Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

Entry of the bacterial pathogen Listeria monocytogenes into mammalian cells

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to meningitis or abortion. Listeria provokes its internalization ('entry') into mammalian cells that are normally non-phagocytic, such as intestinal epithelial cells and hepatocytes. Entry provides access to a nutrient-rich cytosol and allows translocation across anatomical barriers. Here I discuss the two major internalization pathways used by Listeria. These pathways are initiated by binding of the bacterial surface proteins InlA or InlB to their respective host receptors, E-cadherin or Met. InlA mediates traversal of the intestinal barrier, whereas InlB promotes infection of the liver. At the cellular level, both InlA- and InlB-dependent entry require host signalling that promotes cytoskeletal rearrangements and pathogen engulfment. However, many of the specific signalling proteins in the two entry routes differ. InlA-mediated uptake uses components of adherens junctions that are coupled to F-actin and myosin, whereas InlB-dependent entry involves cytosolic adaptors that bridge Met to regulators of F-actin, including phosphoinositide 3-kinase and activators of the Arp2/3 complex. Unexpectedly, entry directed by InlB also involves endocytic components. Future work on InlA and InlB will lead to a better understanding of virulence, and may also provide novel insights into the normal biological functions of E-cadherin and Met.     

Src, cortactin and Arp2/3 complex are required for E-cadherin-mediated internalization of Listeria into cells

Listeria monocytogenes is a food-borne pathogen able to invade non-phagocytic cells. InlA, a L. monocytogenes surface protein, interacts with human E-cadherin to promote bacterial entry. L. monocytogenes internalization is a dynamic process involving co-ordinated actin cytoskeleton rearrangements and host cell membrane remodelling at the site of bacterial attachment. Interaction between E-cadherin and catenins is required to promote Listeria entry, and for the establishment of adherens junctions in epithelial cells. Although several molecular factors promoting E-cadherin-mediated Listeria internalization have been identified, the proteins regulating the transient actin polymerization required at the bacterial entry site are unknown. Here we show that the Arp2/3 complex acts as an actin nucleator during the InlA/E-cadherin-dependent internalization. Using a variety of approaches including siRNA, expression of dominant negative derivatives and pharmacological inhibitors, we demonstrate the crucial role of cortactin in the activation of the Arp2/3 complex during InlA-mediated entry. We also show the requirement of the small GTPase Rac1 and that of Src-tyrosine kinase activity to promote Listeria internalization. Together, these data suggest a model in which Src tyrosine kinase and Rac1 promote recruitment of cortactin and activation of Arp2/3 at Listeria entry site, mimicking events that occur during adherens junction formation.     

Listeria monocytogenes internalin and E-cadherin: from structure to pathogenesis

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.     

Designer bugs: structural engineering to build a better mouse model

Many animal models of bacterial diseases are hampered by differences in tissue tropism and the course of pathogenesis. In a recent issue of Cell, by rationally mutating a surface invasion protein (InlA) to have higher binding affinity for its cognate host receptor (E-cadherin), Wollert et al. were able to "murinize"Listeria monocytogenes, creating a strain capable of invading intestinal epithelial cells in mice, mimicking the route of infection in humans.     

Interaction of Listeria monocytogenes with the intestinal epithelium

Listeria monocytogenes is a food-borne pathogen that must cross the intestinal epithelial barrier to reach its target organs. We have investigated the importance of M cells in translocation using an experimental mouse model and a novel, recently described in vitro coculture system that mimics the follicle-associated epithelium (FAE). Our data demonstrate that L. monocytogenes does not require, nor specifically use, M cells of the FAE to cross the gut. We also show that bacterial translocation is rapid and L. monocytogenes can attach very efficiently to exposed basal lamina of the small intestine indicating an important role for extracellular matrix proteins.     

InlA Promotes Dissemination of Listeria monocytogenes to the Mesenteric Lymph Nodes during Food Borne Infection of Mice

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA^m) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA^m), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA^m significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.     

Candida albicans internalization by host cells is mediated by a clathrin-dependent mechanism

Candida albicans is a major cause of oropharyngeal, vulvovaginal and haematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E- or N-cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes . InlA mediates entry of L. monocytogenes into host cells through binding to E-cadherin. A role in internalization, for a non-classical stimulation of the clathrin-dependent endocytosis machinery, was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans . Using live-cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans , we observed that host E-cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N-cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner. These data indicate that C. albicans , like L. monocytogenes, hijacks the clathrin-dependent endocytic machinery to invade host cells.     

Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection isa 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 Clq (gClq-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.     

