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Mutations in mitochondrial genes impairing energy production cause mitochondrial diseases (MDs), and clinical studies have shown that MD patients are prone to bacterial infections. However, the relationship between mitochondrial (dys)function and infection remains largely unexplored, especially in epithelial cells, the first barrier to many pathogens. Here, we generate an epithelial cell model for one of the most common mitochondrial diseases, Leigh syndrome, by deleting surfeit locus protein 1 (SURF1), an assembly factor for respiratory chain complex IV. We use this genetic model and a complementary, nutrient-based approach to modulate mitochondrial respiration rates and show that impaired mitochondrial respiration favors entry of the human pathogen Listeria monocytogenes, a well-established bacterial infection model. Reversely, enhanced mitochondrial energy metabolism decreases infection efficiency. We further demonstrate that endocytic recycling is reduced in mitochondrial respiration-dependent cells, dampening L. monocytogenes infection by slowing the recycling of its host cell receptor c-Met, highlighting a previously undescribed role of mitochondrial respiration during infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: To investigate the contribution to virulence of the surface protein internalin B (InlB) in the Listeria monocytogenes lineage I strain F2365, which caused a deadly listeriosis outbreak in California in 1985., Methods: The F2365 strain displays a point mutation that hampers expression of InlB. We rescued the expression of InlB in the L. monocytogenes lineage I strain F2365 by introducing a point mutation in the codon 34 (TAA to CAA). We investigated its importance for bacterial virulence using in vitro cell infection systems and a murine intravenous infection model., Results: In HeLa and JEG-3 cells, the F2365 InlB + strain expressing InlB was ≈9-fold and ≈1.5-fold more invasive than F2365, respectively. In livers and spleens of infected mice at 72 hours after infection, bacterial counts for F2365 InlB + were significantly higher compared to the F2365 strain (≈1 log more), and histopathologic assessment showed that the F2365 strain displayed a reduced number of necrotic foci compared to the F2365 InlB + strain (Mann-Whitney test)., Conclusions: InlB plays a critical role during infection of nonpregnant animals by a L. monocytogenes strain from lineage I. A spontaneous mutation in InlB could have prevented more severe human morbidity and mortality during the 1985 California listeriosis outbreak., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Listeria monocytogenes is a pathogenic bacterium that invades epithelial cells by activating host signaling cascades, which promote bacterial engulfment within a phagosome. The pore-forming toxin listeriolysin O (LLO), which is required for bacteria phagosomal escape, has also been associated with the activation of several signaling pathways when secreted by extracellular bacteria, including Ca2+ influx and promotion of L. monocytogenes entry. Quantitative host surfaceome analysis revealed significant quantitative remodeling of a defined set of cell surface glycoproteins upon LLO treatment, including a subset previously identified to play a role in the L. monocytogenes infection process. Our data further shows that the lysosomal-associated membrane proteins LAMP-1 and LAMP-2 are translocated to the cellular surface and those LLO-induced Ca2+ fluxes are required to trigger the surface relocalization of LAMP-1. Finally, we identify late endosomes/lysosomes as the major donor compartments of LAMP-1 upon LLO treatment and by perturbing their function, we suggest that these organelles participate in L. monocytogenes invasion.

Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent to which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we used proteomics to analyse mutants of the intracellular pathogen Listeria monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions, such as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried loss-of-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in a large series of new inlG or vip mutants constructed for this purpose were, however, unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using common genetic procedures or during subculturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components.

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment.

Listeria monocytogenes is a bacterial pathogen that promotes its internalization into host epithelial cells. Interaction between the bacterial surface molecules InlA and InlB and their cellular receptors E-cadherin and Met, respectively, triggers the recruitment of endocytic effectors, the subversion of the phosphoinositide metabolism, and the remodeling of the actin cytoskeleton that lead to bacterial engulfment. Additional bacterial surface and secreted virulence factors also contribute to entry, albeit to a lesser extent. Here we review the increasing number of signaling effectors that are reported as being subverted by L. monocytogenes during invasion of cultured cell lines. We also update the current knowledge of the early steps of in vivo cellular infection, which, as shown recently, challenges previous concepts generated from in vitro data.

