|Director : Philippe SANSONETTI (firstname.lastname@example.org)|
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.
Genetic analysis of the invasive phenotype of Shigella flexneriGroup leader: Claude Parsot. Post-doctoral scientists: Maria Mavris, Kaïs Jamoussi. Graduate student: Anne-Laure Page. Engineer: Hélène d'Hauteville.
Determinants of entry into and dissemination of bacteria within epithelial cells are encoded by a plasmid of 213 kb, pWR100, the sequence and annotation of which have been determined, in collaboration with the Laboratory of Genomics of Pathogenic Microorganisms. This plasmid carries a 30-kb region that encodes a type III secretion apparatus (the Mxi-Spa apparatus), proteins that are secreted by this apparatus (the IpaA-D, IpgB, and IpgD proteins), and cytoplasmic chaperones (IpgC, the chaperone for IpaB and IpaC, and IpgE, the chaperone for IpgD). The secretion apparatus is activated upon contact of bacteria with eukaryotic cells, which induces secretion of IpaA-D, IpgB, and IpgD. The plasmid also encodes approximately 20 other proteins that are secreted by the Mxi-Spa apparatus (the Osp and IpaH proteins), some of these proteins being produced only in conditions of active secretion.
Works performed this year have been focused on three main points: 1) the role of Osp proteins in the pathogenicity of S. flexneri was investigated using a genetic approach, by inactivating the corresponding genes and characterizing the phenotypes of the mutants both in vitro and in vivo; 2) Interactions between secreted proteins and chaperones have been analyzed using the two hybrid system in the yeast and copurification assays in S. flexneri. We have characterized the sites of interactions of IpgC on IpaB and IpaC and identified a new chaperone, Spa15, which is associated with IpaA, IpgB, and OspC3; 3) The mechanism of the control of transcription of osp and ipaH genes by the activity of the Mxi-Spa secretion apparatus has been elucidated and involves a transcriptional activator of the AraC family, MxiE, the activity of which is modulated by the IpgC chaperone.
These results allow us to draw a general framework on the function of the type III secretion pathway in S. flexneri. At 37°C, the Mxi-Spa apparatus is produced and assembled in an inactive form. The IpaA-D, IpgB, and IpgD proteins are produced and stored in the cytoplasm, most of them in association with specific chaperones. Upon contact of bacteria with host cells, the Mxi-Spa apparatus is activated, allowing delivery of bacterial invasins within or beyond the membrane of eukaryotic cells. The IpgC chaperone then activates the transcriptional activator MxiE, leading to expression of a second set of secreted effectors, the function of which has now to be elucidated.Molecules and signals involved in the entry and dissemination of Shigella in epithelial cellsGroup leader: Guy Tran Van Nhieu. Post-doctoral scientist: Nalini Ramarao, Caroline Clair. Graduate student: Laurence Bougnères. Technician: Joëlle Mounier.
We are analysing bacterial signalling to the host cell that causes cytoskeletal rearrangements leading to Shigella macropinocytosis by epithelial cells. A particular emphasis is being put on pathways that involve the small GTPases of the Rho family, Cdc42 and Rac, and the tyrosine kinase p60c-src. These two signalling pathways are engaged simultaneously by Shigella during epithelial cell invasion, and we have obtained evidence that IpaC is directly implicated in the activation of these pathways, following its insertion in the epithelial cell membrane. Cdc42 and Rac activation are required for actin polymerisation mediated by the Arp2/3 complex, that lead to the formation of filopodia and membrane leaflets that engulf the bacterial body. The tyrosine kinase p60c-src regulates actin polymerisation mediated by these GTPases, at a distance from the bacterial-cell membrane interaction site, through the phosphorylation of cortactin, a 80 kDa actin-binding protein. The molecular mechanisms that lead to the activation of these pathways and their coordination during Shigella invasion are currently under study.
We have also demonstrated the essential role plaid by connexins in facilitating cell to cell passage of the bacteria. We are currently identifying the mediators that diffuse through the gap junctions during Shigella invasion of epithelial cells, that facilitate bacterial cell to cell spread in the epithelial layer.
Molecular and cellular bases of the rupture, invasion and inflammatory destruction of the intestinal barrier by Shigella. Group leaders: Philippe Sansonetti and Régis Tournebize. Post-doctoral scientists: Dana Philpott and Stephen Girardin. Engineer: Thierry Pédron.
In our ongoing approach at understanding how Shigella invade and cause the inflammatory destruction of the intestinal epithelial barrier, we have confirmed the essential role plaid by the infected epithelial cell itself in a process in which IL-8, IL-1b and TNFa play a key role. Our strategy combines in vitro and in vivo approaches. Through our in vitro approach, we have identified Nod1, a mammalian homologue of plant resistance proteins, as an intracellular sensor of bacterial LPS that is introduced by invasive shigellae. It appears that this newly recognised signalling system is a cytosolic equivalent of Toll-Like Receptors (TLR). Activation of Nod1 by oligomerization in the presence of LPS, via RICK, activates the NF-kB et Jun-terminal kinase (JNK) pathways, thereby triggering the pro-inflammatory properties of infected cells. Massive production of IL-8 responds to this activation process. We have carried out a global transcriptional analysis of epithelial cells infected by Shigella in the AFFYMETRIX system and defined a transcriptional profile that confirms the essentially pro-inflammatory orientation taken by invaded epithelial cells. In an in vivo model, we have demonstrated that the level of endotoxicity of the lipid A plaid a major role in the rupture of the epithelial barrier. This was achieved following mutagenesis of the two msbB genes whose products carry out additional acylation of the lipid A. We now need to connect in vitro and in vivo observation. For this, we are developing methods for real time analysis of infectious processes based on magnetic resonance imaging and intravital microscopy.
