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 REVIEWS
Listeria monocytogenes:
a multifaceted model
Mélanie Hamon*‡§, Hélène Bierne*‡§ and Pascale Cossart*‡§
Abstract | The opportunistic intracellular pathogen Listeria monocytogenes has become
a paradigm for the study of host–pathogen interactions and bacterial adaptation to
mammalian hosts. Analysis of L. monocytogenes infection has provided considerable insight
into how bacteria invade cells, move intracellularly, and disseminate in tissues, as well as
tools to address fundamental processes in cell biology. Moreover, the vast amount of
knowledge that has been gathered through in-depth comparative genomic analyses and
in vivo studies makes L. monocytogenes one of the most well-studied bacterial pathogens.

*Institut Pasteur, Unité des
Interactions Bactéries
Cellules, Paris 75015, France.
‡
Institut National de la Santé
et de la Recherche Médicale,
U604, Paris 75015, France.
§
Institut National de la
Recherche Agronomique,
USC2020, Paris 75015,
France.
Correspondence to P.C.
e-mail: pcossart@pasteur.fr
doi:10.1038/nrmicro1413
The Gram-positive bacterium Listeria monocytogenes is
a ubiquitous pathogen that thrives in diverse environ-
ments such as soil, water, various food products, humans
and animals. The disease caused by this bacterium,
listeriosis, is acquired by ingesting contaminated food
products and mainly affects immunocompromised
individuals, pregnant women and newborns. Listeriosis
manifests as gastroenteritis, meningitis, encephalitis,
mother-to-fetus infections and septicaemia, resulting in
death in 25–30% of cases. The diverse clinical manifesta-
tions of infection with L. monocytogenes reflect its ability
to cross three tight barriers in the human host. Following
ingestion, L. monocytogenes crosses the intestinal barrier
by invading the intestinal epithelium, thereby gaining
access to internal organs. During severe infections,
crossing the blood–brain barrier results in infection of
the meninges and the brain, and in pregnant women,
crossing the fetoplacental barrier leads to infection of
the fetus1.
L. monocytogenes infection has been a useful model
for evaluation of the cellular interactions that are crucial for
the initiation of the host T-cell response. However, this
aspect of listerial biology is well documented and has
recently been reviewed elsewhere2. This Review highlights
the many ways in which L. monocytogenes manipulates its
mammalian host, and it focuses on the lessons this bact-
erium has taught us in cell biology, bacterial pathophysiol-
ogy, virulence-factor regulation, and bacterial adaptation
to the host cytosol.
Indeed, the ability of L. monocytogenes to invade
and replicate in different cell types has been extensively
studied and has revealed the sophisticated relationship
between the bacterium and its host. The breadth of
information gathered on the elaborate mimicries that
are used by L. monocytogenes to subvert host processes
has made it an exceptional tool for the study of cellular
processes such as actin-based motility, growth-factor-
mediated signalling, endocytosis and cellular adhesion.
Furthermore, available animal models, genetic tools and
genomics have facilitated the compilation of informa-
tion on different aspects of L. monocytogenes biology and
have made this bacterium one of the most useful model
organisms for the study of bacterial pathogenesis and
pathophysiology.
An intracellular bacterium and a cell biologist
L. monocytogenes is a facultative intracellular bacterium. Its
life cycle reflects its remarkable adaptation to intracellular
survival and multiplication in macrophages and other cell
types1,3,4 (FIG. 1). Similar to the situation for most pathogens,
the invasion of macrophages by L. monocytogenes is a pas-
sive process, but entry into non-professional phagocytes
is induced by binding of the bacterial surface proteins
internalin A (InlA) and InlB to receptors on the host cell.
Both of these invasins are necessary and sufficient for bac-
terial entry into cell types such as enterocytes, hepatocytes,
fibroblasts, epithelial cells and endothelial cells, but InlA-
mediated entry is restricted to the smaller number of cell
types that express its receptor. Entry of L. monocytogenes
into mammalian cells is a dynamic process that requires
actin polymerization and membrane remodelling, and
is an excellent example of how a bacterium can manipu-
late host-cell signalling and endocytic pathways to its
advantage. L. monocytogenes can also harness the actin-
polymerization machinery in the cytoplasm to facilitate
intracellular and intercellular movement. New mecha-
nisms by which L. monocytogenes manipulates the host cell
are emerging through the use of microarray analyses that
aim to determine the genes that are specifically activated
by bacterial entry into the host cell5,6.

NATURE REVIEWS | MICROBIOLOGY VOLUME 4 | JUNE 2006 | 423

© 2006 Nature Publishing Group
REVIEWS

Listeria monocytogenes
a

b a

b
c

0.5 µm
c Phagosome

Lysis of
phagosome
and replication d
in cytosol d
0.5 µm
F-actin

0.5 µm

e
e

Double-membraned
vacuole

f 0.5 µm
f

Lysis of vacuole
0.5 µm

Figure 1 | Schematic representation and electron micrographs of the Listeria monocytogenes life cycle.
a | L. monocytogenes induces its entry into a non-professional phagocyte. b | Bacteria are internalized in a vacuole (also
known as a phagosome). c,d | The membrane of the vacuole is disrupted by the secretion of two phospholipases, PlcA and
PlcB, and the pore-forming toxin listeriolysin O. Bacteria are released into the cytoplasm, where they multiply and start to
polymerize actin, as observed by the presence of the characteristic actin tails (see Supplementary information S3 (figure)).
e | Actin polymerization allows bacteria to pass into a neighbouring cell by forming protrusions in the plasma membrane.
f | On entry into the neighbouring cell, bacteria are present in a double-membraned vacuole, from which they can escape to
perpetuate the cycle. F-actin, filamentous actin. Electron micrographs a–c,e–f are reproduced with permission from REF. 113
 (1998) European Molecular Biology Organization, and d is reproduced with permission from REF. 30  (1992) Elsevier.

