Recherche / Départements scientifiques / Biologie cellulaire et infection / Unités et groupes / Unité des Interactions Bactéries-Cellules / Research
Virulence factors of L. monocytogenes: Identification & Characterization
Using gene disruption and classical genetic approaches our unit has identified the major virulence factors of Listeria monocytogenes: LLO, a secreted pore-forming toxin which mediates the escape from the phagocytic vacuole; ActA, a surface protein which allows the intra- and inter-cellular movements by polymerizing cellular actin; the two secreted phospholipases PlcA and PlcB, which are critical for the escape from the secondary vacuole and in some cases from the primary vacuole; the invasion proteins internalin (also called InlA) and InlB, which interact with surface host cell receptors to mediate invasion; and the transcription factor PrfA which acts as a major activator of virulence genes.
Over the years, we have used other approaches to identify new virulence factors: signature tagged-mutagenesis allowed us to identify FbpA as a surface protein which binds fibronectin but also acts as a chaperon for LLO and InlB, and the regulon VirR/VirS which regulates genes involved in the modification of cell wall components. More recently, taking advantage of the sequencing of several Listeria genomes, post-genomic approaches led us to identify Auto as an autolysin involved in entry, Bsh as a bile salt hydrolase allowing persistence in the intestinte or Stp as a serine/threonine phosphatase involved in stress response.
At present, we are interested in the characterization of new L. monocytogenes virulence factors among several secreted proteins like InlC (Edith Gouin) or LntA (Hélène Bierne, Alice Lebreton-Mansuy) and among more than 40 surface proteins including the members of the internalin family InlH (Nicolas Personnic) and InlJ (Serawit Bruck) as well as new proteins of still unknown function (Laurent Dortet, To Tham Nam). Global transcriptional arrays (Olivier Dussurget) have allowed us to identify new regulation netwoks within the L. monocytogenes genome, and small non-coding RNAs emerge as potential new regulators of virulence (Nina Sesto, Cristel Archambaud, Zongfu Wu).
Over the years, we have used other approaches to identify new virulence factors: signature tagged-mutagenesis allowed us to identify FbpA as a surface protein which binds fibronectin but also acts as a chaperon for LLO and InlB, and the regulon VirR/VirS which regulates genes involved in the modification of cell wall components. More recently, taking advantage of the sequencing of several Listeria genomes, post-genomic approaches led us to identify Auto as an autolysin involved in entry, Bsh as a bile salt hydrolase allowing persistence in the intestinte or Stp as a serine/threonine phosphatase involved in stress response.
At present, we are interested in the characterization of new L. monocytogenes virulence factors among several secreted proteins like InlC (Edith Gouin) or LntA (Hélène Bierne, Alice Lebreton-Mansuy) and among more than 40 surface proteins including the members of the internalin family InlH (Nicolas Personnic) and InlJ (Serawit Bruck) as well as new proteins of still unknown function (Laurent Dortet, To Tham Nam). Global transcriptional arrays (Olivier Dussurget) have allowed us to identify new regulation netwoks within the L. monocytogenes genome, and small non-coding RNAs emerge as potential new regulators of virulence (Nina Sesto, Cristel Archambaud, Zongfu Wu).
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Cell cycle of Listeria monocytogenes
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Cell Biology of the Listeria monocytogenes Infection
The discovery of internalin as an invasion protein of L. monocytogenes led us to identify E-cadherin as its receptor, and we demonstrated that a single amino-acid in the first extracellular domain of E-cadherin is suficient to define a species specificity. We also showed that lipid rafts are cellular membrane microdomains required for the initial clustering of E-cadherin at the bacterial entry site. We reported that several members of the catenin family play an import role linking the cytoplasmic tail of E-cadherin to the actin cytoskeleton. Several other molecules including Myosin VIIA, vezatin, ARHGAP10 or Arf6 have also been identified in our laboratory as instrumental for bacterial entry.
Concerning the InlB-invasion pathway, we demonstrated that several protein adaptors including Shc, Gab1 and Cbl are required for recruiting a type I PI3K to the bacterial entry site; the phosphoinosited produced by this lipid kinase is then concentrated in lipid rafts and we showed that this redistribution is critical for the recruitment of the small GTPase Rac, which in turn is involved in the activation of a signaling cascade involving Wave/N-Wasp and ENA/VASP leading to polymerization of the actin filaments by the Arp2/3 complex. We also reported that cofilin and the LIM kinase are also critical for depolymerizing actin during the last steps of bacterial engulfment.