Septins are conserved GTPases that form filaments and are required for cell division. During interphase, septin filaments associate with cellular membrane and cytoskeleton networks, yet the functional significance of these associations have, to our knowledge, remained unknown. We recently discovered that different septins, SEPT2 and SEPT11, regulate the InlB-mediated entry of Listeria monocytogenes into host cells. Here we address the role of SEPT2 and SEPT11 in the InlB-Met interactions underlying Listeria invasion to explore how septins modulate surface receptor function. We observed that differences in InlB-mediated Listeria entry correlated with differences in Met surface expression caused by septin depletion. Using atomic force microscopy on living cells, we show that septin depletion significantly reduced the unbinding force of InlB-Met interaction and the viscosity of membrane tethers at locations where the InlB-Met interaction occurs. Strikingly, the same order of difference was observed for cells in which the actin cytoskeleton was disrupted. Consistent with a proposed role of septins in association with the actin cytoskeleton, we show that cell elasticity is decreased upon septin or actin inactivation. Septins are therefore likely to participate in anchorage of the Met receptor to the actin cytoskeleton, and represent a critical determinant in surface receptor function., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Listeria monocytogenes is a facultative intracellular Gram-positive bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The L. monocytogenes surface protein InlB interacts with c-Met, the hepatocyte growth factor (HGF) receptor, inducing bacterial internalization in numerous non-phagocytic cells. As InlB and HGF are known to trigger similar signaling pathways upon c-Met activation, we investigated the role of CD44, and more specifically its isoform CD44v6, in bacterial internalization in non-phagocytic cells. Indeed, CD44, the hyaluronic acid transmembrane receptor, and more specifically its isoform CD44v6 have been reported as necessary for the activation of c-Met upon the interaction with either the endogenous ligand HGF or the L. monocytogenes surface protein InlB. Our results demonstrate that, in the cell lines that we used, CD44 receptors play no role in the activation of c-Met, neither during L. monocytogenes entry, nor upon HGF activation. Furthermore, none of the CD44 isoforms was recruited at the L. monocytogenes entry site, and depletion by siRNA of total CD44 or of CD44v6 isoform did not reduce bacterial infections. Conversely, the overexpression of CD44 or CD44v6 had no significant effect on L. monocytogenes internalization. Together our results reveal that the activation of c-Met can be largely CD44-independent., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Many bacterial pathogens that invade non-phagocytic cells first interact with host cell surface receptors. Adhesion to the host cell is followed by the activation of specific host signalling pathways that mediate bacterial internalization. The food-borne Gram-positive bacterium Listeria monocytogenes makes use of two surface proteins, internalin (InlA) and InlB to engage in a species-specific manner the adhesion molecule E-cadherin and the hepatocyte growth factor receptor Met, respectively, to induce its internalization. After entry, Listeria has the capacity to spread from cell to cell and disseminate to its target organs after breaching the intestinal, blood-brain and placental barriers in human. InlA but not InlB is critical for the crossing of the intestinal barrier, whereas the conjugated action of both InlA and InlB mediates the crossing of the placental barrier. Here we review the InlA-E-cadherin interaction, the signalling downstream of this interaction, the molecular mechanisms involved in bacterial internalization and the role of InlA-E-cadherin interaction in the breaching of host barriers and the progression to listeriosis. Together, this review illustrates how in vitro data were validated by epidemiological approaches and in vivo studies using both natural hosts and genetically engineered animal models, thereby elucidating key issues of listeriosis pathophysiology., (© 2009 Blackwell Publishing Ltd.)

Septins are filament-forming GTPases implicated in several cellular functions, including cytokinesis. We previously showed that SEPT2, SEPT9, and SEPT11 colocalize with several bacteria entering into mammalian non-phagocytic cells, and SEPT2 was identified as essential for this process. Here, we investigated the function of SEPT11, an interacting partner of SEPT9 whose function is still poorly understood. In uninfected HeLa cells, SEPT11 depletion by siRNA increased cell size but surprisingly did not affect actin filament formation or the colocalization of SEPT9 with actin filaments. SEPT11 depletion increased Listeria invasion, and incubating SEPT11-depleted cells with beads coated with the Listeria surface protein InlB also led to increased entry as compared with control cells. Strikingly, as shown by fluorescence resonance energy transfer, the InlB-mediated stimulation of Met signaling remained intact in SEPT11-depleted cells. Taken together, our results show that SEPT11 is not required for the bacterial entry process and rather restricts its efficacy. Because SEPT2 is essential for the InlB-mediated entry of Listeria, but SEPT11 is not, our findings distinguish the roles of different mammalian septins.

The ability to cross host barriers is an essential virulence determinant of invasive microbial pathogens. Listeria monocytogenes is a model microorganism that crosses human intestinal and placental barriers, and causes severe maternofetal infections by an unknown mechanism. Several studies have helped to characterize the bacterial invasion proteins InlA and InlB. However, their respective species specificity has complicated investigations on their in vivo role. Here we describe two novel and complementary animal models for human listeriosis: the gerbil, a natural host for L. monocytogenes, and a knock-in mouse line ubiquitously expressing humanized E-cadherin. Using these two models, we uncover the essential and interdependent roles of InlA and InlB in fetoplacental listeriosis, and thereby decipher the molecular mechanism underlying the ability of a microbe to target and cross the placental barrier.