InlB: subverting cellular-signalling and endocytic
pathways. Binding of InlB to its cellular receptor
Met results in the entry of L. monocytogenes into
different cell types. Met is a protein tyrosine kinase,
and the endogenous ligand of this receptor is hepato-
cyte growth factor (HGF) 7 (FIG. 2) . In vivo, Met is
expressed mainly by cells of epithelial origin, whereas
HGF is produced mainly by fibroblasts and stromal
cells. The binding of HGF to Met activates cellular
survival and proliferation signals, and it induces
cytoskeletal rearrangements that function in cellular
motility and differentiation. Binding of InlB activates
the protein-tyrosine-kinase activity of Met, as well
as the phosphatidylinositol 3-kinase (PI3K) and
the Ras–mitogen-activated protein kinase (MAPK)
pathways, all of which are required for the uptake
process7–9. Interestingly, although InlB and HGF both
bind and activate Met, InlB does not strictly mimic
HGF. Indeed, the kinetics of InlB-induced signalling
are different from those of HGF-induced signalling7,
and at an equal concentration, InlB seems to induce
a more potent activation of the Ras–MAPK pathway
than does HGF10. Differences in signalling might be
explained by the finding that InlB also binds gC1qR

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 REVIEWS

a HGF-mediated signalling b InlB-mediated signalling

Listeria monocytogenes
InlB
HGF
GAGs
Met
Out gC1qR Met
Out
PtdIns(4,5)P2 PtdIns(3,4,5)P3
PtdIns(4,5)P2 PtdIns(3,4,5)P3
In
Recycling Cbl Akt PLCγ In Dynamin
PI3K Akt PLCγ
Rac1

Cortactin
Ub CD2AP PI3K ABI
Clathrin- Cbl
mediated Survival CIN85 Cbl Cbl CDC42
GAB1 Rac1

WAVE
endocytosis EPS15 GAB1
SHC CDC42 Ub
SHC
Ras

SP
GRB2

HR
A3

A
GRB2

N-W
GG
SOS
Cytoskeletal
rearrangements F-actin
Clathrin Ras
Degradation
Arp2/3

Nucleus Proliferation

Figure 2 | Met signalling induced by hepatocyte growth factor (HGF) and internalin B (InlB). a | Phosphorylation
of Met leads to the recruitment and activation of many transducers, which in turn recruit cytosolic signalling proteins.
Signalling mediated by HGF activates survival and proliferation signals, and it induces cytoskeletal rearrangements that
are important for cellular motility and differentiation. On stimulation with HGF, the endocytosis of Met, similar to most
signalling receptors, is an important regulatory mechanism that downregulates the cell-surface expression of the activated
receptor. b | Met signalling mediated by the Listeria monocytogenes protein InlB induces cytoskeletal rearrangements that
are important for bacterial entry into non-phagocytic cells. Clathrin components of the endocytic machinery are also
recruited to the site of entry. The link between the cytoskeletal machinery (shown on the right) and the endocytic
machinery (shown on the left) is still unclear. InlB, through the GW repeats at its C terminus, also binds gC1qR (the receptor
for the globular part of complement component C1q) and glucosaminoglycans (GAGs), which are negatively charged
polysaccharides that are present at cell surfaces. Both components might contribute to entry of L. monocytogenes by
modulating the interaction of InlB with Met. ABI, Abl interactor 1; Arp, actin-related protein; CD2AP, CD2-associated protein;
CIN85, Cbl-interacting protein of 85 kDa; EPS15, epidermal-growth-factor-receptor substrate 15; F-actin, filamentous
actin; GAB1, GRB2-associated binding protein 1; GGA3, Golgi-localized, γ-ear-containing, ADP-ribosylation-factor-binding
protein 3; GRB2, growth-factor-receptor-bound protein 2; HRS, HGF-regulated tyrosine-kinase substrate; N-WASP, neural
Wiskott–Aldrich syndrome protein; PI3K, phosphatidylinositol 3-kinase; PLCγ, phospholipase C-γ; PtdIns(3,4,5)P3,
phosphatidylinositol-3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; SHC, SRC-homology-2-
domain-containing transforming protein C; SOS, son of sevenless; Ub, ubiquitin; WAVE, Wiskott–Aldrich syndrome protein
(WASP)-family verprolin homologous protein.

(the receptor for the globular part of complement
component C1q), which might therefore function as
a co-receptor for InlB11. Furthermore, InlB and HGF
do not compete for binding to Met7, indicating that
they might bind distinct sites on Met. These results
are consistent with the fact that InlB and HGF have
no sequence homology and are structurally unrelated.
Crystal structures of InlB bound to Met could provide
valuable information about the molecular mechanism
that underlies the activation of Met by InlB.
The signalling pathways that are activated by InlB
ultimately lead to cytoskeletal rearrangements and
entry of L. monocytogenes. How activation of the
PI3K and the Ras–MAPK pathways leads to cytoskel-
etal rearrangements has been extensively studied, and
many of the proteins that are crucial for invagination
and internalization have been identified. Local actin
remodelling at the site of InlB attachment is mediated
by the recruitment and activation of the actin-nucleation
complex, Arp2/3, which promotes actin nucleation and
polymerization (discussed later). The mechanism of
Arp2/3 activation seems to be cell-type dependent, but
it involves a combination of the small GTPases Rac and
CDC42 and proteins of the Wiskott–Aldrich syndrome
protein (WASP) family, which includes neural WASP
(N-WASP) and WAVE 12. Proteins of the Ena/VASP
family (enabled homologue/vasodilator-stimulated-
phosphoprotein family), which promote actin-filament
elongation, are also central to the process. In addition,
cofilin, which is essential for depolymerization of actin
filaments, functions successively as a stimulator and
a downregulator of actin rearrangements that occur
during the internalization process 12,13. All of these
components that are recruited by the binding of InlB
to Met also have a role in growth-factor-receptor
activation. Therefore, although there are differences
between the kinetics of signalling mediated by InlB
and HGF, the machinery that is recruited to the site
of activation seems to be the same for both molecules,
showing the utility of L. monocytogenes as a tool to
study cellular signalling by Met or other growth-factor
receptors.

