|Director : Pascale Cossart (email@example.com)|
Our Unit is studying the molecular and cellular basis of the pathogenicity of Listeria monocytogenes, a bacterium responsible for severe foodborne infections which is now a paradigm for the study of bacterial intracellular parasitism and the exploitation of cellular functions by microbial pathogens. L. monocytogenes is responsible for gastroenteritis, septicemia, meningitis and abortions in humans and animals. In humans, it has the capacity to cross the intestinal barrier, and the blood brain and the foeto-placental barriers. In vitro, L. monocytogenes has the ability to enter into many cell types and replicate therein. After lysis of the phagocytic vacuole, bacteria move inside the cytosol and spread from cell to cell by polymerizing the host cell's actin at one pole of the bacteria. Our activity in 2001 has focused on the study of bacterial entry into cultured cells and in the host in vivo, on the study of intra- and intercellular movements, of the regulation of virulence gene expression, and on the identification of new virulence genes and on the comparison of the sequences of the genomes of L. monocytgenes and L. innocua. We have started post-genomic projects (functional genomics, transcriptomics). We are also studying the actin-based motility of Rickettsia conorii, another intracellular bacterium.
I. Entry of L. monocytogenes into mammalian cells
We are studying two bacterial proteins involved in entry, internalin (also called InlA) and InlB.
Internalin is involved in entry into cells expressing E-cadherin, in particular cells from epithelial origin. InlB promotes entry into most cell types.
Internalin and its receptor E-cadherin : from in vitro data to in vivo relevance
(M. Lecuit, S. Sousa)
The internalin receptor was identified by an affinity chromatography approach. It is E-cadherin. Internalin interacts with human, chicken or guinea pig E cadherin but not with the mouse or rat E-cadherins. This specificity relies on a single in E-cadherin, the 16th, which is a proline in permissive species and a glutamic acid in non permissive species. These data explain why internalin plays no role in murine listeriosis. We have analyzed the role of internalin in guinea pigs and in transgenic mice (generated in collaboration with C. Babinet) expressing human E-cadherin. Using these two models, we have demonstrated the role of internalin in crossing the intestinal barrier. This is the first transgenic model used to demonstrate the role of a virulence factor in a human bacterial disease. We are in the process of analyzing the role of internalin in the crossing of the blood brain barrier and in the crossing of the foeto placental barrier.
We have also analyzed the role of the cytoplasmic domain of E-cadherin. We have demonstrated that E-cadherin must be connected to the actin cytoskeleton via a and b catenins to promote entry.
We are also analyzing the early events of the entry process including the actin cytoskeleton rearrangements and the membrane extensions events that lead to entry. We investigate the role of vezatin and myosin VIIA which are both recruited to adherens junctions by a-catenin.
The InlB protein and its receptors gC1qR and Met
(H. Bierne, S. Dramsi, R. Jonquières, N. Khelef)
InlB is loosely associated to the bacterial surface by C-terminal repeats that start by GW (GW repeats). This domain interacts with lipotechoic acids. One of the InlB receptors is gC1qR, the receptor for C1q the first component of the complement cascade. This protein has no transmembrane domain and no cytoplasmic tail, implying that there should be a coreceptor to mediate intracellular signals. A second receptor for InlB is Met, the receptor for the hepatocyte growth factor or " scatter factor ", a tyrosine kinase receptor. This receptor interacts with the N-terminal part of InlB, the LRRs. We are studying the role of each of these two receptors during and after the entry process. We are also analyzing the synergy between internalin and InlB during the entry process. The contribution of the fraction of gC1qR present at the mitochondira is also studied. We have recently shown that the GW repeats can interact directly with glycosaminoglycans. The role of this interaction during the infectious process is under investigation.
Signaling during InlB-mediated entry
(H. Bierne, S. Dramsi, R. Jonquières, N. Khelef)
We have shown that InlB activates PI 3-kinase via Met by stimulating the phosphorylation of the three adaptor proteins Gab1, Cbl and Shc. InlB is the first bacterial agonist of PI 3 kinase. While bacteria enter cells without important actin cytoskeleton rearrangements, soluble InlB trigger spectacular membrane ruffles which are actin rich. We have analyzed the mechanisms underlying these rearrangments and have shown a role for the Arp2/3 complex, cofilin, LIM kinase and small GTPases in the induction of the actin polymerisation process. We are searching for the downstream targets of PI 3-kinase. PLC gamma is one of them. It is activated but has no direct role in entry. InlB also activates NF-KB and could have anti-apoptotic effect that we are investigating. InlB is thus a potent signaling molecule.
