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  Director : Cossart Pascale (pcossart@pasteur.fr)


  abstract

 

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 enters into many cell types and replicates 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. In 2002, our activity focused on the analysis of the entry of the bacteria into cells, on the regulation of virulence genes and on the identification of new virulence factors. We are also studying the cell biology of Rickettsia infections



  report

cale

I. Entry of L. monocytogenes into mammalian cells.

We have pursued the study of internalin(InlA) and InlB, two proteins involved in the entry into cells.

Internalin and its receptor E-cadherin, a couple critical for the crossing of the intestinal barrier.(E. Huillet, M. Lecuit, S. Sousa)

The internalin receptor is E-cadherin. Internalin interacts with human E-cadherin, and chicken and guinea pig E-cadherin but not rat or mouse E-cadherin. This species specificty explains why internalin does not play a role in the murine model. The analysis of the role of internalin in guinea pigs and transgenic mice expressing human E-cadherin in the intestine (in collaboration with C. Babinet, Unité de Biologie du Développement, Institut Pasteur) has demonstrated its critical role in the crossing of the intestinal barrier by L. monocytogenes. It is the first transgenic model for the study of a bacterial disease. To analyze the possible role of internalin in the crossing of the hemato-encephalic and foeto-placental barriers, we have undertaken to generate other transgenic mice that express human E-cadherin in place of murine E-cadherin.

We have anlyzed the role of the cytoplasmic domain of E-cadherin : E-cadherin must be connected to the actin cytoskeleton by catenins to allow entry. We are trying to identify all the molecules paticipating to the actin cytoskeleton rearrangements and the remodeling of the membrane. We have identified a non conventional myosin, myosin VIIA which is normally recruited to the adherens junctions via catenins and an adaptor protein named vezatin. It is the first time that a non conventional myosin is involved in entry of a bacterial pathogen into epithelial cells.

The InlB protein and its recptors gC1qR and Met.(H. Bierne, S. Dramsi, N. Khelef)

InlB is loosely attached to the bacterial surface. One of its receptors is gC1qR, the receptor for C1q, the first component of the complement cascade. This protein has no transmembrane domain and no cytoplasmic domain, predicting the presence of a co receptor for a link with the cytosol. Another receptor for InlB and a possible co receptor for gC1qR is Met, the receptor for HGF, a tyrosine kine receptor. This receptor interacts with the N-terminal part of InlB. We have shown that the GW repeats can interact directly with cells via the glycosaminoglycans. They also interact with gC1qR. We are studying the respective roles of Met and of gC1qR during entry, the interaction between these receptors and the events that occur after entry. We have also undertaken the analysis of the putative synergy between internalin and InlB during entry.

Signaling and cytoskeleton rearrangements during InlB mediated entry (H. Bierne, S. Dramsi, 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. This activation is medited via Ras and Akt. The consequences of this activation are investigated. InlB is thus a potent signaling molecule.

Other factors controlling entry(S. Dramsi, S. Seveau)

We have recently shown that the Listeria hemolysin named listeriolysinO which up to now was considered to be mainly essential for the escape from the vacuole also plays a role in the entry of extracellular calcium into cells, an event that is critical for bacterial internalisation. We are also investigating the role of lipid microdomains during entry.

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 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. The protein composition of the phagosome is analyzed by 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. Regulation

Discovery of an RNA thermosensor (J. Johansson, P. Mandin)

The regulator PrfA is a pleitropic activator of transcription of most virulence genes. The regulation of expression of the protein is controlled by several factors. The PrfA levels are high at high temperature (37°C) and low at lower temperatures (30°C). We have shown that at low temperatures, the 5'end of the mRNA encoding PrfA can form a stem and loop structure that sequesters the Shine and Dalgarno sequence and prevents translation of the prfA gene. At high temperature, this structure is not made, translation proceeds and virulence genes are expressed.

Transcriptomic analysis of the PrfA regulon (E. Milohanic in collaboration with C. Buchrieser, P. Glaser, and the Génopole)

The sequence of the Listeria genome has allowed to generate membranes carrying all the Listeria genes. We have analyzed expression of all these genes in wild type strains and prfA mutant strains. The results indicate that in addition to the ten already known PrfA regulated virulence genes, two other genes appear directly controlled by PrfA, some genes are negatively regulated and other seem to be indirectly regulated by PrfA. Some of these genes are currently analyzed to address their function.