NATURE REVIEWS | MICROBIOLOGY VOLUME 4 | JUNE 2006 | 425

© 2006 Nature Publishing Group
REVIEWS
Adherens junctions
Together with tight junctions
and desmosomes, these are
specialized structures that
allow epithelial cells to adhere
to each other, and they have
epithelial cadherin (E-cadherin)
as a major component.
426 | JUNE 2006 | VOLUME 4
Recently, the study of InlB-induced internaliza-
tion revealed an unexpected mechanism used by
L. monocytogenes during host-cell entry. L. monocytogenes
invades epithelial cells by subverting clathrin-mediated
endocytosis14 (FIG. 2b; see Supplementary information
S1 (figure)), a process that is used by mammalian cells
to take up nutrients and to recycle membrane proteins.
Owing to their size, which is usually 1–3 µm, bacteria
were thought to enter cells through a mechanism related
to phagocytosis (that is, an actin-dependent mecha-
nism), which differs from ‘endocytosis’, a process that
is thought to internalize particles no larger than 120 nm
and that was considered to be actin independent until
recently. Therefore, the finding that L. monocytogenes
can induce its internalization by using clathrin-depend-
ent machinery was surprising and indicated that clath-
rin-coated structures can engulf much larger particles
than previously thought. Cell-surface expression of
Met is downregulated by HGF, and similarly, soluble
InlB induces the degradation of Met, through monou-
biquitylation and clathrin-mediated endo cytosis 14.
Furthermore, as shown using RNA-interference-
mediated knockdown, important components of the
endocytic machinery are required for internalization
of L. monocytogenes. Although the underlying molecu-
lar mechanisms have not been defined, other invasive
bacteria had previously been reported to use clathrin-
mediated entry, implying that this mechanism is not
restricted to Listeria species and could be a commonly
used mechanism for bacterial entry 15–17. Although
the endocytic machinery is important for entry of
L. monocytogenes, this bacterium might exploit other
mechanisms, because inhibitors of endocytosis reduce
bacterial entry but do not completely abolish it14.
Plasma-membrane microdomains known as lipid rafts
have also been shown to be important for the entry of
L. monocytogenes18. Because lipid rafts are usually asso-
ciated with clathrin-independent endocytic pathways,
this indicates either that L. monocytogenes exploits
more than one endocytic mechanism or that the sepa-
ration among the classes of endocytosis is not as well
demarcated as conventionally thought. Further work is
necessary to decipher how lipid rafts, clathrin-mediated
endocytosis and actin-mediated phagocytosis combine
to enable listerial entry and cellular invasion.
InlA: exploiting intercellular junctions. Similar to InlB,
InlA induces local cytoskeletal rearrangements in the
host cell to stimulate uptake of L. monocytogenes by
epithelial cells. InlA is a covalently linked bacterial
cell-wall protein that binds the host epithelial-cell
protein E-cadherin19 (FIG. 3). E-cadherin is a transmem-
brane protein that belongs to a large family of cell–cell
adhesion molecules that are required for the correct
formation of adherens junctions between epithelial
cells. E-cadherin is localized at these cellular junc-
tions, where its intracellular domain forms a complex
with the cytoskeleton through the catenins (which
are cadherin-binding proteins), and its extracellular
domain is in contact with E-cadherin molecules on
neighbouring cells 20. InlA binds the extracellular
© 2006 Nature Publishing Group
domain of E-cadherin, but it is the intracellular domain
of E-cadherin that is essential for the cytoskeletal
rearrangements that are required for bacterial entry21.
Because all of the components of the endogenous
machinery of cell–cell junctions seem to be recruited
on binding of InlA, L. monocytogenes is a good system
for the study of cellular adhesion and identification of
components that are involved in this process.
Although it is known that assembly and attachment
of the E-cadherin, α-catenin and β-catenin complex
to the cytoskeleton is central to both intercellular
adhesion and L. monocytogenes entry21, neither the
mechanisms that hold cells together nor the molecu-
lar mechanisms that are required for InlA-dependent
entry are as yet fully understood. Until recently, the
accepted model of intercellular adhesion proposed
that α-catenin anchors the E-cadherin–β-catenin
complex to the actin cytoskeleton, providing a stable
structure that maintains tissue integrity. However, it
has become apparent that the dynamics of this process
are more complex. As was recently shown, α-catenin
cannot simultaneously interact with actin filaments
and the E-cadherin–β-catenin complex, indicating
that α-catenin is not an actin anchor but is, instead,
an actin-filament organizer22,23. Further investigation is
required to understand this mechanism fully; however,
the dynamic interactions between the cadherin–catenin
complex and the underlying actin cytoskeleton are
consistent with the findings that L. monocytogenes can
regulate and rearrange actin structures at intercellular
junctions through adhesion to E-cadherin, and further
emphasize the validity of the listerial model for analysing
events at intercellular junctions.
The validity of using L. monocytogenes as a tool to
study cell–cell junction formation was shown by a
recent study of InlA-dependent entry that identified
the protein ARHGAP10 (Rho GTPase-activating pro-
tein 10) as a novel cellular component that is involved
in the recruitment of α-catenin to cell–cell junctions.
This study also showed that ARHGAP10 was essential
for listerial entry (see Supplementary information S2
(figure))24. Overexpression of ARHGAP10 disrupted
the cytoskeleton and increased the local concentration
of α-catenin, indicating that ARHGAP10 has a direct
role in regulation of the dynamics of cell–cell junction
formation. Furthermore, ARHGAP10 was shown to
control the activity of RhoA and CDC42, two proteins
that regulate cell–cell junction formation.
Components that generate the tension that is required
to hold neighbouring cells together, that is, myosin VIIA
and its ligand vezatin25, have been found to be impor-
tant for L. monocytogenes entry, indicating that these
components might generate the force that is neces-
sary for engulfment of the bacterium through phago-
cytosis26. These findings also indicate that the tension
that holds two cells together could be similar to the
tension that is exerted during phagocytosis, as if each
cell is attempting to engulf its neighbour. An analogous
process known as ‘frustrated phagocytosis’ occurs
when macrophages adhere to immune-complex-coated
surfaces27.
www.nature.com/reviews/micro
 REVIEWS

