Unit: Bacteria-Cell Interactions

Director: Cossart Pascale

Our Unit investigates the molecular and cellular basis of the pathogenicity of Listeria monocytogenes, a model microorganism for studying intracellular parasitism. L. monocytogenes is responsible listeriosis, a severe human foodborne infection with a mortality rate around 30%. L. monocytogenes crosses the intestinal, blood-brain or feto-placental barriers and is responsible for gastroenteritis, meningo-encephalitis and maternofetal infections. It is able to invade several cell types including non-phagocytic cells in which it can survive and multiply. L. monocytogenes propels itself in the cytosol by polymerizing actin at one of its pole and spread from cell to cell.

During the last year, we have identified new virulence factors of L. monocytogenes, we have improved our knowledge of the cell biology of the internalization process, as well as the pathophysiology of listeriosis, including the analysis of the mechanisms allowing the crossing of the host barriers. We have also made major progress in understanding the cell biology of Rickettsia conorii, another intracellular bacteria, responsible for Mediterranean spotted fever.

Here are summarized the main scientific achievements of our laboratory in the last decade:

- Discovery and/or characterization of the main virulence factors of L. monocytogenes,
- Discovery of a new type of regulation mediated by an RNA thermosensor,
- Determination of the complete genome sequences of L. monocytogenes and of L. innocua, a closely related non-pathogenic species,
- Discovery of the internalin receptor, E-cadherin,
- Discovery of several key cellular components allowing the internalin-mediated entry,
- Demonstration of the internalin/E-cadherin and InlB/Met species specificities,
- Deciphering of the signaling pathways triggered during the InlB-mediated entry, and identification of two InlB receptors,
- Elucidation of the role of rafts in Listeria internalization,
- Identification of the role of the endocytic machinery in bacterial entry,
- Discovery of the mechanism used by Listeria to polymerize actin and move intracellularly,
- Discovery of the vaccinia virus capacity to polymerize actin,
- Identification of a novel actin nucleator produced by Rickettsia conorii,
- Identification of the role of internalin in the crossing of the intestinal barrier, after generating the first transgenic model used to analyze a bacterial human disease,
- Demonstration of the role of internalin in human listeriosis by an epidemiological survey of a large collection of food and clinical isolates,
- Identification of the role of internalin in targeting and crossing the placental barrier,
- Identification of the first Rickettsia receptor and of its bacterial ligand.

In 2005, we have focused our investigations on the identification of new L. monocytogenes virulence factors using post genomic approaches, on the study of the cell biology of L. monocytogenes infection (mainly the entry into mammlian cells), and on the pathophysiology of listeriosis. We have also improved our knowledge of the cell biology of Rickettsia conorii infection.

Identification of new Listeria monocytogenes virulence factors

Our laboratory had identified the major virulence factors of L. monocytogenes, including LLO which mediates the escape of the vacuole, ActA, that allows the intra- and inter-cellular movements, the two phospholipases PlcA and PlcB, which are critical for the escape from the secondary vacuole and in some cases from the primary vacuole and the invasion proteins internalin (also called InlA) and InlB. We also had identifed PrfA as the major virulence activator protein.

More recently, by using signature tagged mutagenesis, we discovered and characterized FbpA, a surface protein which binds fibronectin but also acts as a chaperon for LLO and InlB, and this past year the regulon VirR/VirS, which regulates genes involved in the modification of membrane or cell wall components (Mandin, Mol. Microbiol, 2005).

By post-genomic approaches, we identifed Bsh, a bile salt hydrolase that allows persistence in the intestine, and Auto, an autolysin that is involved in entry. By mutating the sortase A, we highlighted that LPXTG proteins other than internalin are important virulence determinants in oral listeriosis. This year, we have identifed one such protein that we called Vip, and which is involved in entry and survival in the host (Cabanes, EMBO J. 2005), as well as InlJ, a surface protein, which plays a role in virulence that is currently being investigated (Sabet, Infect. Immun. 2005). We also identified Stp, a serine/threonine phosphatase involved in the stress response, which dephosphorylates EF-Tu (Archambaud, Mol. Microbiol. 2005). We also demonstrated the role in virulence of Listeria monocytogenes ferritin (Dussurget, FEMS Microbiol Lett. 2005). The identification of additional virulence determinants and their characterization is under way in the laboratory.

Cell biology of Listeria monocytogenes infection

The InlA and InlB pathways of entry are studied in the laboratory.

The InlA pathway: We have previously shown that E-cadherin is the receptor for InlA, and that E-cadherin connection to catenins and the actin cytoskeleton are both critical for entry to occur. We next showed the role in the entry process of an unconventional myosin VIIA, that we had contributed to identify as an adherent junction protein, as well as one of its ligand, the transmembrane protein vezatin.