How invasive bacteria exploit mammalian host cell components to induce their entry into cells has received a lot of attention in the last two decades. Model organisms have emerged and helped understanding the various mechanisms that are used. Among those, Listeria monocytogenes is one of the most documented organisms. It enters into cells via two bacterial proteins, internalin (also called InlA) and InlB, which interact with cell surface receptors, E-cadherin and the hepatocyte growth factor receptor, Met, respectively. These interactions initiate a series of events that leads to actin polymerization, membrane invagination and bacterial internalization. Investigations on internalin- and InlB-mediated entries have repeatedly shown that Listeria fully usurps the host cell machinery. Moreover, they have also shown that previously unknown components discovered during the study of Listeria invasion play a role either in E-cadherin-mediated cell-cell adhesion or Met signalling. Unexpectedly, recent studies have highlighted a role for clathrin in Listeria InlB-mediated actin polymerization and entry, revealing a new role for this endocytic protein, i.e. in bacterial-induced internalization. Furthermore, comparative studies have demonstrated that the clathrin-mediated endocytosis machinery is also used in the internalin-E-cadherin pathway, and for the entry of other bacteria that enter by a 'zipper' mechanism. By contrast, the clathrin-mediated endocytic machinery is not used by bacteria that inject effectors into mammalian cells via the type III secretion system and enter by the so-called trigger mechanism, characterized by enormous membrane ruflles that result in the macropinocytosis of the corresponding bacteria. Finally, adherent bacteria, for example enteropathogenic Escherichia coli (EPEC), also co-opt clathrin to induce the formation of actin-rich pedestals. Together, these new data illuminate our view on how actin rearrangements may be coupled to clathrin recruitment during bacterial infection. They also shed light on a new function for clathrin in mammalian cells, i.e. internalization of objects much larger than previously accepted.

Listeria monocytogenes cross the intestinal barrier causing systemic infections with high mortality rates. Intestinal infection triggers release of intestinal mucus. We show that three L. monocytogenes internalins, InlB, InlC and InlJ all bound to MUC2 (the major component of intestinal mucus), but not to the cell surface mucin MUC1. Binding was strongest to InlB>InlC>InlJ (P < 0.001). Listerial internalins are characterized by their internalin domain, composed by leucine rich repeats (LRR) followed by an immunogloblin-like region. We report here that the internalin domain of the InlJ protein also bound MUC2, suggesting that an internalin domain is sufficient to bind to MUC2.

Interaction of the Listeria surface protein InlB with the hepatocyte growth factor receptor Met activates signalling events that trigger bacterial internalization into mammalian epithelial cells. We show here that purified phagosomes containing InlB-coated beads display type II phosphatidylinositol 4-kinase (PI4K) activity. In human epithelial HeLa cells, both PI4KIIalpha and PI4KIIbeta isoforms are corecruited with Met around InlB-coated beads or wild-type Listeria during the early steps of internalization, and phosphatidylinositol 4-phosphate [PI(4)P] is detected at the entry site. We demonstrate that PI4KIIalpha or PI4KIIbeta knockdown, but not type III PI4Kbeta knockdown, inhibits Listeria internalization. Production of PI(4)P derivatives such as phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P(3)] upon InlB stimulation is not affected by PI4KIIalpha or beta knockdown, suggesting that these phosphoinositides are generated by a type III PI4K. Strikingly, knockdown of the PI(4)P ligand and clathrin adaptor AP-1 strongly inhibits bacterial entry. Together, our results reveal a yet non-described role for type II PI4Ks in phagocytosis.

InlB, a surface protein of the human bacterial pathogen Listeria monocytogenes, interacts with the receptor tyrosine kinase Met on host cells to enable bacterial invasion. In this issue, Niemann et al. (2007) provide the first structural evidence that InlB does not compete for the same interaction site on Met as the natural ligand HGF.

The genome of the human food-borne pathogen Listeria monocytogenes is predicted to encode a high number of surface proteins. This abundance likely reflects the ability of this bacterium to survive in diverse environments, including soil, food, and the human host. This review focuses on the various mechanisms by which listerial proteins are attached at the bacterial surface and their many functions, including peptidoglycan metabolism, protein processing, adhesion to host cells, and invasion of host tissues. Extensive in silico analysis of the domains or motifs present in these mosaic proteins reveals that diverse structural features allow the surface proteome to interact with diverse bacterial or host components. This diversity offers new clues about the molecular bases of Listeria pathogenesis.

The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.

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.

InlA and InlB mediate L. monocytogenes entry into eukaryotic cells. InlA is required for the crossing of the intestinal and placental barriers. InlA uses E-cadherin as receptor in a species-specific manner. The human E-cadherin but not the mouse E-cadherin is a receptor for InlA. In human cells, InlB uses Met and gC1qR as receptors. By studying the role of InlB in vivo, we found that activation of Met by InlB is species-specific. In mice, InlB is important for liver and spleen colonization, but not for the crossing of the intestinal epithelium. Strikingly, the virulence of a DeltainlB deletion mutant is not attenuated in guinea pigs and rabbits. Guinea pig and rabbit cell lines do not respond to InlB, although expressing Met and gC1qR, but support InlB-mediated responses upon human Met gene transfection, indicating that InlB does not recognize or stimulate guinea pig and rabbit Met. In guinea pig cells, the effect of human Met gene transfection on InlB-dependent entry is increased upon cotransfection with human gc1qr gene, showing the additive roles of gC1qR and Met. These results unravel a second L. monocytogenes species specificity critical for understanding human listeriosis and emphasize the need for developing new animal models for studying InlA and InlB functions in the same animal model.