Recently, we showed that clathrin is required for L. monocytogenes invasion. We observed in the InlB-invasion pathway that Cbl mediates the ubiquitination of Met and the recruitmenet of clathrin, dynamin, Grb2 and Eps15 among others effectors. For the internalin-dependent invasion pathway, we reported that E-cadherin present in caveolin-rich domains is phosphorylated by Src and recruits Hakai, which promotes E-cadherin ubiquitination and activation of the endocytic machinery. At present, we are pursuing these studies to understand how clathrin is organized at the bacterial entry sites (Matteo Bonazzi). We also investigate the implication during invasion of novel elements such as septin filaments (Serge Mostowy) or the tetraspanin CD81 (Javier Pizarro-Cerda).
Recently, we showed that clathrin is required for L. monocytogenes invasion. We observed in the InlB-invasion pathway that Cbl mediates the ubiquitination of Met and the recruitmenet of clathrin, dynamin, Grb2 and Eps15 among others effectors. For the internalin-dependent invasion pathway, we reported that E-cadherin present in caveolin-rich domains is phosphorylated by Src and recruits Hakai, which promotes E-cadherin ubiquitination and activation of the endocytic machinery. At present, we are pursuing these studies to understand how clathrin is organized at the bacterial entry sites (Matteo Bonazzi). We also investigate the implication during invasion of novel elements such as septin filaments (Serge Mostowy) or the tetraspanin CD81 (Javier Pizarro-Cerda).
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Clathrin recruitment by Listeria monocytogenes
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Listeriosis Physiopathology, Host Responses and Immunity
Listeriosis is a pleitropic food-borne disease characterized by gastroenteritis, bacteremia, meningitis in the new born and eventually abortion in pregnant women. The complex evolutionary interplay between L. monocytogenes and its human host had led, on one hand, to immune responses from the host to control the bacterial infection, and on the other side to bacteria adaptations to evade the immune responses.
As mentioned above, we have shown that L. monocytogenes produces a bile salt hydrolase (Bsh) required for resistance to the bile, and a serine/threonine phosphatase (Stp) which regulates the anti-oxidant activity of the superoxide dismutase. More recently, we demonstrated that the peptidoglycan deacetylase PdgA modifies the bacterial peptidoglycan allowing resistance to lysozyme and survival in macrophages. We continue to investigate other modifications of the L. monocytogenes peptidoglycan which allow the bacterial escape from the host innate immune responses (Camille Aubry, Olivier Dussurget).
We have also reported that the L. monocytogenes pore-forming toxin LLO decreases the levels of histone phosphorylation, reducing the transcriptional activity of a subset of key immune response genes. We pursue these studies concerning the effects of LLO on histone modifications (Mélanie Hamon, Alexander Eskandarian), on the deSUMOylation of host proteins (David Ribet) and in the reorganization of host organelles (Fabrizia Stavru) during infection.
As mentioned above, we have shown that L. monocytogenes produces a bile salt hydrolase (Bsh) required for resistance to the bile, and a serine/threonine phosphatase (Stp) which regulates the anti-oxidant activity of the superoxide dismutase. More recently, we demonstrated that the peptidoglycan deacetylase PdgA modifies the bacterial peptidoglycan allowing resistance to lysozyme and survival in macrophages. We continue to investigate other modifications of the L. monocytogenes peptidoglycan which allow the bacterial escape from the host innate immune responses (Camille Aubry, Olivier Dussurget).
We have also reported that the L. monocytogenes pore-forming toxin LLO decreases the levels of histone phosphorylation, reducing the transcriptional activity of a subset of key immune response genes. We pursue these studies concerning the effects of LLO on histone modifications (Mélanie Hamon, Alexander Eskandarian), on the deSUMOylation of host proteins (David Ribet) and in the reorganization of host organelles (Fabrizia Stavru) during infection.
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Bioluminescent imaging
of mice infected with Listeria monocytogenes |
Cell Biology of the Rickettsia conorii Infection
Rickettsia conorii is an obligate bacterial intracellular pathogen with a cell cycle similar to the one of L. monocytogenes. We have shown that the host cell protein Ku70 is a cellular receptor for R. conorii, and that the Cbl-dependent ubiquitination of this receptor is required for bacterial invasion. We also demonstrated that the bacterial protein RickA activates the Arp2/3 complex to promote actin polymerization and R. conorii movement inside host cells. We continue to characterize the early steps of host cell invasion and the actin-dependent movement of R. conorii (Edith Gouin, Veronique Villiers).
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Actin polymerization by Rickettsia conorii
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