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  Director : Philippe SANSONETTI (psanson@pasteur.fr )


  abstract

 

Our Research Unit is involved in studying the molecular, cellular and tissular basis of the rupture, invasion and inflammatory destruction of the intestinal barrier by invasive bacteria, as well as the defense and protective mechanisms against these infections. Our major model is Shigella, the agent responsible for bacillary dysentery. For this study, we apply a multidisciplinary approach that encompasses molecular genetics, functional genomics, cell biology, experimental medicine and immunology. The regulatory mechanisms that control expression of bacterial virulence genes are investigated. Their products (i.e. effectors of pathogenicity) modify the behaviour of both epithelial cells and phagocytic cells interacting with their cellular targets. These cross talks between bacteria and cells lead to bacterial internalisation, intracellular motility and cell to cell spread of bacteria. Interaction of invasive shigellae with phagocytic or epithelial cells elicits a cascade of pro-inflammatory signals that causes disruption of the epithelial barrier and, eventually, its destruction. We are also studying the nature of the immune protection against Shigella and how the innate immune response, dominated by strong inflammation, influences the quality of the adaptive immune response. These studies are applied to the development of vaccine candidates against bacillary dysentery.



  report

cale

Genetic bases of the invasive phenotype of Shigella.Permanent staff: Claude Parsot. Post-doctoral scientists : Ariel Blocker, Maria Mavris, Kirsten Niebuhr, Kaïs Jamoussi. Graduate student: Anne-Laure Page. Engineer: Hélène d'Hauteville.

In collaboration with the group of Philippe Glaser and Franck Kunst, the 220 kb virulence plasmid of Shigella flexneri 5a has been sequenced and annotated. The plasmid carries the 30 kb pathogenicity island encoding the type III secreton (TTSS) and its secreted effector proteins (IpaA-D and IpgD) which are responsible for entry into epithelial cells and apoptotic killing of macrophages. In addition, about fifteen additional genes encode proteins that are potentially secreted by the TTSS. Protein targets for the various chaperones that characterise this system have been identified by a two-hybrid strategy and the molecular mechanisms that regulate the transcription of the genes encoding the proteins secreted via the TTSS in function of its state of activation have been established. Finally, in collaboration with Bernard Payrastre (INSERM, Toulouse), we have characterised the phosphatidyl-inositol phosphatase activity of IpgD that hydrolyses PIns(4,5)P2 into PIns(5)P. In collaboration with Mike Sheetz's group in New York, we have shown thay IpgD was able to disconnect the cell membrane from the actin subcortical cytoskeleton, thereby dramatically lowering the membrane tension force and facilitating extension of filopodial structures.


Molecular signals involved in Shigella entry into epithelial cells and cell to cell spread.Permanent staff: Guy Tran Van Nhieu. Graduate student: Laurence Bougnères. Technician: Joëlle Mounier.

Signalling elicited by IpaC, a Shigella protein inserted into host cell membrane during the invasion process that leads to actin nucleation/polymerisation via small GTPases of the Rho family was investigated further. In addition, the role of the proto-oncogene c-src in remodelling the macropinocytic entry focus of Shigella has been confirmed, its recruitment and activation by RhoA and IpaC have been shown and the major role of cortactin as a tyrosine phosphorylation target for c-src and amplification factor for actin polymerisation has been demonstrated.
The major role of connexins, the gap junction components, in the efficiency of Shigella cell to cell spread has been demonstrated. Connexin expression modulates the pattern of calcium fluxes that are induced by invasive Shigella, by allowing the diffusion of a second messenger from the infected cell to neighbouring cells. The role of connexins in the paracrine transmission of the second messenger is currently under investigation.


Molecular and cellular basis of the inflammation and invasion of the intestinal epithelial barrier by Shigella.Permanent staff: Philippe Sansonetti, Régis Tournebize. Post-doctoral scientist: Dana Philpott.

The dual role of Caspase-1, following its activation by IpaB, in triggering apoptotic death of infected macrophages and maturation of two pro-inflammatory cytokines, IL-1band IL-18, has been demonstrated. At the early stage of infection, triggering of early inflammation by IL-1bdestabilises the epithelial barrier and facilitates bacterial invasion. Conversely, initiation of the g-IFN-mediated innate response via IL-18 causes anti-bacterial immunity. The molecular basis of NF-kB and JNK activation, which turns the invaded epithelial cell into a potent pro-inflammatory cell through the production of IL-8 is being studied. We have shown that Nod1/CARD4, a homologue of plant resistance proteins, accounts for this signalling in the presence of intracellular LPS. Components of the signalling cascade, particularly RICK and IKKa,have been identified. Finally, we have demonstrated the role of the lipid A, especially when saturated by fatty acids in acyl-oxy-acyl bounds, in the inflammatory rupture of the epithelial barrier.


Specific intestinal immunity in bacillary dysentery.Permanent staff: Armelle Phalipon. Post-doctoral scientist: Maria-Isabel Fernandez-Martinez. Graduate student : Florence Rivenet. Technician: Audrey Thuizat. Permanent collaboration with Laurence Mulard, Unité de Chimie Organique.

In a murine model of shigellosis, we have demonstrated, in collaboration with Blaise Corthésy, the major role played by the secretory component in the protective efficiency of secretory IgAs directed against the bacterial somatic antigen. We have developed in vitro models in polarised intestinal epithelial cells and in vivo models such as the intragastric infection of new born mice, in order to analyse the anti-infectious and anti-inflammatory capacity of anti-LPS secretory IgAs.
In collaboration with Franco Felici, we have pursued the study of the immunogenicity of new peptides mimicking the polysaccharide subunits that constitute the protective somatic antigen of Shigella. Finally, in collaboration with Laurence Mulard, the protective saccharidic epitopes on the somatic antigen have been recently characterized. These two last approaches may open the way to innovative vaccine strategies.

Photos:
1: The 214 kb-virulence plasmid of Shigella flexneri.

2: Actin-dependent entry and cell-to-cell spread of Shigella flexneri.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
   

Claude Parsot

Guy Tran Van Nhieu

Philippe Sansonetti

Régis Tournebize

Armelle Phalipon

Ariel Blocker

Maria Mavris

Kirsten Niebuhr

Kaïs Jamoussi

Anne-Laure Page

Dana Philpott

Maria-Isabel Fernandez-Martinez

Florence Rivenet

Laurence Bougnères

Hélène d'Hauteville

Joëlle Mounier

Thierry Pedron

Audrey Thuizat


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