Listeria monocytogenes expresses surface proteins covalently anchored to the peptidoglycan by sortase enzymes. Inactivation of srtA attenuates Listeria virulence in mice (H. Bierne, S. K. Mazmanian, M. Trost, M. G. Pucciarelli, G. Liu, P. Dehoux, L. Jansch, F. Garcia-del Portillo, O. Schneewind, and P. Cossart, Mol. Microbiol. 43:869-881, 2002). We show here that an srtA mutant is more attenuated than an internalin mutant in orally infected guinea pigs and transgenic mice expressing human E-cadherin (hEcad mice), indicating the involvement of other SrtA substrates, LPXTG proteins, in food-borne listeriosis. Data recently generated with a listerial DNA macroarray identified two LPXTG protein-encoding genes present in the genomes of L. monocytogenes strains and absent from all other Listeria species, inlI (lmo0333) and inlJ (lmo2821). They also revealed two other LPXTG protein-encoding genes, ORF29 and ORF2568, present only in a subclass of L. monocytogenes serovars, including the epidemic serovar 4b. We report here that an inlJ deletion mutant, in contrast to inlI and ORF29 mutants, is significantly attenuated in virulence after intravenous infection of mice or oral inoculation of hEcad mice. Interestingly, a DeltaORF2568 strain showed a slight increase in virulence. inlJ encodes a leucine-rich repeat (LRR) protein that is structurally related to the listerial invasion factor internalin. However, the consensus sequence of the InlJ LRR defines a novel subfamily of cysteine-containing LRRs in bacteria. In conclusion, this postgenomic approach identified InlJ as a new virulence factor among the proteins belonging to the internalin family in L. monocytogenes.

By comparative genomics, we have identified a gene of the intracellular pathogen Listeria monocytogenes that encodes an LPXTG surface protein absent from nonpathogenic Listeria species. This gene, vip, is positively regulated by PrfA, the transcriptional activator of the major Listeria virulence factors. Vip is anchored to the Listeria cell wall by sortase A and is required for entry into some mammalian cells. Using a ligand overlay approach, we identified a cellular receptor for Vip, the endoplasmic reticulum (ER) resident chaperone Gp96 recently shown to interact with TLRs. The Vip-Gp96 interaction is critical for bacterial entry into some cells. Comparative infection studies using oral and intravenous inoculation of nontransgenic and transgenic mice expressing human E-cadherin demonstrated a role for Vip in Listeria virulence, not only at the intestine level but also in late stages of the infectious process. Vip thus appears as a new virulence factor exploiting Gp96 as a receptor for cell invasion and/or signalling events that may interfere with the host immune response in the course of the infection.

Listeria monocytogenes is a bacterial pathogen with the capacity to invade non-phagocytic cells. This dynamic process involves coordinated membrane remodelling and actin cytoskeleton rearrangements. Although some of the molecular factors promoting these events have been identified, the driving force allowing internalization is unknown. One of the receptors for L. monocytogenes on epithelial cells is E-cadherin, a transmembrane protein normally involved in homophilic interactions that allow cell-cell contacts at the adherens junctions. E-cadherin has to be connected to the actin cytoskeleton to mediate strong cell-cell adhesion and to trigger Listeria entry; alpha- and beta-catenins play key roles in these processes. We have recently identified an unconventional myosin, myosin VIIa and its ligand vezatin, at the adherens junctions of polarized epithelial cells. Here, we demonstrate by pharmacological and genetic approaches that both myosin VIIa and vezatin are crucial for Listeria internalization. These results provide the first evidence for the role of an unconventional myosin in bacterial internalization and a novel example of the exploitation of mammalian proteins, by a pathogen, to establish a successful infection.

The Listeria monocytogenes protein InlB promotes invasion of mammalian cells through activation of the receptor tyrosine kinase Met. The InlB N-cap, a approximately 40 residue part of the domain that binds Met, was previously observed to bind two calcium ions in a novel and unusually exposed manner. Because subsequent work raised questions about the existence of these calcium-binding sites, we assayed calcium binding in solution to the InlB N-cap. We show that calcium ions are bound with dissociation constants in the low micromolar range at the two identified sites, and that the sites interact with one another. We demonstrate that the calcium ions are not required for structure, and also find that they have no appreciable effect on Met activation or intracellular invasion. Therefore, our results indicate that the sites are fortuitous in InlB, but also suggest that the simple architecture of the sites may be adaptable for protein engineering purposes.