a Intercellular junctions b InlA-induced entry

F-actin
Arp2/3

α G-actin α Formin
α
β β β
Listeria monocytogenes

In
InlA

Out E-cadherin
E-cadherin

Out

In
Out Vezatin
Catenins
β β
In Myosin α β α G-actin
ARHG
VII AP10 α
ARF6

β β β
Catenins
α α α Arp2/3
Formin F-actin

Figure 3 | Adherens junction and internalin A (InlA)-induced bacterial entry. a | Adherens junctions hold adjacent
cells together through the transmembrane protein epithelial cadherin (E-cadherin). The intracellular domain of E-cadherin
recruits α-catenin and β-catenin, and α-catenin bridges the actin cytoskeleton and E-cadherin. Formins, which interact
directly with α-catenin, are also essential for forming actin cables at cell–cell junctions, although the mechanism by which
they achieve this is not understood. b | The receptor for the Listeria monocytogenes protein InlA is E-cadherin. Many
components that are important for adherens junctions are recruited to the site of bacterial entry, where the cytoskeletal
rearrangements that are required for invasion occur. ARF6, ADP-ribosylation factor 6; ARHGAP10, Rho GTPase-activating
protein 10; Arp, actin-related protein; F-actin, filamentous actin; G-actin, globular actin.

Harnessing the actin-polymerization machinery. information S3 (figure)). Polymerization of host actin is

Following internalization into a host-cell vacuole,
L. monocytogenes lyses the membrane-bound phago-
some (discussed later) and escapes into the cytoplasm,
where it can polymerize host actin and propel itself
through the cell and into neighbouring cells28. The abil-
ity to spread from cell to cell without coming in contact
with the extracellular milieu allows the bacterium to
propagate through tissues and avoid contact with cir-
culating antibodies or other extracellular bactericidal
compounds. At the leading edge of moving cells, the
control of actin polymerization is a complex mechanism
triggered by intricate signalling pathways that take place
at the plasma membrane. L. monocytogenes bypasses
these signalling pathways and directly nucleates actin
constitutively, making it a simple and effective model
for studying the dynamics of actin polymerization.
L. monocytogenes polymerizes actin asymmetrically
along its surface, producing an actin tail that propels the
bacterium through the cytoplasm28,29 (see Supplementary
mediated by the bacterial surface protein ActA, the first
protein that was identified to have functions that promote
actin nucleation30–32. It was later found that ActA mimics
its eukaryotic counterparts, proteins of the WASP family
(which includes N-WASP and WAVE)33. The N-terminal
region of ActA, which is essential for actin-based motil-
ity 34, has homology to the C-terminal region of WASP,
which binds the actin-nucleation complex Arp2/3
(REF. 35). Accordingly, both ActA and WASP-family pro-
teins function as nucleation-promoting factors (NPFs)
for the Arp2/3 complex and are involved in forming a
ternary complex that is composed of Arp2/3, an NPF and
actin. The Arp2/3 complex consists of seven proteins,
including two proteins that are related to actin, Arp2 and
Arp3, which (as a result of their structural similarity to
actin) are thought to function as a template for polymeri-
zation. Discovery of the Arp2/3 complex ensued from
studies of ActA partners, thus the role of Arp2/3 as a key
actin-nucleating complex was consequently revealed.

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REVIEWS
a b N
Listeria
monocytogenes
InlB InlA
Pro16
C nity 3. However, recent molecular evidence showed that
Out
In Met E-cadherin Met E-cadherin Met E-cadherin
Guinea pig Mouse Human
and rabbit
Figure 4 | Host specificity of Listeria monocytogenes proteins internalin A (InlA)
and InlB. a | InlA and InlB can bind and induce entry of L. monocytogenes into human
cells that express the respective cell-surface receptors, epithelial cadherin (E-cadherin)
or Met. However, a single amino-acid change in E-cadherin (at position 16; see b)
prevents InlA from binding mouse E-cadherin, and for unknown reasons, InlB cannot
recognize or activate guinea pig or rabbit Met. b | A diagrammatic representation of the
crystal structure of the leucine-rich-repeat region of InlA (purple) bound to E-cadherin
(green) is shown. The position of the crucial proline residue at position 16 of E-cadherin is
indicated. It is this residue that determines intermolecular recognition and therefore
host specificity. The crystal-structure representation is reproduced with permission from
REF. 114  (2002) Elsevier.
The L. monocytogenes model has also been useful for
understanding the importance and the function of VASP,
the other main ligand of ActA. VASP binds the central
proline-rich domain of ActA and promotes efficient
actin-based motility34,36–38, highlighting the importance
of this protein in actin polymerization. Only recently,
however, have the complex molecular mechanisms of
VASP activity begun to emerge. VASP seems to promote
listerial motility by recruiting the actin-binding protein
profilin, which promotes polymerization at actin-filament
barbed ends39. VASP also seems to induce faster growth
of the actin network at the bacterial surface by causing
the release of Arp2/3 from ActA40. In addition, VASP
seems to decrease Y-branch formation, thereby increas-
ing parallel alignment of actin filaments41. Although their
mechanistic role is not fully elucidated, proteins of the
Ena/VASP family are important in the formation of actin
fibres, filopodial tips and the lamellipodial leading edge42.
Intracellular and intercellular movement using actin
polymerization is not restricted to L. monocytogenes.
A growing number of intracellular pathogens, includ-
ing Rickettsia species, Shigella species, mycobacteria,
Filopodia Burkholderia pseudomallei and vaccinia virus, show this
Rod-like cell-surface feature during infection (see Supplementary information
projections that are composed S3 (figure), and for recent reviews, see REFS 43,44).
of actin filaments. They are
found on various cell types and
have sensory or exploratory A paradigm in pathophysiology
functions. As a pathogen that displays such interesting features
as strong T-cell activation, a sophisticated relation-
Lamellipodia ship with its host and crossing of protective barriers,
Thin actin-rich structures that
form protrusions at the edge of
L. monocytogenes has more recently also emerged as a
the cell and are essential for model to study the pathophysiology of a complex bac-
cellular motility. terial infection. Findings from in vitro work have been
428 | JUNE 2006 | VOLUME 4
© 2006 Nature Publishing Group
used to bypass stringent host species specificity and gen-
erate relevant model systems to study infection in vivo.
In this respect, L. monocytogenes is a good example of
how in vitro studies can help to generate animal models
that more closely reflect human infection.
For many years, the animal model used to study
L. monocytogenes infection was intravenous infection
of mice. This model provides a dose-dependent infec-
tion with dissemination of the bacteria into organs and
was crucial in the discovery of cell-mediated immu-
the mouse model is inadequate to study the crossing of
barriers that is characteristic of listeriosis. It had long
been known that oral inoculation of mice (rather than
intravenous infection), which most closely reflects the
human mode of infection, is not efficient because only
small numbers of L. monocytogenes cross the mouse
intestinal barrier. The reason for this was uncovered
by molecular in vitro studies that showed that a single
amino-acid difference in the mouse cellular recep-
tor for InlA, E-cadherin, prevented it from binding
InlA, thereby showing the inadequacy of the mouse
model for study of the invasive role of InlA45 (FIG. 4).
Consequently, a novel animal system was developed to
study the crossing of the intestinal barrier: a transgenic
mouse that expresses human E-cadherin on the surface
of enterocytes46. This model showed that InlA has a
key role in disease, because it is essential for crossing
of the intestinal barrier. In this model system, InlA
could interact with E-cadherin, and a wild-type strain
of L. monocytogenes was able to cause disease through
oral inoculation.
So far, E-cadherin has been found only at cell–cell
junctions and on the basolateral face of epithelial cells,
so the mechanism by which L. monocytogenes gains
access to E-cadherin was enigmatic. Two hypotheses
were put forward to explain how InlA could target
E-cadherin 46. The first hypothesis proposed that
L. monocytogenes could gain access to E-cadherin
at the tips of intestinal microvilli, where apoptotic
epithelial cells slough off. The second hypothesis pro-
posed a synergy between InlA- and InlB-dependent
internalization, because activation of Met by HGF has
been shown to stimulate the disassembly of junctions
between epithelial cells46. Recent results have shown
that L. monocytogenes invades the intestinal epithelium
at sites of cell extrusion at the tips of villi47 and that the
contribution of InlB to crossing of the intestinal bar-
rier is insignificant in vivo48. Whether the mechanism
of intestinal invasion is used to cross other barriers is
unknown. Synergy between InlA and InlB could still be
important for crossing of the placental barrier49.
Because the transgenic mice described here express
human E-cadherin only on enterocytes, it is not pos-
sible to study the role of InlA in deeper tissues. The
generation of transgenic mice that express E-cadherin
on all cells is in progress. At present, the role of InlA
can also be studied in guinea pigs or rabbits, because
E-cadherin is recognized by InlA in these animals.
However, recent studies show a species specificity for
InlB: InlB does not recognize or activate guinea-pig
www.nature.com/reviews/micro

