Unit: Bacteria-Cell Interactions
Director: Cossart Pascale
Our Unit investigates the molecular and cellular basis of the pathogenicity of Listeria monocytogenes, a foodborne pathogen which has become a model study for intracellular parasitism. L. monocytogenes is responsible for gastroenteritis, septicemia, meningitides and abortions in humans, with 30% of mortality. Susceptible individuals include pregnant women and fetuses, newborns, elderly and immunocompromised persons. L. monocytogenes is an intracellular bacterium, which can cross the intestinal barrier, then blood-brain or feto-placental barriers, invade and multiply within several cell types. Bacteria move in the cytosol and spread from cell to cell using an original propelling process: cell actin polymerization at one pole of the bacteria. In 2003, our activity concerned the study of the bacterial and cellular components controlling bacterial entry into cells, the identification of new virulence genes, their regulation and their anchoring mechanism at the bacterial surface. We also performed studies on the molecular epidemiology and the biodiversity of L. monocytogenes strains . In parallel, we investigate the actin-dependent motility of another intracellular bacterium, Rickettsia conorii, as well as its mechanism of entry into epithelial cells.
I. Entry of L. monocytogenes into epithelial cells:
Role of the internalins, InlA and InlB, two bacterial proteins involved in cell entry
InlA and its receptor, E-cadherin (M. Lecuit, S. Sousa).
We demonstrated that entry into cells requires the interaction of the cytoplasmic domain of E-cadherin with the actin cytoskeleton via catenins as well as the participation of the unconventional myosin VIIa and of vezatin, a transmembrane adaptor molecule. Myosin VIIa is maintained at adherens junctions through alpha-catenin and vezatin. As for catenins, myosin VIIa and vezatin are recruited at the L. monocytogenes entry site. Investigations are now oriented towards other molecules regulating actin polymerization, such as GTPases and different actin activators or nucleators, and towards the study of the motor forces generated by myosins .
InlB and its receptors gC1q-R and Met (H. Bierne, N. Khelef)
InlB is non-covalently associated to the bacterial surface and possesses several receptors: gC1q-R, the receptor for the globular form of the complement component C1q, Met, the HGF receptor, and the glycosaminoglycans. Met belongs to the family of growth factor receptors with tyrosine kinase activity, allowing transduction of cell signals required for InlB-dependent entry. gC1q-R has no transmembrane domain and no cytoplasmic domain. Its role in cell invasion, especially as a co-receptor of Met is currently studied.
Signaling and cytoskeletal rearrangements during InlB-dependent entry (H. Bierne, N. Khelef)
InlB-dependent entry involves cytoskeletal rearrangements mediated by the Arp2/3 complex, the cofilin-LIM-kinase couple and Rac, a Rho-GTPase, which regulate actin polymerization. Using dominant negative mutants, we showed that WAVE and ENA/VASP are involved in the actin cup formation at the entry site. Analysis of further events regulating actin polymerization is in progress.
InlB activates PI 3-kinase, which itself stimulates PLC-gamma, inducing the production of IP3 and the release of calcium. InlB also stimulates NF-KB via Ras and Akt. The consequences of the activation of these signaling pathways, especially for the mechanisms of cell survival, are studied .
Other mechanisms involved in cell entry (S. Dramsi, S. Dupuis, J. Pizarro-Cerda, S. Seveau)
We have established that listeriolysin, which is important for bacterial escape from the phagocytic vacuole, generates an extracellular calcium influx, which regulates epithelial cell invasion. We have also demonstrated that membrane cholesterol plays an important role in L. monocytogenes cell invasion, suggesting an involvement of membrane lipid microdomains in cell entry. In parallel, we showed that PI 4-K is recruited at the entry site of beads coated with InlA or InlB. Finally, we have discovered that septin 9, a GTPase, which forms filaments colocalizing with filamentous actin and microtubules, is recruited at the entry site of L. monocytogenes and of beads coated with InlA or InlB. The exact contribution of PI 4-K and septin 9 is currently evaluated using dominant negative mutants and the technique of gene inactivation by RNA interference.