Sortases are transamidases that covalently link proteins to the peptidoglycan of gram-positive bacteria. The genome of the pathogenic bacterium Listeria monocytogenes encodes two sortases genes, srtA and srtB. The srtA gene product anchors internalin and some other LPXTG-containing proteins to the listerial surface. Here, we focus on the role of the second sortase, SrtB. Whereas SrtA acts on most of the proteins in the peptidoglycan fraction, SrtB appears to target minor amounts of surface polypeptides. We identified one of the SrtB-anchored proteins as the virulence factor SvpA, a surface-exposed protein which does not contain the LPXTG motif. Therefore, as in Staphylococcus aureus, the listerial SrtB represents a second class of sortase in L. monocytogenes, involved in the attachment of a subset of proteins to the cell wall, most likely by recognizing an NXZTN sorting motif. The DeltasrtB mutant strain does not have defects in bacterial entry, growth, or motility in tissue-cultured cells and does not show attenuated virulence in mice. SrtB-mediated anchoring could therefore be required to anchor surface proteins involved in the adaptation of this microorganism to different environmental conditions.

Listeria monocytogenes is an opportunistic food-borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non-phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection is a key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions. Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB. Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor C1q (gC1q-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans (including heparan sulphate). The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation. Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells, including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway). At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined. Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.

InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion.

Molecules from some pathogenic bacteria mimic natural host cell ligands and trigger engulfment of the bacterium after specifically interacting with cell-surface receptors. The leucine-rich repeat (LRR)-containing protein InlB of Listeria monocytogenes is one such molecule. It triggers bacterial entry by interacting with the hepatocyte growth factor receptor (HGF-R or Met) and two other cellular components: gC1q-R and proteoglycans. Recent studies point to significant similarities between the molecular mechanisms underlying InlB-mediated entry into cells and classic phagocytosis. In addition, InlB, in common with HGF, activates signaling cascades that are not involved in bacterial entry. Therefore, studies of InlB may help us to analyze the previously noticed similarities between growth factor receptor activation and phagocytosis.

On the basis of the recently determined genome sequence of Listeria monocytogenes, we performed a global analysis of the surface-protein-encoding genes. Only proteins displaying a signal peptide were taken into account. Forty-one genes encoding LPXTG proteins, including the previously known internalin gene family, were detected. Several genes encoding proteins that, like InlB and Ami, possess GW modules that attach them to lipoteichoic acids were also identified. Additionally, the completed genome sequence revealed genes encoding proteins potentially anchored in the cell membrane by a hydrophobic tail as well as genes encoding P60-like proteins and lipoproteins. We describe these families and discuss their putative implications for host-pathogen interactions.

Internalization of Listeria monocytogenes into non-phagocytic cells is mediated by the interactions between the two bacterial invasion proteins InlA (internalin) and InlB and their cellular surface receptors E-cadherin and c-Met. To get an insight into all the cellular components necessary for uptake and early intracellular life, we undertook a global proteomic characterization of the early listerial phagosome in the human epithelial cell line LoVo. First, we proceeded to an immunocytochemical characterization of intracellular marker recruitment to phagosomes containing latex beads coated with InlA or InlB. E-cadherin and c-Met were, as expected, rapidly recruited to the phagosomal formation site. Phagosomes subsequently acquired the early endosomal antigen 1 (EEA1) and the lysosomal-associated membrane protein 1 (LAMP1), while presenting a more delayed enrichment of the lysosomal hydrolase cathepsin D. Early phagosomes devoid of lysosomal, endoplasmic reticulum and Golgi enzymatic activities could then be isolated by subcellular fractionation of LoVo cells. Two-dimensional gel electrophoresis (2DPAGE) revealed differences between the protein profiles of InlA- or InlB-phagosomes and those of early/late endosomes or lysosomes of naive LoVo cells. One major protein specifically recruited on the InlB-phagosomes was identified by mass spectrometry as MSF, a previously reported member of the septin family of GTPases. MSF forms filaments that co-localize with the actin cytoskeleton in resting cells and it is recruited to the entry site of InlB-coated beads. These results suggest that MSF is a putative effector of the InlB-mediated internalization of L. monocytogenes into host cells.

Internalin B (InlB), a 630-amino acid protein loosely attached to the surface of Listeria monocytogenes, participates in the entry of the bacterium into mammalian cells. This process requires the activation of phosphoinositide (PI) 3-kinase by InlB. Previously, we demonstrated that InlB activates the transcription factor Nuclear Factor-kappaB in murine J774 macrophage-like cells, an event that also requires PI 3-kinase. Here we have further investigated this phenomenon. InlB activated the small G-protein Ras in J774 cells. Inhibition of Ras with the farnesyltransferase inhibitor manumycin A inhibited NF-kappaB activation and the recruitment of the p85 subunit of PI 3-kinase, implying that Ras is required for PI 3-kinase activation. InlB also activated the PI 3-kinase downstream effector, Akt, as assessed by increased phosphorylation of Akt on serine 473. Transfection of Hep2 cells with dominant negative Ras N17 or dominant negative Akt inhibited the induction of a reporter gene linked to the interleukin-8 promoter by InlB. Furthermore, the Ras inhibitor manumycin A, the PI 3-kinase inhibitor LY294002, and an Akt inhibitor all blocked the induction of interleukin-8 by InlB. Our study is the first report of a bacterial product activating a pathway involving Ras, PI 3-kinase, and Akt, which leads to NF-kappaB activation. This process could be involved in host defense or the inhibition of apoptosis during infection.