or rabbit Met. Therefore, the role of InlA and InlB in
infection cannot be studied using these animal mod-
els48. A human model remains the best model system
for studying listerial infection, and human explants have
been successfully used to determine the mechanism by
which L. monocytogenes crosses the maternofetal bar-
rier49. Human placental villus explants, together with
primary or immortalized trophoblastic cells, were
used to show that InlA is a key bacterial protein that is
required for crossing of the human maternofetal bar-
rier, although InlA is not essential for this role in the
pregnant guinea-pig model49,50. Crossing of the blood–
brain barrier is still poorly understood. However, the
optimization of animal models should help to decipher
this crucial step.
Other model systems are being developed to identify
the host factors that are required for the intracellular sur-
vival of L. monocytogenes and possibly of other intracel-
lular pathogens. Drosophila melanogaster has attracted
attention as a model because of the many genetic and
immunological studies that have been carried out using
this organism, and it has been successfully used to test
L. monocytogenes virulence51. Moreover, genome-wide
RNA-interference screens in D. melanogaster S2 cells
(which are macrophage-like cells) have revealed many
new host factors that are important for entry into the
host cell, escape from the vacuole and intracellular
growth of L. monocytogenes52,53. Another noteworthy
organism that has been shown to support a listerial
infection is Caenorhabditis elegans54.
a resulted in thermoregulation of GFP. This mechanism
plcA prfA
P P1 and P2
b rapid expression of the encoded transcription factor and
Ribosome
subunits
SD
High
temperature
prfA ATG
mRNA prfA
Low
temperature
No PrfA PrfA
Virulence-gene
expression
Figure 5 | The PrfA regulator. a | Schematic representation of the prfA region.
During exponential phase, prfA is mostly transcribed as a bicistronic product from
the promoter (P) upstream of plcA. By contrast, during stationary phase, prfA is mostly
transcribed as a monocistronic product from P1 and/or P2. b | Mechanism that controls
the thermoregulated expression of PrfA in the promoter upstream of prfA. At low
temperatures (≤30°C), a secondary structure forms in the untranslated region of prfA,
and this prevents ribosome binding and therefore expression of PrfA. At high
temperatures (≥37°C), melting of the prfA untranslated region allows ribosomes
to bind and PrfA expression to occur. SD, Shine–Dalgarno sequence. This figure is
modified with permission from REF. 58  (2002) Elsevier.
NATURE REVIEWS | MICROBIOLOGY
© 2006 Nature Publishing Group
REVIEWS
Novel regulatory mechanisms
L. monocytogenes is a facultative intracellular pathogen
that can live both inside and outside its host. This bac-
terium has therefore evolved sophisticated regulatory
mechanisms to ensure that virulence factors are opti-
mally expressed when they are required. These regula-
tory mechanisms might prove to be general mechanisms
used by other bacteria.
PrfA: a tightly regulated protein. Most of the virulence
proteins that have been identified in L. monocytogenes
are under the control of one transcriptional regulator,
PrfA, which itself is tightly regulated by environmen-
tal conditions. During exponential growth, prfA is
mainly transcribed as a bicistronic mRNA from the
plcA promoter. By contrast, during stationary phase,
a monocistronic mRNA is preferentially transcribed
from a promoter upstream of prfA55–57 (FIG. 5a). Similar
to many other pathogens, L. monocytogenes can sense
conditions in the mammalian host and respond by
expressing virulence genes. A novel regulation of PrfA
by temperature, owing to the structure of the upstream
untranslated region of the prfA mRNA, was recently dis-
covered58 (FIG. 5b). At low temperature (30°C), the prfA
leader transcript controls translation of the downstream
mRNA by forming a secondary structure that masks the
ribosome-binding site. At mammalian host tempera-
ture (37°C), this structure partially melts to expose the
ribosome-binding site, thereby allowing translation to
occur. Fusion of the prfA leader transcript to the gene
that encodes green fluorescent protein (GFP) also
might be used by other bacteria, as has previously been
suggested for the Yersinia pestis activator protein LcrF59.
Such post-transcriptional regulation of prfA allows
therefore efficient transcription of virulence factors as
soon as the bacterium enters the host.
Other environmental conditions — such as osmol-
arity, iron concentrations, pH, the presence of fermentable
sugars, stress (through σB), and conditions in the host-cell
intracellular compartment — have been shown to regu-
late prfA and PrfA-controlled genes through mechanisms
that are not completely understood60,61. Post-translational
regulation of PrfA by a putative cofactor is suggested by
its structure, which resembles that of the cyclic-AMP
receptor62,63. The number of mechanisms that regulate
PrfA is probably indicative of the importance of this
crucial virulence factor during infection.
Listeriolysin O: a pH-sensing protein. L. monocytogenes
thrives in the cytoplasm of numerous cell types. Following
internalization, bacteria escape from membrane-bound
phagosomes by secreting two phospholipases, PlcA and
PlcB, and the pore-forming toxin listeriolysin O (LLO),
thereby gaining access to the cytoplasm (FIG. 1).
Although LLO is a member of a large family of
cholesterol-dependent cytolysins that are secreted by
numerous Gram-positive bacteria, L. monocytogenes
is the only pathogen that secretes this type of toxin
inside the host cell. Therefore, secretion of LLO must
VOLUME 4 | JUNE 2006 | 429
REVIEWS