IV. Identification of novel virulence genes(D. Cabanes, S. Dramsi,J. Johansson, P. Mandin)

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 surface protein which has no signal sequence, revealing a novel type of targeting at the plasma membrane.This protein binds fibronectin and may play a role in the interaction with the extra cellular mattrix. The second candidate encodes for a two component system that we analyze via transcriptomic. The signals that activate this two component system are also analyzed.

V. Post genomic analysis of Listeria virulence ( C. Archambaud, H. Bierne, D. Cabanes, O. Dussurget, P. Dehoux, E. Milohanic)

In collaboration with the Laboratoire de génomique des pathogènes (P. Glaser, C. Buchrieser and L. Frangeul ) and a European consortium, we have sequenced, annotated and compared the genomes of L. monocytogenes and that of L. innocua, a non pathogenic species of the genus Listeria. Exploitation of these sequences has allowed to undertake :

A transcriptomic analysis (in collaboration with the laboratories of P. Glaser and T. Msadek and other groups in France) : in addition to the analysis carried out on strains inactivated for different transcriptional regulators, a transcriptional analysis is carried out on strains grown in different conditions encountered by Listeria in the environment.( Biofilms…)

A biodiversity analysis (in collaboration with the laboratoies of P. Glaser and P. Martin) : the analysis of various Listeria strains of different serovars and species is carried out in order to identify new specific features of virulent strains.

A search for new virulence genes by gene targeting.

We have inactivated genes present in L. monocytogenes and absent in L. innocua and genes encoding surface proteins. Work in progress indicate that some surface proteins play a role in entry and in virulence in various animal models. We have also inactivated a gene encoding a bile salt hydrolase. This new type of virulence factor plays a role in resistance to bile and consequently in bacterial persistence in the intestinal lumen.

Inactivation of srtA encoding a protein that anchor LPXTG containing proteins to the peptidoglycan affects virulence predicting that in addition to internalin other LPXTG proteins are involved in infection. These are actively searched.

VI. Rickettsia conorii : another model for the analysis of actin-dependent movements and entry of bacteria into cells (E. Gouin, J. Martinez, V. Villiers)

We have during many years analyzed the actin-based motility of Listeria. We are now analyzing that of R. conorii, a strict intracellular bacterium which possesses an actin-based motility. We have identified a gene that is present in the R. conorii genome and absent from that of R. prowasekii, another rickettsia but which does not polymerize actin. R. conorii uses a novel mechanism that we are investigating in detail. We have also started to analyze the other stages of the infectious process.

Fig. 1 Listeria in rolling motion. Example of the actin-based motility process.

Fig. 2Recruitment of the hepatocyte growth factor (Met) at the entry site of bacteria during InlB-mediated entry.

Keywords:



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Carton Isabelle, icarton@pasteur.fr Cossart, Pascale, Institut Pasteur, Professeur, pcossart@pasteur.fr

Dussurget, Olivier, Institut Pasteur, Chargé de Recherches, odussur@pasteur.fr

Khelef, Nadia, Institut Pasteur, Chargée de Recherches, nkhelef@pasteur.fr

Pizarro-Cerda, Javier, Institut Pasteur, Chargé de Recherches, pizarroj@pasteur.fr

Bierne, Hélène, INRA, Chargée de Recherches, hbierne@pasteur.fr

Huillet, Eugénie, INRA, Chargée de Recherches, ehuillet@pasteur.fr

Cabanes, Didier, Stagiaire post-doctorant IP/CEE ; dcabanes@pasteur.fr

Dupuis,Stéphanie, Stagiaire post-doctorante AFRT, sdupuis@pasteur.fr

Johansson, Jorgen, Stagiaire post-doctorant Werner Gren Foundation, jjohanss@pasteur.fr

Martinez, Juan, Stagiaire post doctorant EMBO, jmartine@pasteur.fr

Seveau, Stéphanie, Stagiaire post doctorante ARC, sseveau@pasteur.fr

Archambaud, Cristel, Doctorante, Boursière du Ministère de la Recherche, carcham@pasteur.fr

Mandin, Pierre, Doctorant Boursier du Ministère de la Recherche, pmandin@pasteur.fr

Sousa, Sandra, Doctorante, Boursière de la Fundaçao para a Ciencia e Tecnologia, ssousa@pasteur.fr

Sabet, Christophe, DEA, csabet@pasteur.fr

Gouin Edith, Institut Pasteur, Ingénieur position 3 egouin@pasteur.fr

Villiers Véronique, Institut Pasteur, Technicienne vvilliers@pasteur.fr


Activity Reports 2002 - Institut Pasteur
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