InlB is a Listeria monocytogenes protein promoting entry in non-phagocytic cells, and has been shown recently to activate the hepatocyte growth factor receptor (HGFR or Met). The N-terminal domain of InlB (LRRs) binds and activates Met, whereas the C-terminal domain of InlB (GW modules) mediates loose attachment of InlB to the listerial surface. As HGF activation of Met is tightly controlled by glycosaminoglycans (GAGs), we tested if GAGs also modulate the Met-InlB interactions. We show that InlB-dependent invasion of non-phagocytic cells decreases up to 10 times in the absence of GAGs, and that soluble heparin releases InlB from the bacterial surface and promotes its clustering. Furthermore, we demonstrate that InlB binds cellular GAGs by its GW modules, and that this interaction is required for efficient InlB-mediated invasion. Therefore, GW modules have an unsuspected dual function: they attach InlB to the bacterial surface and enhance entry triggered by the LRRs domain. Our results thus provide the first evidence for a synergy between two host factor-binding domains of a bacterial invasion protein, and reinforce similarities between InlB and mammalian growth factors.

Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (41)DEWEEE(46) and basic (146)KKRRK(150) regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.

Entry of the bacterial pathogen Listeria monocytogenes into non-phagocytic mammalian cells is mainly mediated by the InlB protein. Here we show that in the human epithelial cell line HEp-2, the invasion protein InlB activates sequentially a p85beta-p110 class I(A) PI 3-kinase and the phospholipase C-gamma1 (PLC-gamma1) without detectable tyrosine phosphorylation of PLC-gamma1. Purified InlB stimulates association of PLC-gamma1 with one or more tyrosine-phosphorylated proteins, followed by a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels and a release of intracellular Ca2+ in a PI 3-kinase-dependent manner. Infection of HEp-2 cells with wild-type L. monocytogenes bacteria also induces association of PLC-gamma1 with phosphotyrosyl proteins. This interaction is undetectable upon infection with a deltainlB mutant revealing an InlB specific signal. Interestingly, pharmacological or genetic inactivation of PLC-gamma1 does not significantly affect InlB-mediated bacterial uptake, suggesting that InlB-mediated PLC-gamma1 activation and calcium mobilization are involved in post-internalization steps.

Defects in myosin VIIA are responsible for deafness in the human and mouse. The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood. Here we show that the C-terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identified by a yeast two-hybrid screen. Vezatin is a ubiquitous protein of adherens cell-cell junctions, where it interacts with both myosin VIIA and the cadherin-catenins complex. Its recruitment to adherens junctions implicates the C-terminal region of alpha-catenin. Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin-catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell-cell adhesion. In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane-membrane interaction site, namely at the fibrillar links interconnecting the bases of adjacent stereocilia. In myosin VIIA-defective mutants, inactivity of the vezatin-myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia.

InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells.

Listeria monocytogenes causes a pro-inflammatory response on adhesion to macrophages. Upregulation of inflammation genes involves the transcription factor NF-kappaB. Several components of L. monocytogenes, including lipoteichoic acid (LTA), phospholipases and listeriolysin O (LLO), have since been shown to mediate NF-kappaB activation. Here, we report that purified recombinant InlB, but not internalin (InlA), is a potent activator of NF-kappaB in the mouse macrophage-like cell line J774. Expression of InlB in Listeria innocua enhances its ability to activate NF-kappaB, while deletion of InlB from L. monocytogenes marginally decreases its effect on NF-kappaB, possibly because of the presence of NF-kappaB activators such as LTA and LLO. The effect correlates with the rapid degradation of IkappaBalpha, a sustained degradation of IkappaBbeta and increases in tumour necrosis factor alpha (TNF-alpha) and interleukin (IL) 6 production, two cytokines controlled by NF-kappaB. Using a series of anti-InlB monoclonal antibodies and domains of InlB, NF-kappaB activation was shown to be dependent upon the N-terminal 213-amino-acid leucine-rich repeat (LRR) domain of InlB, recently demonstrated to be responsible for InlB-mediated L. monocytogenes invasion and phosphoinositide-3 (PI-3) kinase activation. The effect of InlB was blocked by PI-3 kinase inhibitors, indicating the involvement of PI-3 kinase in this response. This report thus illustrates that InlB not only promotes invasion, but also contributes to the macrophage pro-inflammatory response.