II. Identification of phagosomal components
In order to identify key molecules involved in entry of L. monocytogenes and in the maturation of the phagocytic vacuole, we use a lipido/proteomic approach and have undertaken the analysis of the composition of the internalization vacuole. Vacuoles are isolated by a subcellular fractionation after interaction of cells with latex beads coated with internalin or InlB, or by FACS analysis after interaction of cells with L. monocytogenes or beads coated with internalin or InlB. The protein composition of the phagosome is analyzed after electrophoresis in bidimensional gels and mass spectrometry. Candidate proteins (the septin MSF -a GTPase-, and a PI 4-kinase) have been isolated and their function in the entry/maturation of phagosome is under investigation.
III. The actin based movements of L . monocytogenes and R. conorii
(E. Gouin, V. Villiers)
L . monocytogenes motility
By genetic and biochemical approaches (in collaboration with MF Carlier) we have analysed the interaction of ActA with the Arp2/3 complex, the general actin nucleator, and shown that ActA mimics WASP family proteins.
Rickettsia conorii motility
R. conorii is a strict intracellular bacterium which is, as L. monocytogenes or Shigella, able to move inside cells using an actin-based motility. Rickettsia seem to have an original mechanism for actin based movement : the actin filaments in the actin tail are long, not branched and do not contain the Arp2/3 complex. The genome of R. conorii has been sequenced at the " Génoscope " in Evry. A candidate gene possibly encoding the protein able to polymerize actin has been identified. Its function is currently being characterized.
IV. Regulation of virulence gene expression
(J. Johansson, A. Renzoni)
The PrfA protein is a pleiotropic activator of most idientified virulence genes. The regulation of expression of this protein is complex. In some environmental conditions where virulence genes are repressed, the protein can be present but inactive. The expression of this protein is increased in presence of host cells or cell extracts. We are searching for the signals responsible for this induction. We are also analyzing the molecular mechanisms underlying the regulation by temperature as well as the possible post translational modifications (association with a cofactor ). Other transcriptional regulators are also studied.
V. Identification of novel virulence genes
(F. Bourdichon, S. Dramsi, H. Fsihi)
To identify new virulence genes critical for the infectious process in vivo, we have used a genetic approach called signature tagged mutagenesis or STM. We have constructed a bank of mutants tagged with 96 different transposons. These mutants have been used to infect mice intraveinously and the mutants unable to survive in the mouse have been further characterized. One of them encodes a transcriptional regulator, another encodes a potential surface protein.
VI. Sequencing of the genomes of L. monocytogenes and L. innocua and post genomic analysis
(D. Cabanes, O. Dussurget, P. Dehoux, E. Milohanic and the Listeria European genome consortium)
In collaboration with the " Laboratoire de Génomique des Pathogènes " (P. Glaser, C. Buchreiser, and L. Frangeul) and the Listeria European genome consortium, we have annotated and compared the genomes of L. monocytogenes and L. innocua, a non pathogenic species of the genus Listeria. A transcriptomic analysis following growth in various conditions is performed to identify L. monocytogenes genes critical for survival in different conditions encountered by Listeria. The same analysis is performed with various isolates. We also analyze by this technique the genes regulated by PrfA and which are potential virulence genes. Finally, we inactivate various genes that are present in L. monocytogenes and absent in L. innocua in order to identify new virulence genes. Ongoing mutational analysis concern genes encoding surface proteins. Recent results indicate that some of these proteins may be involved in entry into host cells.
We have also inactivated a gene endoding a bile-salt hydrolase, which seems to play a role in bacterial survival within the host. Inactivation of the srtA gene that encodes a sortase anchoring LPXTG proteins to the cell wall has also been performed. The respective roles of srtA and of the paralogous gene srtB is under current investigation.
In collaboration with the " Laboratoire de Génomique des Pathogènes " and the " Laboratoire des Listeria " we have undertaken the analysis of a large number of L. monocytogenes strains of various serovars in order to identify all the genes involved in virulence.
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
Isabelle Carton, Secrétaire IP
KHELEF Nadia, CR1 IP
BIERNE Hélène, CR1 INRA
DRAMSI Shaynoor, CR1 IP
DUSSURGET Olivier, CR1 IP
ARCHAMBAUD Christel, Etudiante en DEA
HUILLET Eugénie, Stagiaire post-doctorant, CR2 INRA
CABANES Didier, Stagiaire post-doctorant CEE
JOHANSSON Jorgen, Stagiaire post-doctorant, Bourse du gouvernement suédois
LECUIT Marc, Stagiaire post-doctorant
MILOHANIC Eliane, Stagiaire post-doctorant CEE
MANDIN Pierre, Etudiant en DEA
MARTINEZ Juan, Stagaire post Doctorant EMBO
PIZARRO-CERDA Javier, Stagiaire post-doctorant ARC
SEVEAU Stéphanie, Stagiaire post-doctorant ARC
SOUSA Sandra, Etudiante en thèse, Bousière du gouvernement portugais
DEHOUX Pierre, Ingénieur IP
GOUIN Edith, Ingénieur IP
VILLIERS Véronique, Technicienne IP