Pettersson et al. were able to visualize photon
emission from bacteria that had adhered to a macro-
phage, but not from neighboring bacteria attached to
the glass surface 8. In this way, they demonstrated that
the transcription of genes encoding Yersinia outer-
membrane proteins (Yops) increases upon bacterial
contact with the target cell surface.
Other reporter systems, such as those using chlor-
amphenicol acyltransferase (the cat gene product), pro-
vide, in addition to colorimetric assays for enzyme ac-
tivity, a selectable phenotype. Gene selections have been
performed in which bacteria bearing cat gene fusions
that are transcriptionally active inside cells are isolated
by their resistance to chloramphenicol9. Heithoff et al.
have used gene fusions to a tandem cat-lacZ to isolate
bacteria that are Lac- under laboratory conditions but
are resistant to chloramphenicol when present inside
macrophages ~°. Sequence analysis of some of these
macrophage-isolated in vivo-induced (ivi) fusions shows
homology to gene products involved in Mg 2÷and Cu 2+
uptake. The predicted functions of these ivi genes help
define some of the stimuli to which microorganisms
are exposed and provide a glimpse into the complex
ecology of the host cell.
An alternative method of monitoring gene expres-
sion involves the creation of protein fusions to specific
epitope tags that can be identified immunologically. For
example, Rhen et al. have used transcriptional fusions
between the spv genes of the S. typhimurium virulence
plasmid and the fimbrial gene cluster of Escherichia
coli KS71A to demonstrate transcriptional activation of
the spv genes during growth in murine macrophages.
In contrast to those grown in normal laboratory media,
bacteria grown in macrophages showed a high degree
of fimbriation and were readily agglutinated with anti-
fimbrial antibodies u.
Bacterial gene expression within host cells can be
monitored directly by identifying (or displaying) differ-
entially expressed mRNA transcripts. Briefly, RNA is
isolated from intracellular bacteria and treated with re-
verse transcriptase (RT) to synthesize bacterial cDNA.
This cDNA is then used as a template for PCR with sets
of primers specific for the gene of interest. The prod-
ucts of the PCR reaction are then subjected to polyacryl-
amide gel electrophoresis and compared with RT-PCR
products from bacteria grown under laboratory con-
ditions. Kwaik and Pederson, for example, have used
differential display PCR to identify L. pneumophila
genes that are induced during residence in macrophage-
like cells 12. Zhang and Normark have used a similar
methodology to identify genes from uropathogenic E.
coli that are induced upon P-pilus-mediated attachment
to human red blood cells 13.
TRENI) SIN MICR()BIOLO(;Y 361
R E V I E W S
'New age' gene reporters
In the past three years, green fluorescent protein (GFP)
has become a standard in the study of in vivo gene ex-
pression and protein localization in eukaryotic and,
more recently, prokaryotic systems 14. GFP expression
can be monitored by spectrofluorimetry, fluorescence
microscopy or fluorescence-based flow cytometry. Un-
like spectrofluorimetry and fluorescence microscopy,
which give either an average fluorescence of all the
bacteria in a sample or information on small sample
sizes (< 100), flow cytometry provides a multiparameter
measure of each cell in very large samples (>10 000).
Thus, the percentage of bacteria within a population
that are synthesizing a particular gene product and the
amount of gene product that is being made by each cell
can be determined. This feature allows microbiologists
to move away from measuring bacterial gene expression
as the average of a population and into the measure-
ment of the genetic responses by individual bacteria in
large samples. As a result, many important questions
concerning the interactions between the pathogen and
its host can now be addressed. Do all intracellular bac-
teria respond identically to the host environment? Can
we identify different host cell microenvironments by the
patterns of bacterial gene expression? What is the fate
of bacteria that do not activate intracellular-specific
genes at the correct time or in the correct compartment ?
The ability of fluorescence-activated cell sorters
(FACS) to detect and separate single bacteria has ex-
tended the use of GFP. We have devised a selection
scheme, termed differential fluorescence induction (DFI),
that uses gfp as a selectable marker to isolate bacteria
bearing gfp gene fusions that are differentially expressed
in response to environmental changes (see Fig. 1). For
example, we have isolated seven different acid-inducible
gfp gene fusions by DFI (Fig. 1) and have tested them
for induction in macrophages is. These include fusions
to genes encoding muhidrug-efflux pumps, cell-surface
repair enzymes and stress-response proteins. Four of
these acid-inducible gfp gene fusions are expressed in-
side cells. However, macrophage-induction levels are
not similar to acid induction in vitro, suggesting that,
although low pH is a relevant inducer of gene expres-
sion within host cells, other stimuli can either prevent or
potentiate the induction of pH-dependent genes.
We have determined the kinetics of gene induction
after bacterial entry into the host cell for the acid-
inducible genes inside macrophages. In this way, we
have identified at least two classes of macrophage-
Questions for future research
• Is there a correlation between the level of expression of a viru-
lence factor and the ability to survive inside a cell?
• What are the stimuli that bacteria sense inside the host cell and
that activate intracellular gene expression?
• Can we identify bacterial genes that are induced at specific
stages during intracellular trafficking?
• What bacterial gene products are required for the rerouting of
the intracellular trafficking of bacteria-containing vacuoles?
• Why does Salmonella typhimurium specifically begin to assemble
components of a secretory apparatus inside a cell?
VOL. 5 NO. 9 SH'TEMBER199V
REVIEWS