The metalloprotease of Listeria monocytogenes controls cell wall translocation of the broad-range phospholipase C

Listeria monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from secondary vacuoles. PC-PLC requires cleavage of an N-terminal propeptide for activation, and Mpl, a metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. Previously, we showed that cell wall translocation of PC-PLC is inefficient, resulting in accumulation of PC-PLC at the membrane-cell wall interface. In infected cells, rapid cell wall translocation of PC-PLC is triggered by a decrease in pH and correlates with cleavage of the propeptide in an Mpl-dependent manner. To address the role of the propeptide and of Mpl in cell wall translocation of PC-PLC, we generated a cleavage site mutant and a propeptide deletion mutant. The intracellular behavior of these mutants was assessed in pulse-chase experiments. We observed efficient translocation of the proform of the PC-PLC cleavage site mutant in a manner that was pH sensitive and Mpl dependent. However, the propeptide deletion mutant was efficiently translocated into host cells independent of Mpl and pH. Overall, these results suggest that Mpl regulates PC-PLC translocation across the bacterial cell wall in a manner that is dependent on the presence of the propeptide but independent of propeptide cleavage. In addition, similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediated translocation of PC-PLC across the bacterial cell wall is pH sensitive.     

Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells

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.     

Extending the host range of Listeria monocytogenes by rational protein design

In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis.     

Distinct protein patterns associated with Listeria monocytogenes InlA- or InlB-phagosomes

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.     

Species specificity of the Listeria monocytogenes InlB protein

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.     

Listerial invasion protein internalin B promotes entry into ileal Peyer's patches in vivo

Listeria monocytogenes (Lm) invades the host intestine using listerial invasion proteins, internalins. The in vivo role of internalin A (InlA) and internalin B (InlB) is reported here. Intragastric (i.g.) administration and ligated loop assays with ΔinlB-Lm demonstrated that a lack of InlB significantly attenuates the invasive ability of Lm into various organs. On the other hand, InlA(m)-Lm expressing a mutant InlA with two substitutions, S192N and Y369S, which has been reported to increase the affinity of InlA to mouse E-cadherin, resulted in little increase in intestinal infection according to both ligated loop and i.g. infection assays. Lm preferentially enters ileal Peyer's patch (PP) via M cells and ΔinlB-Lm showed severely reduced ability to invade though these cells. The present results reveal the importance of InlB, which accelerates listerial invasion into M cells on ileal PPs in vivo.     

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.     

FbpA, a novel multifunctional Listeria monocytogenes virulence factor

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.     

The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.     

Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence

During infection of their hosts, Gram-positive bacteria express surface proteins that serve multiple biological functions. Surface proteins harbouring a C-terminal sorting signal with an LPXTG motif are covalently linked to the cell wall peptidoglycan by a transamidase named sortase. Two genes encoding putative sortases, termed srtA and srtB, were identified in the genome of the intracellular pathogenic bacterium Listeria monocytogenes. Inactivation of srtA abolishes anchoring of the invasion protein InlA to the bacterial surface. It also prevents the proper sorting of several other peptidoglycan-associated LPXTG proteins. Three were identified by a mass spectrometry approach. The DeltasrtA mutant strain is defective in entering epithelial cells, similar to a DeltainlA mutant. In contrast to a DeltainlA mutant, the DeltasrtA mutant is impaired for colonization of the liver and spleen after oral inoculation in mice. Thus, L. monocytogenes srtA is required for the cell wall anchoring of InlA and, presumably, for the anchoring of other LPXTG-containing proteins that are involved in listerial infections.     

Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes

Listeria monocytogenes is an intracellular bacterium that causes systemic infections after traversing the intestinal mucosa. Clearance of infection and long term protective immunity are mediated by L. monocytogenes-specific CD8 T lymphocytes. In this report, we characterize the murine CD8 T cell response in the lamina propria and intestinal epithelium after enteric L. monocytogenes infection. We find that the frequency of MHC class Ia-restricted, L. monocytogenes-specific T cells is approximately 4- to 5-fold greater in the lamina propria than in the spleen of mice after oral or i.v. infection. Although the kinetics of T cell expansion and contraction are similar in spleen, lamina propria, and intestinal epithelium, high frequencies of Ag-specific T cells are detected only in the lamina propria 1 mo after infection. In contrast to MHC class Ia-restricted T cells, the frequency of H2-M3-restricted, L. monocytogenes-specific T cells is decreased in the intestinal mucosa relative to that found in the spleen. In addition to this disparity, we find that MHC class Ia-restricted CD8 T cells specific for a dominant L. monocytogenes epitope have different TCR V beta repertoires in the spleen and intestinal mucosa of individual mice. These findings indicate that the intestinal mucosa is a depot where L. monocytogenes-specific effector CD8 T cells accumulate during and after infection irrespective of immunization route. Furthermore, our results demonstrate that CD8 T cell populations in these two sites, although overlapping in Ag specificity, are distinct in terms of their repertoire.