This year, by use of a two-hybrid screen, we have identified and characterized ARHGAP10 as a new ligand of alpha-catenin, which recruits it to the adherent junctions and plays a critical role in Listeria internalization (Sousa, Nat. Cell Biol., 2005).

The InlB pathway: InlB is a potent signaling molecule that triggers entry into a variety of cell types. Its three dimensional structure was determined in collaboration with P. Ghosh. We had shown following pharmacological inhibition experiments that the PI3-kinase pathway is a key signaling cascade required for entry. This led to the identification of Met, the hepatocyte growth factor receptor as one of the three receptors for InlB. The two other InlB ligands are gC1qR and glycosaminoglycans (or GAGs). A series of cytoskeleton molecules, such as the small GTPases Rac and Cdc42, Arp2/3 and cofilin, are critical for entry. This year, we have shown that WASP-related proteins, Abi1 and Ena/VASP, are also key mediators of the InlB pathway (Bierne, J. Cell. Sci. 2005). In addition, we have identified the critical role of the lipid rafts and the endocytic machinery (Veiga, Nat. Cell Biol. 2005) in the internalization process. These latter findings highlight that clathrin-dependent endocytosis may be used for internalization of particles bigger than previously reported and illustrate the power of the Listeria model for addressing basic cell biology questions.

Work in the laboratory is pursued in order to gain a more detailed insight in the cell biology of L. monocytogenes infection, at the entry level and beyond. In addition, the cell response to Listeria infection is being studied in great detail.

Pathophysiology of listeriosis

We had shown that InlA interaction with its receptor E-cadherin is species-specific, and that mouse E-cadherin, in contrast to human E-cadherin, does not interact with InlA. By generating transgenic mice expressing human E-cadherin at the intestinal barrier level, we showed that orally-acquired listeriosis can be lethal and that internalin is a key virulence factor, by mediating in vivo entry in enterocytes and translocation across the intestinal barrier. In contract to what observed in non-transgenic mice, guinea pigs are permissive to orally-acquired listeriosis, and we had shown that this correlates with the ability of guinea pig E-cadherin to interact with internalin. We have also shown, by combining an epidemiological approach and the use of human infected placenta, the key role of InlA in Listeria monocytogenes targeting and crossing of the placental barrier.

This year, we have shown that InlB interaction with its receptors is also species-specific, and that this interaction does not occur in guinea pigs and rabbits, although these species are naturally permissive to L. monocytogenes (Khelef, Cell. Microbiol, 2006). In this study, we have also shown that, in transgenic mice expressing human E-cadherin at the small intestine level, InlB is not involved in bacterial translocation across the intestinal barrier.

Work in the laboratory currently focuses on the molecular mechanisms underlying the central nervous system tropism of L. monocytogenes. The host response to L. monocytogenes is also studied, as well as real time in vivo imaging of listeriosis.

Cell biology of Rickettsia conorii infection

In 2005, we have made important contributions to the understanding of R. conorii cell infection. We had last year identified RickA, the rickettsial protein involved in the actin-based movement of this microorganism. We focused this year in the entry of the bacterium into cells and have identified the R. conorii cell surface receptor, Ku70, a subunit of the DNA-dependent protein kinase, a protein normally involved in a variety of nuclear functions (Martinez, Cell, 2005). Current efforts focus on the study of the cell biology of the internalization process.

For a more detailed description of the current research in the three fields listed above, please visit our laboratory website at http://www.pasteur.fr/recherche/unites/uibc/, which is updated on a regular basis and on which each researcher presents his/her own research project.

Photos :

Figure 1: 3D reconstruction of confocal images showing localization of endogenous clathrin and Listeria during infection. Extracellular bacteria appear in cyan and intracellular bacteria in green. Endogenous clathrin (detected using the anti-clathrin heavy chain mAb X-22; Affinity BioReagents) is shown in red. 3D reconstructions were performed from 0.17 μm confocal slice images using the Osirix program. Note that clathrin surrounds internalizing bacteria. (For more detail, see Veiga et al, Nat. Cell Biol, 2005, 7:894-900)

Figure 2: Confocal images of ARHGAP10, alpha-catenin and actin labellings in Caco-2 cells. Top pannel : ARHGAP10 localizes at the cell-cell junctions as well as alpha-catenin and actin, and at the nucleus and perinuclear region. Bottom pannel : ARHGAP10 is recruited and colocalizes with alpha-catenin and actin at the entry site of L. innocua expressing InlA, and at adherent junctions. Arrows indicate the accumulation of proteins around entering bacteria. (For more detail, see Sousa et al, Nat. Cell Biol, 2005, 7:954-60)

Keywords: Listeria, Rickettsia, Phagocytosis, Endocytosis, Barriers, Actin, Mutagenesis, Genomics, Post-genomics, Cellular microbiology


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