Innate and adaptive immunity in bacillary dysentery.Group leader: Armelle Phalipon. Post-doctoral scientists: Maria-Isabel Fernandez-Martinez, Karine Le Barillec. Graduate student: Florence Rivenet. Technician: Audrey Thuizat.
We study the effectors involved in the eradication of primary infection and in the protection against re-infection as well as the influence of innate immunity on the orientation of the adaptive immune response. In collaboration with James Di Santo, we have shown, using a murine model, that NK and TCD4+(a/b) lymphocytes are both involved in the eradication of primary infection via the production of interferon g ( INF-g). In addition, it seems that Shigella, at the very early stages of primary infection, modulate the INF-gproduction. This may constitute a key step in the induction and orientation of the protective humoral response.
The humoral response specific for the polysaccharidic part of LPS (O-Ag) is crucial for protection against re-infection. We have shown that the anti-O-Ag IgG-mediated immune response may contribute to protection only if the response is induced at the mucosal level in order to ensure the production of the effectors at the site of bacterial infection. Concerning the secretory IgA (S-IgA)-mediated response required to protect the mucosal surface from bacterial infection, we have shown, in collaboration with Blaise Corthésy, that the secretory component is directly involved in the protective function of S-IgA by ensuring through its glycosylated residues the appropriate localization of IgA for optimal immune exclusion function. Moreover, we have reported that LPS internalized by epithelial cells activates NF-k B with a slower kinetics than that observed with the invasive bacterium. Interestingly, LPS-mediated NF-k B activation is inhibited by a monoclonal IgA specific for LPS that intercepts during, its transcytosis, the bacterial product. This is a newly and unexpected anti-inflammatory protective function of S-IgA. Therefore, S-IgA-mediated mucosal protection seems to occur via a double mechanism comprising immune exclusion of bacteria from the epithelial surface and intracellular neutralization of pro-inflammatory bacterial product.
Towards a live, attenuated vaccine against shigellosis.
In collaboration with the Walter Reed Army Institute of Research in the USA and the ICDDR,B in Bangladesh, we continue the phase I and II trials of strain SC602, a live attenuated mutant of S. flexneri 2a. With the support of DGA, we are initiating a phase 1 study of strain SC599, a live attenuated mutant of S. dysenteriae 1. Our aim is to develop a pentavalent vaccine comprising 3 serotypes of S. flexneri, S. dysenteriae 1 and S. sonnei.
Photo 1: The 214 kb-virulence plasmid pWR100 of Shigella flexneri.
Photo 2: Effects of Src kinase activity on Shigella-induced cytoskeletal reorganisation during Shigella entry into epithelial cells. HeLa cells were challenged with Shigella and cytoskeletal reorganization were analyzed by immunofluorescence labeling of cortactin (red), F-actin (green), and bacterial LPS (blue). a-c: parental HeLa cells; d-f: HeLa cells transfected with a dominant-negative form of Src; g-i: HeLa cells transfected constitutively active Src.
Photo 3: In vivo analysis of the role of secretory component (SC) in the secretory IgA-mediated protection at mucosal surface. Radiolabeled dimeric IgA devoid of SC administered intranasally diffuse within the pulmonary tissue (a) but remain localized in restricted areas when bound to SC (b) ( b -imager analysis of lung-histological sections). This in turn impacts on bacterial localisation (c and d, green fluorescent protein (GFP)-expressing bacteria). Therefore, SC ensures the appropriate localisation of antibody for optimal immune exclusion function.
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
JACQUEMIN Colette, email@example.com
PARSOT Claude, IP, Chef de Laboratoire, firstname.lastname@example.org
PHALIPON Armelle, IP, Chargé de Recherche, email@example.com
TRAN VAN NHIEU Guy, INSERM, DR2, firstname.lastname@example.org
TOURNEBIZE Régis, INSERM, CR2, email@example.com
CLAIR Caroline, Post-doc
FERNANDEZ-MARTINEZ Maria-Isabel, Post-doc
GIRARDIN Stephen, Post-doc
JAMOUSSI Kaïs, Post-doc
MAVRIS Maria, Post-doc
PHILPOTT Dana, Post-doc
RAMARAO Nalini, Post-doc
BOUGNÈRES Laurence, PhD Student
GAMELAS MAGALHAES Joao, PhD Student
PAGE Anne-Laure, PhD Student
RIVENET Florence, PhD Student
d'HAUTEVILLE Hélène, Ingineer, firstname.lastname@example.org
PEDRON Thierry, Ingineer, email@example.com
MOUNIER Joëlle, Technician, firstname.lastname@example.org
THUIZAT Audrey, Technician, email@example.com