inducible genes: the first class is

(a) Px gfp ~ (b) induced within I h of cell entry,
and the second is induced 4 h after
pH7
entry is. It would not be surpris-
O ing to find other classes of intra-
0 0 ~ 0 cellular-induced genes that are ex-
pressed in a temporal hierarchy.
Presumably, this type of gene in-
1 duction cascade is important for
AcifduisndoU:ible ~ p H 4.5)Sort pool bacterial adaptation to the chang-
ing microenvironments within the

-..- [. ).=_ p H 4.5

host cell and allows the pathogen
to continue the infection process.
DFI selection has also been per-
formed directly on infected macro-
Sort pool 3 ~
phages to isolate gfp gene fusions
,, ( p H 4 . 5 ) ~ 2
that are induced inside cells (R.H.
0 (pH 7) A Valdivia and S. Falkow, submit-
ted). We have characterized 14
different gfp gene fusions. Ap-
Sort pool ~1~ proximately half of these genes
are previously described S. typhi-
Fig. 1. Gene isolation by differential fluorescence induction (DFI). (a) The identification of differentially murium genes or are close homo-
expressed genes can be achieved by separating bacteria bearing random gfp gene fusions using a
fluorescence-activated cell sorter (FACS) in response to stimulus-dependent expression of the fluor-
logs of bacterial genes of known
escent protein~5. Briefly, a pool of bacteria bearing gfp gene fusions to random bacterial DNA fragments function. Two macrophage-
(Px) is subjected to a stimulus (e.g. low pH), and all fluorescent bacteria (green) are collected by inducible genes (mig) encode pre-
FACS (step 1). This sorted pool contains bacteria bearing both constitutive and acid-inducible pro- viously described virulence factors.
moter fusions. False positives (constitutive promoter fusions) are removed by growing the collected The other mig encode small, highly
pool at neutral pH and sorting all non-fluorescent bacteria (white) (step 2). A final passage through low
pH and sorting for fluorescent bacteria (green) removes false negatives and enriches the pool with hydrophobic proteins with lipid-
bacteria bearing gfpgene fusions that are acid-inducible (step 3). By adjustingthe sorting parameters, binding motifs and are therefore
'windows' of differential expression can be isolated, such that highly induced gfp gene fusions can be likely to be membrane-associated.
distinguished from moderately and weakly induced gene fusions. (b) Salmonella typhimuriumbearing Approximately half of the mig re-
an acid-inducible gfpgene fusion isolated by DFI before (top) and after (bottom) exposure to L-broth
quire PhoP/PhoQ for intracellular
at pH 4.5 for 2 h.
induction. However, neither of the
two established virulence factors
is controlled by PhoP/PhoQ. One of these virulence
factors, SsaH, is a member of the YscF/MxiH/Prg!
family of virulence proteins that is present in type III
secretion systems and maps within the recently de-
scribed S. typhimurium pathogenicity island II (SPI-2)
(Ref. 16). An ssaH-gfp fusion is specifically induced
inside macrophages (Fig. 2) and is dependent on the
presence of the putative two-component regulatory
system (SsrA/SsrB) that is also present within SPI-2
(Ref. 16).
Reporter genes, such as gfp, permit the investigation
of the dynamics of bacterial gene expression with un-
precedented resolution. Thus, investigators can now
monitor the temporal expression of genes in the highly
complex and changing environment of the host cell.
In the near future, we can expect that new, spectrally
distinct fluorescent reporter proteins14 will be used sim-
Fig. 2. Intracellular-specific gene expression. Macrophage-like ultaneously to study the expression and interaction of
ceil lines (RAW264.7) were infected with Salmonellatyphimurium multiple bacterial genes inside host cells and infected
bearing an ssaH-gfpfusion. Four hours after infection, the cells
were fixed with 2% paraformaldehyde. Extracellular bacteria were
tissues.
detected by immunofluorescence with anti-Salmonella poly-
clonal antibodies followed by a second stage phycoerythrin (PE) Conclusions
conjugate (orange). Expression of the gfp fusion was detected by A variety of methodologies to measure bacterial gene ex-
emitted fluorescence (green). Fluorescence images were super- pression within eukaryotic hosts exist. These measure-
imposed on a direct interference contrast (DIC) image of the in-
fected cells to show the relative topology of bacteria with respect
ments have provided invaluable information regarding
to host cells. the microenvironments to which intracellular patho-
gens are exposed and the way bacteria respond to and