II. Identification of new virulence mechanisms for L. monocytogenes
Identification of new virulence genes by random mutagenesis (D. Cabanes, J. Johansson, P. Mandin)
By signature tagged mutagenesis, we identified new L. monocytogenes genes, important for the infectious process in the murine model. They encode the FbpA protein and a two component system, VirR/VirS. FbpA is surface protein with no signal sequence, suggesting a new mechanism to address proteins at the bacterial surface. FbpA binds fibronectin and allows bacterial adhesion to the cells. Expression of FbpA is important for that of two other virulence factors, the listeriolysin and InlB, suggesting that FbpA may also act as a chaperone. Transcriptome analysis of the VirR/VirS system showed that it regulates several genes, some of which are involved in virulence. These genes, their regulation mechanism, and the signals controlling expression of the VirR/VirS regulon are currently studied.
Post-genomic approach to study L. monocytogenes virulence (C. Archambaud, H. Bierne, D. Cabanes, O. Dussurget, C. Sabet)
Exploitation of the complete genome sequences of L. monocytogenes and of the non-pathogenic strain Listeria innocua, allowed us to identify new virulence genes par targeted mutagenesis. We have inactivated genes present in L. monocytogenes and absent in L. innocua, coding for factors involved in infection:
- a bile salt hydrolase (Bsh), whose expression is regulated by PrfA, and which favors bacterial resistance to bile and persistence in the intestinal lumen. The gene encoding BSH is absent from L. innocua, appearing as a new tool to identify Listeria.
- several surface proteins involved in bacterial entry into cells and in virulence in different animal models, among which a new autolysin (Auto).
- a serine/threonine phosphatase, whose role in virulence, activity on other factors and bacterial and cellular targets are under investigation.
- two sortases, which anchor surface proteins through a LPXTG motif for sortase A and a newly identified motif (NXXTX) for sortase B. Deletion of the gene encoding sortase A abolishes the anchoring of several LPXTG surface proteins and attenuates the virulence during the early steps of infection. Deletion of the gene encoding sortase B has no effect on virulence. The precise contribution of sortase B and of its potential targets is currently evaluated.
Studies on the biodiversity of Listeria have been developed using comparative genomics between two L. monocytogenes strains of different serovars (4b and 1/2a) and the genomic analysis of 113 other strains (Collaboration with M. Doumith and P. Martin, Centre de Référence des Listeria, and C. Buchrieser, Laboratoire des Microorganismes Pathogènes, Institut Pasteur). Thirty markers specific for L. monocytogenes and for different serovars have been identified, opening the way to the development of new identification tools and to the study of Listeria evolution.
III. Regulation of Listeria monocytogenes virulence gene expression
Discovery of a thermosensor RNAand non coding small RNAs (J. Johansson, P. Mandin)
PrfA is a pleiotropic activator of the transcription of most of the virulence genes. Its expression is high at 37°C and inhibited at 20°C. Under certain environmental conditions, PrfA is present on its inactive form. We showed that at 20°C, but not at 37°C, the 5' end of the mRNA encoding PrfA forms a stem and loop that sequesters the Shine and Dalgarno sequence, inhibiting the transcription of prfA transcripts. In parallel, we have initiated the study of small non coding des ARN non codants et de leur rôle dans la régulation.
Transcriptome analysis of the PrfA regulon (E. Milohanic in collaboration with C. Buchrieser and P. Glaser, Laboratoire de Génomique des Microorgnismes Pathogènes and the Génopole)
Transcriptome analysis of the genes regulated by PrfA in different growth conditions allowed us to identify 3 groups of genes comprising 10 genes already known and 2 new genes directly activated by PrfA (Group I), 8 genes negatively regulated (Group II) and 53 genes indirectly controlled by PrfA (Group III). Addiction of charcoal to growth medium stimulates expression of the group I genes and abolishes that of most of the genes from group III, while addition of de cellobiose has the inverse effect. Genes from group II are always repressed. The absence of a PrfA box and the presence of sequences similar to sigma B-dependent promoters upstream of certain genes regulated by PrfA suggest that they be controlled by a sigma B factor, which controls stress responses.