Premature In addition to its pore-forming ability, LLO has the

unfolding surprising ability to induce potent signalling in the host
cell. It has been shown to activate NF-κB67, MAPK68,
phosphatidylinositol 69,70, calcium 71–73 and protein-
Neutral kinase-C 74 signalling pathways. As a pore-forming
pH toxin with cholesterol as the only identified ligand, this
signalling function is puzzling. LLO could have a signal-
Soluble ling function at neutral pH, as has been suggested for
monomer
Formation of another member of the cholesterol-dependent-cytolysin
D1 Membrane-bound Pre-pore the pre-insertion family, Streptococcus intermedius intermedilysin, which
monomers oligomer β-sheet binds the host cellular receptor CD59 (REF. 75). However,
it has been difficult to separate the signalling function of
D2 D3 Pore
oligomer LLO from its pore-forming ability. For example, MAPK
activation occurs on treatment of host cells with LLO,
Low but it also occurs after exposure to a mild detergent,
D4 pH indicating that signalling could be the result of mem-
brane damage rather than a specific activity of LLO68.
Clearly, the signalling activity of LLO requires further
Out investigation.

A model of bacterial adaptation

In
Figure 6 | Listeriolysin O (LLO) pore-forming mechanism. At low (acidic) pH, the
soluble LLO monomer interacts with the host-cell plasma membrane, presumably by
binding cholesterol. On contact with the membrane, structural rearrangements in one
monomer expose residues that can form hydrogen bonds with other monomers, thereby
allowing oligomerization into a pre-pore complex. Following oligomerization, two
α-helical bundles from each monomer extend to form transmembrane β-hairpins (red)
that punch through the membrane. Pores formed by LLO and other cholesterol-binding
proteins can be 250–300 Å in diameter. At neutral pH, domain 3 (D3) of the monomer
prematurely unfolds, rendering the protein unable to form pores. This figure is modified
with permission from REF. 115  (2005) American Society for Microbiology.
be tightly regulated, because the bacterium needs to
balance efficient escape from the vacuole against pre-
vention of host-cell damage to allow its intracellular
survival.
Unlike other pore-forming toxins, the activity of
LLO is optimal at acidic pH (<6). So, LLO is fully active
in the acidic environment of the phagosome, but it is
less active at the neutral pH of the host-cell cytoplasm.
An L. monocytogenes strain that was constructed to
express the pore-forming toxin perfringolysin O, which
is active at neutral pH, can also escape the vacuole
but is toxic to the host cell and is avirulent in mice,
Two-component regulatory highlighting the importance of pH regulation of LLO
system activity 64. The molecular mechanism that underlies
A two-protein signal-
transduction system that is
the optimum pH for LLO activity was only recently
important for the bacterial identified: LLO was found to be stable at acidic pH,
response to environmental and to unfold and aggregate at neutral pH65 (FIG. 6). At
changes. It consists of a acidic pH, pore formation occurs when LLO monomers
membrane-bound sensor
oligomerize into large complexes that penetrate the
protein kinase and a
transcriptional-response membrane by extending transmembrane β-hairpins66.
regulator. At neutral pH, acidic amino-acid residues in the trans-
membrane domain initiate irreversible denaturation of
Elongation factor the β-hairpins of LLO, leading to inactivation of the
A protein that allows tRNAs
to bind the ribosome and is
pore-forming function of LLO65. The elaborate struc-
essential for elongation of the ture of LLO therefore allows this protein to sense and
polypeptide chain. respond to its environment.
One particularly interesting feature of L. monocytogenes
is its capacity to thrive both inside and outside the host.
This ability to colonize a broad range of ecosystems is
reflected in the genome of the organism, which contains an
unusually large number of regulatory and transport pro-
teins (11.6% of all predicted genes)76. With one-quarter
of its transport proteins dedicated to carbohydrate
transport, L. monocytogenes has the largest number of
phosphotransferase systems that have been described
for bacteria so far. Furthermore, the genome contains 16
putative two-component regulatory systems, more than are
found in other pathogenic bacteria and comparable to
the number found in ubiquitous bacteria such as Bacillus
subtilis. Inactivation of several of these two-component
regulatory systems has revealed their importance for
the resistance of L. monocytogenes to various stresses
and for survival in the host77–82. In addition to these
conventional bacterial two-component regulatory sys-
tems, which phosphorylate histidine and aspartic-acid
residues, L. monocytogenes also uses a eukaryotic-like
serine/threonine protein phosphatase system, known as
Stp, to regulate the translation elongation factor EF-Tu
and, consequently, virulence83.
Outside the host, L. monocytogenes is particularly
well adapted to grow at low temperatures, making it
difficult for the food industry to rely on refrigeration
to control listerial contamination84–87. More remark-
able is the ability of L. monocytogenes to survive in
the host. The resistance of L. monocytogenes to acidic
conditions88,89 and to bile salts90–92 makes this patho-
gen particularly adept at infecting the gastrointestinal
tract. Consistent with the many bile-resistance genes
in the L. monocytogenes genome, a recent study using
in vivo bioluminescence imaging (a non-invasive pro-
cedure that allows bioluminescent L. monocytogenes
to be traced through the body of a mouse) has shown
an intriguing new reservoir for L. monocytogenes, the
gall bladder, where bile is stored and concentrated93.
Whether this organ is indeed colonized during human
listeriosis is unknown.