The N-terminal region of the Listeria monocytogenes ActA protein, in conjunction with host cell factors, is sufficient for actin polymerization at the bacterial surface. Previous data suggested that ActA could protect barbed ends from capping proteins. We tested this hypothesis by actin polymerization experiments in the presence of the ActA N-terminal fragment and capping protein. ActA does not protect barbed ends from capping protein. In contrast, this polypeptide prevents PIP(2) from inhibiting the capping activity of capping protein. Gel filtration and tryptophan fluorescence experiments showed that the purified ActA N-terminal fragment binds to PIP(2) and PIP, defining phosphoinositides as novels ligands for this functional domain of ActA. Phosphoinositide binding to the N-terminal region of ActA may induce conformational changes in ActA and/or facilitate binding of other cell components, important for ActA-induced actin polymerization., (Copyright 2000 Wiley-Liss, Inc.)

The L. monocytogenes protein lnlB activates phosphoinositide 3-kinase and induces phagocytosis in several mammalian cell types. The 1.86 A resolution X-ray crystal structure of the leucine-rich repeat domain of lnlB that is both necessary and sufficient to induce phagocytosis is presented here. The structure supports a crucial role for calcium in host cell invasion by L. monocytogenes and supplies a rationale for its function. Calciums are bound to the protein in an unusually exposed manner that suggests that the metals may act as a bridge between lnlB and mammalian cell surface receptors. The structure also identifies surfaces on the curved and elongated molecule that may constitute additional interaction sites in forming a bacterial-mammalian signaling complex.

InlB is a Listeria monocytogenes protein that is sufficient to promote entry in a variety of mammalian cells. The last 232-amino-acid domain (Csa) of InlB has been shown to mediate attachment on the listerial surface, although its sequence does not suggest any known mechanism of association to the bacterial surface. InlB is present both on the bacterial surface and in culture supernatants. As has been recently demonstrated, both forms of InlB, soluble and surface-bound, can trigger signalling in host cells. To elucidate the specific role of each of the two forms, it was important to understand how InlB associates with the bacterial surface. Using microscopy, we find evidence that InlB is partially buried in the cell wall layer, and using fractionation experiments we demonstrate that InlB associates with the bacterial cytoplasmic membrane. Moreover, using purified lipoteichoic acid (LTA) and the three polypeptides InlB, Csa, or InlBDeltaCsa (InlB lacking the last 232 amino acids), we demonstrate that LTA is a ligand for the Csa domain of InlB. These results provide the first evidence of an interaction between lipoteichoic acids and a bacterial protein involved in adhesion and signalling, and highlight a new mechanism of protein association on the surface of Gram-positive bacteria.

The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.

The Gram-positive pathogen Listeria monocytogenes induces its own internalization into some non-phagocytic mammalian cells by stimulating host tyrosine phosphorylation, phosphoinositide (PI) 3-kinase activity, and rearrangements in the actin cytoskeleton. Entry into many cultured cell lines is mediated by the bacterial protein InlB. Here we investigate the role of InlB in regulating mammalian signal transduction and cytoskeletal structure. Treatment of Vero cells with purified InlB caused rapid and transient increases in the lipid products of the PI 3-kinase p85-p110, tyrosine phosphorylation of the mammalian adaptor proteins Gab1, Cbl, and Shc, and association of these proteins with p85. InlB also stimulated large scale changes in the actin cytoskeleton (membrane ruffling), which were PI 3-kinase-dependent. These results identify InlB as the first reported non-mammalian agonist of PI 3-kinase and demonstrate similarities in the signal transduction events elicited by this bacterial protein and known agonists such as epidermal growth factor.

InIB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco-2 requires InIA, whereas InIB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp-2 and HeLa cells. InIB-mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InIB is sufficient to promote internalization. Indeed, coating of normally non-invasive bacteria or inert latex beads with InIB leads to internalization into mammalian cells. In addition, a soluble form of InIB also appears to promote uptake of non-invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes, uptake of InIB-coated beads required tyrosine phosphorylation in the host cell, PI 3-kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InIB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97-126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.