TRENDS , N M,cROB,O' OOY 362 VOL.. NO. 9 SEPTEMBER1997

 R E V I E W S

exploit such habitats. The development of new fluor-
escence-based reporters permits the analysis of live bac-
terial gene responses with single-cell resolution.
Acknowledgements
We thank Evi Strauss,Nina Salamaand Tim McDanielfor their input
and editingexpertise, and J. Shea and D. Holdenfor sharingstrains
and unpublished results. This work was supported by PHS grant
AI 26195 and by unrestrictedgifts from Lederle-PraxisBiologicals
and Bristol-MyersSquibb.
References
1 Garciadel Portillo,F. and Finlay,B.B.(1995) Trends MicrobioI.
3, 373-380
2 Finlay,B.B.and Cossart,P. (1997) Science 276, 718-725
3 Garciadel Portillo,F. et al. (1992) Mol. Microbiol. 6, 3289-3297
4 GarcfaVdscovi,E., Soncini,F.C. and Groisman,E.A.(1996) Cell
84, 165-174
5 Fields,P.I., Groisman,E.A.and Heffron,F. (1989) Science 243,
1059-1062
6 Meighen,E. (1993)FASEBJ. 7, 1016-1022
7 Contag,C.H. et al. (1995) Mol. MicrobioI. 18, 593-603
8 Pettersson,J. et al. (1996) Science 273, 1231-1233
9 Mahan,M.J. etal. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,
669-673
10 Heitboff,D.M. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,
934-939
11 Rhen,M., Riikonen,P. and Taira, S. (1993) Mol. Microbiol. 10,
45-56
12 Kwaik,Y.A.and Pederson,Li. (1996) Mol. Microbiol. 21,
543-556
13 Zhang,J.P. and Normark,S. (1996) Science 273, 1234-1236
14 Cubitt,A.B.et al. (1995) Trends Biochem. Sci. 20, 448-455
15 Valdivia,R.H. and Falkow,S. (1996) Mol. Microbiol. 22,
367-389
16 Shea,J.E. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,
2593-2597

Pore-forming proteins in
pathogenic protozoan parasites
M. Fbtima Horta

lthough pathogenic pro- Pore-forming proteins (PFPs) may play inside the cell generates osmotic

A tozoa differ in many

ways, many of them pro-
duce cytolytic proteins that dis-
important roles in pathogenesis by
protozoan parasites by either directly
damaging the plasma membrane of the
pressure, causing a water influx.
The cell disrupts through a pro-
cess known as colloid-osmotic
rupt target cell membranes by host cells or ensuring intraceUular survival lysis3,4.
forming discrete channels in of the parasites by promoting their exit Death of host cells mediated
the lipid bilayer. These pore- from lysosomal vacuoles. The L e i s h m a n i a by PFPs from bacterial patho-
forming proteins (PFPs) are amazonensis pore-forming cytolysin, gens has frequently been de-
thought to play a significant leishporin, may play a crucial role in the scribed (for reviews, see Refs
role in the pathogenesis of pathogenesis of leishmaniasis. 4-6). Protozoan pathogens can
many protozoan infections 1,2. now be added to the list of PFP-
Channel formation by PFPs is M.F. Horta is at the producing organisms, and the
Dept de Bioquimica e Imunologia, notion of a cause-and-effect re-
a well-defined mechanism of Instituto de CiSncias Biol6gicas,
membrane damage used in bio- Universidade Federal de Minas Gerais, lationship between cytolysins
logical systems ranging from 31270-010 Belo Horizonte, MG, Brazil. and host tissue damage can
bacteria to vertebrates: the C9 tel: +55 31 441 5 7 7 7 , fax: +55 31 441 5963, now be extended to parasitic
component of the complement e-mail: phorta@icb.ufmg.br diseases.
system and perforin from cyto-
lytic lymphocytes in vertebrates are good examples. The outsider's attack
PFPs bind to the plasma membrane of a target cell E n t a m o e b a histolytica, which is responsible for human
and insert into the lipid bilayer by changing their con- amoebiasis, was the first protozoan in which PFPs were
formation and exposing hydrophobic domains. A discovered 7,s. Amoebiasis is an enteric illness that may
chain reaction follows in which the altered molecules spread to multiple organs when the parasite invades
bind others, oligomerizing around a central axis to form the intestinal mucosa. The main manifestation of the
transmembrane pores that grow in diameter by the disease is the destruction of host tissues, which results
successive annexation of new monomers. Some PFPs from parasite contact-mediated cytolysis. The cytolytic
act as monomers that adopt circular or semicircular effect has been attributed to a family of pore-forming
structures without mutual association. Ions and small peptides, termed amoebapores, that are localized within
molecules can then pass freely across the lipid bilayer, cytoplasmic granular vesicles of the trophozoites 9.
and the high concentration of macromolecules trapped Amoebapores form transmembrane channels with an
Copyright © 1997 Elsevier Science Ltd. All rights reserved. 0966 842X/97/$17.00 PII: S0966-842X(97)01109-8

TRENI)S IN MICP,()BI()LOGY 363 W,L 5 NO. 9 SEPTEMBER 1 997