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 REVIEWS

InlG 490 identified: the sugar-uptake system (Hpt) and lipoate

protein ligase A1 (LplA1). Hpt (which is homologous
InlE 499
to the mammalian translocase of glucose-6-phosphate)
Lmo1136 539 is required for optimal intracellular replication 95.
InlH 548 Interestingly, transcription of hpt is regulated by PrfA
Lmo0610 589 and is therefore activated upon entry into the cytosol of
Lmo1289 593 the host cell. Expression of Hpt allows the bacterium to
use glucose-1-phosphate, which is an available carbon
Lmo1290 598
source in the host-cell cytoplasm. Other bacterial patho-
Lmo0514 605 gens such as Escherichia coli, Salmonella enterica, Shigella
Lmo2026 626 flexneri and Chlamydia trachomatis have a homologue
LPXTG

Lmo0331 633 of hpt, indicating that this transporter system could have
Lmo0732 638 a general role in intracellular survival. The other factor
that has been identified to be important for intracellular
Lmo0801 646
growth, LplA1, catalyses the formation of a covalent
InlA 800 link between lipoic acid and specific protein targets96.
InIF 821 A mutant that lacks LplA1 cannot replicate in the cyto-
Lmo0171 832 plasm of macrophages and has a 300-fold decrease in
InlJ 851 virulence96. LplA1 seems to be important for lipoylation
and full activity of the pyruvate-dehydrogenase enzyme
Lmo2396 940
complex in the host-cell cytosol, where lipoic acid is
Lmo0327 1349 mainly absent. LplA1 has therefore been proposed to be
InlI important for the scavenging of lipoyl groups from host
1778 molecules.
GW

InlB 630
Interestingly, several reports indicate that the expres-
sion of many L. monocytogenes genes is upregulated in
InlC 296 the cytosol. A library of Tn917-lacZ L. monocytogenes
mutants was screened for higher lacZ expression when
Lmo2445 300
present in the cytosol of macrophages than when grown
Secreted

Lmo2027 367 in rich broth. This approach allowed the identification

Lmo2470 388
Lmo0549 673
Signal peptide LPXTG motif LRR region Sorting signal
Conserved inter-repeat region GW module
Figure 7 | The internalin family of proteins. Homologous regions of internalin-family
members include an N-terminal signal-peptide sequence, followed by several leucine-
rich tandem repeats (LRRs). Several internalins contain an inter-repeat region, which is
structurally related to an immunoglobulin-like domain. The internalin family can be
divided into three subfamilies on the basis of their association with bacteria. The first
subfamily includes internalins that contain an LPXTG amino-acid motif (where X denotes
any amino acid), and these are covalently anchored to the cell wall. The second subfamily
includes internalin B (InlB), which is loosely associated with the bacterial surface through
its GW modules (which contain the amino-acid motif GW). The third subfamily contains
five internalins that are predicted to be secreted, because they do not have any surface-
anchoring domains. It should be noted that the number of LRRs present in the proteins
Lmo2445 and Lmo2470 is unclear, because these proteins contain degenerate LRRs.
The number of amino acids in each protein is indicated. This figure is modified with
permission from REF. 101  (2002) Elsevier.
Survival and replication in the cytosol of many types
of host cells, including macrophages and epithelial cells,
is one of the distinguishing features of infection with
L. monocytogenes3,4. Other extracellular bacteria and
intracellular pathogens that normally reside inside a
vacuole cannot replicate in the cytosol of the host cell94,
highlighting that L. monocytogenes has evolved specific
mechanisms to grow in the host-cell cytosol. Although
intracytosolic survival remains poorly understood,
two key proteins for intracellular growth have been
of several L. monocytogenes genes, including plcA and
prfA, that are activated after infection of host cells.
Recently, two transcriptomic studies have advanced
the understanding of adaptation to the host-cell cytosol
by showing that L. monocytogenes turns on ~500 genes
for survival in this cellular compartment98,99. Further
analysis of the genes that were identified could
elucidate the metabolism of this bacterium in the
host-cell cytosol.
Genomics reveals new virulence factors
Major listerial virulence factors have been identified
by classical genetics; that is, the search for a mutant
with a particular phenotype, followed by gene iden-
tification and characterization 1. The genetic basis
of L. monocytogenes virulence can now be deci-
phered by exploitation of the genome sequences
of L. monocytogenes and the closely related non-
pathogenic species Listeria innocua76, using three main
strategies: first, the analysis of genes that are present
in L. monocytogenes and absent from L. innocua;
second, the study of genes that encode potentially
interesting proteins; and third, the use of whole-
genome DNA arrays to screen a large population of
strains from different Listeria species and to identify
genes that are present in, and specific to, all virulent
L. monocytogenes strains100.
A first glance at the global genome structure indi-
cates that, in contrast to many other bacterial genomes,
the L. monocytogenes genome (strain EGD-e) contains
only three insertion sequences and five bacteriophages,