IV. In vivo studies of L. monocytogenes infection and host responses
Role of InlA in the crossing tissue barriers (E. Huillet, M. Lecuit)
InlA interacts with the E-cadherin from human or guinea pigs, but not from mice or rats. The use of transgenic mice expressing the human E-cadherin at the intestinal level revealed the critical role of InlA in the crossing of the intestinal barrier by L. monocytogenes. We are generating transgenic mice expressing the human E-cadherin in place of the murine E-cadherin in all tissues of the organism to evaluate the potential contribution of InlA in the crossing of the blood-brain and feto-placental barriers (Collaboration with C. Babinet, Unité de Biologie du Développement, Institut Pasteur).
Host responses to L. monocytogenes infection (O. Dussurget, M. Lecuit)
We have started a global analysis of the host response to infection by a transcriptomic approach (Collaborations with J.I. Gordon, Washington University, St Louis, MO, and P. Ricciardi-Castagnoli, University of Milano-Bicocca, Italy). We studied the intestinal response of germ-free mice expressing the human E-cadherin at the intestinal level and of epithelial and dendritic cell lines. The comparison of the gene expression patterns induced by wild type and mutants strains of L. monocytogenes is currently analyzed .
Real time analysis of murine listeriosis (O. Dussurget)
We are initiating the real time analysis of L. monocytogenes infection using a non invasive technique of imaging based on bioluminescence (Collaboration with J. Hardy and C. Contag from Stanford University, CA). This approach should allow us to perform a spatio-temporal analysis of the progression of murine listeriosis.
V. Epidemiological studies
(M. Lecuit in collaboration with C. Jacquet, Centre de Référence des Listeria)
Certain strains of L. monocytogenes express a truncated form of InlA, affecting their invasive capacity. An epidemiological study designed to evaluate the frequency and the distribution of this type of strains showed that 96% of the clinical strains but only 65% of the strains from food origin express a functional InlA. Strikingly, all strains responsible for feto-placental infections express a functional InlA. These results demonstrate the involvement of InlA in the development of human listeriosis and strongly suggest that it play a critical role in the crossing of the feto-placental barrier.
VI. Rickettsia conorii:
Another model to study actin-dependent motility and entry into epithelial cells (E. Gouin, J. Martinez, V. Villiers)
As L. monocytogenes, Rickettsia conorii, an intracellular bacterium, has an intracytoplasmic motility dependent on actin polymerization. Contrary to L. monocytogenes, actin filaments polymerized by R. conorii are long, unbranched, and attached to the bacteria. The sequence of the genome of R. conorii and of Rickettsia prowazekii, which does not polymerize actin allowed the identification of a rickA gene present in R. conorii but absent from R. prowazekii. We have purified the protein RickA encoded by the rickA gene and showed that it induces actin polymerization and branching of actin filaments in vitro. RickA is expressed at the surface of intracellular bacteria, revealing that the protein, which does not posses the usual signal peptide or an anchoring signal, is secreted. We also established that the actin comets generated by R. conorii do not contain the Arp2/3 complex, while it is recruited around the bacteria, suggesting a new polymerization mechanism. In parallel, we analyzed other undocumented steps of R. conorii infection, such as the nature of the R. conorii receptors or the signaling resulting from R. conorii interaction with epithelial cells. Finally, we identified a phospholipase D, which could be a new virulence factor since specific antibodies reduce their cytotoxicity towards Vero cells (Collaboration with D. Raoult, Unité des Rickettsies, Marseille, France).
Photo 1. Scheme of the factors and events involved in InlA-dependent entry.
Photo 2. Scheme of the factors and events involved in InlB-dependent entry.
Photo 3. Labeling of the unconventional myosin VIIa (red), at the adherens junctions of epithelial cells, which is also recruited with actin (green) at the Listeria monocytogenes entry site.
Keywords: bacteria, virulence, cell biology, transcriptomics, mutagenesis