I. Entry of L. monocytogenes into mammalian cells ( H. Bierne, R. Jonquières, N. Khelef, M. Lecuit, P. Mandin, S. Sousa)
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
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 position in E-cadherin position 16 which is a proline in permissive species and a glutamic 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 study 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 to promote entry via a and b catenins
. We analyze 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
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 cytoplamsic tail implying that there should be a coreceptor to mediate intracellular signals. A second receptor for InlB has recently been identifed. It is Met, the receptor for the hepatocyte growth or Scattor 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 entry
We have shown that InlB activates PI 3-kinase via Met by stimulating the phsophorylation 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 analyze the mechanisms underlying these rearrangemnts 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 a potent signaling molecule.
II. Identification of phagosomal components (J Pizarro-Cerda)
In order to identify key molecules involved in entry of L. monocytogenes and in the maturation of the phagocytic vacuole, we use a proteomic approach and have undertaken to analyze the proteic and lipidic composition of the internalization vacuole. Vacuoles are isolated by a subcellular fractionation after interaction of bacteria (L. monocytogenes or L . innocua expressing internalin or InlB) or beads coated with internalin or InlB with cells . The proteic composition of the phagosome is analyzed after electrophoresis in bidimensional gels and mass spectrometry. The role of candidate proteins 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 study the interaction of ActA with the Arp2/3 complex, the general actin nucleator.
Rickettsia conorii motility .R. conorii is a strict intracellular bacterium which is 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 filamnets in the actin tail are long, not branched and do not contain the Arp2/3 complex. The genome of R. conorii has been achieved at the Génoscope in Evry. A candidate gene possibly able to polymerize actin has been identified. Its function is currently characterized.
IV. Régulation 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, 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 transcritptional regulator, the other 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 European genome consortium)
In collaboration with the 'Labotratoire de Génomique des Pathogènes' (P. Glaser, C. Buchreiser, and L. Frangeul) and the Listeria genome European 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 including genes encoding different surface proteins in order to identify new virulence genes.