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© 2006 Nature Publishing Group
REVIEWS

Chaperone
A protein that assists other
proteins to fold correctly.
none of which seems to have a role in acquisition of
virulence genes. Moreover, except for genes of the
major virulence locus, virulence genes seem to be dis-
persed on the chromosome and are not concentrated in
pathogenicity islands. One of the most striking features
of the L. monocytogenes genome with respect to virulence
is the exceptionally large number of genes that encode
surface proteins (4.7% of all predicted genes101). These
proteins are among the most likely candidates to interact
with the host and therefore to be virulence factors. These
proteins can be divided into three families. The largest
family is composed of 68 lipoproteins, and the second
largest family contains 41 LPXTG proteins (including
InlA), which are anchored to the cell wall by a sortase (an
enzyme that is involved in the covalent linkage of Gram-
positive bacterial proteins to the bacterial surface)102–104.
Inactivation of sortase A (SrtA) or Lsp, a signal peptidase
that is involved in maturation of lipoproteins, attenu-
ates virulence, thereby pinpointing the importance of
these two classes of surface protein in L. monocytogenes
infection103,105,106. The third family of surface proteins
includes proteins that are non-covalently attached to
the bacterial surface by their C-terminal domains. This
family includes GW proteins (such as InlB and Ami),
which contain modules of 80 amino acids that contain
the dipeptide Gly–Trp (also known as GW modules107)
in their C-terminal region. An important outcome from
genome-sequence analysis was the discovery of a SecA2-
dependent pathway, which allows proteins that lack a
typical signal-peptide sequence to be targeted to the
bacterial surface or to be secreted108.
Comparison of the L. monocytogenes and L. innocua
genomes has proved an efficient approach to identify new
virulence factors, including bile-salt hydrolase (Bsh)90,
two LPXTG proteins (Vip and InlJ) and a GW protein
(Auto)105,109,110. The GW protein Auto is a novel surface-
associated cell-wall hydrolase that is required for entry
into eukaryotic cells. Its contribution to listerial infection
is still unclear but might also be linked to the release of
immunologically active cell-wall components that could
interact with components of the innate immune sys-
tem109. The LPXTG protein Vip, which is positively regu-
lated by the transcriptional activator PrfA, is required for
bacterial entry into some eukaryotic cells in vitro and
for the infectious process in vivo110. Vip interacts with
the host-cell endoplasmic-reticulum chaperone gp96, a
protein that is involved in Toll-like-receptor signalling111.
The LPXTG protein InlJ, the function of which remains
to be elucidated, is a leucine-rich-repeat-containing
protein that is structurally related to InlA and InlB105.
These three proteins are members of the internalin
family, which is a large family of proteins that contain
leucine-rich repeats (FIG. 7). The gene that encodes InlJ,
similar to the genes that encode five other proteins of
the internalin family (InlA, InlB, InlE, InlH and InlI),
is conserved in the genomes of pathogenic serovars
of L. monocytogenes (that is, 1/2a, 1/2b, 1/2c and 4b)
and absent from all other Listeria species100,105, and this
is consistent with a role for InlJ in virulence.
Conclusions and future perspectives
The study of L. monocytogenes infection highlights the
sophisticated relationship between this bacterium and its
host, and reveals their long co-evolution and reciprocal
adaptation. For decades, L. monocytogenes has been a tool
for immunologists. It has now also become a paradigm in
bacterial pathogenesis and cellular microbiology.
The availability of the genome sequence of five
L. monocytogenes serovars76,100,112 and L. innocua76, and
soon of Listeria ivanovii and Listeria welshimeri, will pro-
vide further insight into the molecular basis of the patho-
genesis determinants of Listeria species. Comparative
genomics could also reveal genetic loci that confer
specific pathogenic traits to epidemic strains: for exam-
ple, loci that facilitate adaptation to different environ-
ments and that influence the onset of infection. It should
be noted that it is often difficult to unravel the function
of virulence factors that are identified by post-genomic
methods and reverse genetics. To this end, more sophisti-
cated in vivo studies — such as measurement of cytokine
production, non-invasive in vivo imaging techniques or
infection of ex vivo tissue explants — need to be used to
understand the role of these bacterial factors in the inter-
play between the bacterium and the host. Applications
of these novel techniques to the study of Listeria species
will probably continue to show that this bacterium is an
invaluable model in the fields of cellular microbiology,
bacterial pathogenesis and cell biology.

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Acknowledgements
Our sincere apologies to colleagues whose work has not been
cited in this manuscript because of space constraints and
because of the focus of this Review, which does not claim to
be comprehensive. We thank E. Gouin for critical assessment
of the manuscript and E. Veiga for help with FIG. 2. Work in
the laboratory of P.C. is supported by the Institut National de
la Recherche Agronomique, the Institut Pasteur, the Institut
National de la Santé et de la Recherche Médicale, the
Ministère de l’Education Nationale et de la Recherche
Scientifique et Technique, and the Association par la
Recherche sur le Cancer. M.H. is supported by a Pasteur
Foundation fellowship. P.C. is an international research
scholar of the Howard Hughes Medical Institute.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Bacillus subtilis | Burkholderia pseudomallei | Chlamydia
trachomatis | Escherichia coli | Listeria innocua | Listeria
ivanovii | Listeria monocytogenes | Listeria welshimeri | Shigella
flexneri | Yersinia pestis
UniProtKB: http://ca.expasy.org/sprot
InlA | InlB | LLO | PrfA
FURTHER INFORMATION
Bacteria–Cells Interactions Unit, Institut Pasteur:
http://www.pasteur.fr/recherche/unites/uibc/welcome.html
ListiList: http://genolist.pasteur.fr/ListiList/
Microbiology in Motion:
http://www.nature.com/nrmicro/animation/index.html
The Prokaryotes: An Evolving Electronic Resource for the
Microbiological Community:
http://link.springer-ny.com/link/service/books/10125/
SUPPLEMENTARY INFORMATION
See online article: S1 (figure) | S2 (figure) | S3 (figure)
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