Listeria monocytogenes is an intracellular bacterial pathogen that expresses several surface proteins critical for the infectious process. Such proteins include InlA (internalin) and InlB, involved in bacterial entry into the host cell, and ActA, required for bacterially induced actin-based motility. Although the molecular mechanisms of attachment of InlA and ActA have been characterized, essentially nothing is known about how InlB is anchored to the bacterial surface. Using a genetic approach, we demonstrate that the last 232 amino acids of InlB are both necessary and sufficient for anchoring this protein to the bacterial surface. An InlB mutant protein deleted for the last 232 amino acids was secreted and not detected at the cell surface. A 'domain-swapping' strategy in which these 232 amino acids were used to replace the normal cell wall-anchoring domain of InlA resulted in a chimeric protein that was anchored to the cell surface and able to confer entry. Interestingly, surface association of InlB also occurred when InlB was added externally to bacteria, suggesting that association may be able to occur after secretion. This association was productive for invasion, as it conferred bacterial entry into host cells. The C-terminal anchoring region in InlB contains 80-amino-acid repeats beginning with the sequence GW that is also present in a newly identified surface-associated bacteriolysin of L. monocytogenes, called Ami. Addition of GW repeats to the C-terminal of InlB improves anchoring of the protein to the cell surface. These and other data suggest that such 'GW' repeats may constitute a novel motif for cell-surface anchoring in Listeria and other Gram-positive bacteria. This motif may have important consequences for the release of surface proteins involved in interactions with eukaryotic cells.

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N-terminus of ActA (residues 1-234) to the omega fragment of beta-galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region T, amino acids 117-121) is critical for filament elongation, as shown by the absence of actin tail in a 117-121 deletion mutant or when motility assays are performed in the presence of anti-region T antibodies. The second region (region C, amino acids 21-97), is more specifically involved in maintenance of the continuity of the process, probably by F-actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21-97) leading to 'discontinuous' actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33-74 can interact with F-actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N-terminal domain of ActA.

The intracellular bacterial pathogen Listeria monocytogenes moves inside the host-cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin-driven bacterial propulsion, we have now generated several L. monocytogenes strains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino-terminal part is critical for F-actin assembly and movement. The internal proline-rich repeats and the carboxy-terminal domains are not essential. However, in vitro motility assays have demonstrated that mutants lacking the proline-rich repeats domain of ActA moved two times slower (6+/-2 micrometers min(-1)) than the wild type (13 +/-3 micrometers min(-1)). In addition, phosphatase treatment of protein extracts of cells infected with the L. monocytogenes strains expressing the ActA variants suggested that phosphorylation may not be essential for ActA activity.

Listeria monocytogenes and Shigella flexneri are two unrelated facultative intracellular pathogens which spread from cell to cell by using a similar mode of intracellular movement based on continuous actin assembly at one pole of the bacterium. This process requires the asymmetrical expression of the ActA surface protein in L. monocytogenes and the IcsA (VirG) surface protein in S. flexneri. ActA and IcsA share no sequence homology. To assess the role of the two proteins in the generation of actin-based movement, we expressed them in the genetic context of two non-actin polymerizing, non-pathogenic bacterial species, Listeria innocua and Escherichia coli. In the absence of any additional bacterial pathogenicity determinants, both proteins induced actin assembly and propulsion of the bacteria in cytoplasmic extracts from Xenopus eggs, as visualized by the formation of characteristic actin comet tails. E. coli expressing IcsA moved about two times faster than Listeria and displayed longer actin tails. However, actin dynamics (actin filament distribution and filament half-lives) were similar in IcsA- and ActA-induced actin tails suggesting that by using unrelated surface molecules, L. monocytogenes and S. flexneri move intracellularly by interacting with the same host cytoskeleton components or by interfering with the same host cell signal transduction pathway.

A gene homologous to the actA gene of Listeria monocytogenes was cloned from Listeria ivanovii (strain CLIP257) by chromosome walking starting from the ilo gene that encodes the pore-forming toxin ivanolysin. The nucleotide sequence revealed that this gene, named iactA, encodes a protein of 1,044 amino acids (IactA) comprising a central region with seven highly conserved tandem proline-rich repeats of 47 amino acids. Although IactA and ActA share an overall similar structure, these two proteins are only distantly related. Like ActA, IactA migrates aberrantly on sodium dodecyl sulfate gels. When expressed in an L. monocytogenes actA deletion mutant strain, iactA restored actin polymerization.

Actin assembly on the surface of Listeria monocytogenes in the cytoplasm of infected cells provides a model to study actin-based motility and changes in cell shape. We have shown previously that the ActA protein, exposed on the bacterial surface, is required for polarized nucleation of actin filaments. To investigate whether plasma membrane-associated ActA can control the organization of microfilaments and cell shape, variants of ActA, in which the bacterial membrane signal had been replaced by a plasma membrane anchor sequence, were produced in mammalian cells. While both cytoplasmic and membrane-bound forms of ActA increased the F-actin content, only membrane-associated ActA caused the formation of plasma membrane extensions. This finding suggests that ActA acts as an actin filament nucleator and shows that permanent association with the inner face of the plasma membrane is required for changes in cell shape. Based on the observation that the amino-terminal segment of ActA and the remaining portion which includes the proline-rich repeats cause distinct phenotypic modifications in transfected cells, we propose a model in which two functional domains of ActA cooperate in the nucleation and dynamic turnover of actin filaments. The present approach is a new model system to dissect the mechanism of action of ActA and to further investigate interactions of the plasma membrane and the actin cytoskeleton during dynamic changes of cell shape.