Unit: Molecular Microbial Pathogenesis
Director: Philippe SANSONETTI
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 aetiological agent of 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 develop innovative imaging techniques to vizualize and analyze the initiation and development of infection and inflammation at cell and tissue levels. We also develop animal models of intestinal inflammation. We are also studying the mechanisms of induction and the effectors of the adaptive immune protection against Shigella and how the innate immune response, dominated by strong inflammation, influences the quality of this adaptive response. These studies are applied to the development of vaccine candidates against bacillary dysentery. Phase 1 and 2 clinical trials are currently underway.
Genetic analysis of the invasive phenotype of Shigella flexneri
Group leader: Claude Parsot. Post-doctoral scientists: John Rhode Student: Christophe Penno. Technician: Elisabeth Ageron.
Determinants of entry into and dissemination of bacteria within epithelial cells are encoded by a plasmid of 213 kb, pWR100. 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, IpgE, the chaperone for IpgD and Spa15, the chaperon for IpaA, IpgB and OspC3). 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.
Work is focussed on three main points: 1) the role of Osp proteins in the pathogenicity of S. flexneri is investigated using a genetic approach, by inactivating the corresponding genes and characterizing the phenotypes of the mutants both in vitro and in vivo. The opsG mutant, for instance, shows an exacerbated virulence phenotype. The OspG protein playing and anti-inflammatory role by inhibiting protein ubiquitination, thus the degradation of I-KB. Interactions between secreted proteins and chaperones is analyzed using the two hybrid system in the yeast, copurification assays in S. flexneri and structural analysis following cristallography. We have characterized the sites of interactions of IpgC on IpaB and IpaC. Structure of these chaperons is currently studied in collaboration with cristallographers ; 3) The mechanism of transcription control 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 and responds to the presence of a consensus operator sequence upstream of these genes. This regulon appears to play a major role in the control of the innate inflammatory response by Shigella at mucosal level. For instance, OspG binds members of the ubiquitin conjugating enzymes family, thereby blocking ubiquitination, thus degradation of I-kB, causing an anti-inflammatory effect.
Picture #1 : The 214 kb-virulence plasmid pWR100 of Shigella flexneri.
Molecules and signals involved in the entry and dissemination of Shigella in epithelial cells
Group leader: Guy Tran Van Nhieu. Post-doctoral scientist: Gianfranco Grompone, Jost Enninga. Graduate students: Laurence Bougnères, Valentin Jaumouillé. Ingineer: 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, IpaC being 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 which, thereby gets recruited to the cell membrane via the adaptor protein Crk, and activates the Arp2/3 nucleator. The IpaA prtotein achieves complete internalization of the bacteria and the repair process of the entry focus via activation of vinculin that act as an actin filamentous capper achieving depolymerization.
We have also demonstrated the important role played by connexins in cell invasion. Connexin hemi-channels open in epithelial cells infected by Shigella and release ATP which, in a paracrine manner activates non-infected adjacent cells. This original mechanism of cell communication causes recurrent calcium fluxes in non-infected cells and is associated with a major increase in the permissiveness of these adjacent cells to bacterial entry and cell to cell spread. ATP plays a critical role in the ultra-precocious steps of entry by capturing bacteria through the formation of cellular extension called nanopodes.
Figure#2. Recruitment of the Src tyrosine kinase at Shigella entry site in HeLa cells. Red : actin, blue : bacteria, green : Src-GFP
Molecular and cellular bases of the rupture, invasion and inflammatory destruction of the intestinal barrier by Shigella.
Group leaders: Philippe Sansonetti, Régis Tournebize, Laurence Arbibe, Stephen Girardin. Post-doctoral scientist: Irit Paz. PhD Student: Jean Bergounioux, Tien Meng-Tsung. Engineer: Thierry Pédron.
In the framework of our project aimed at understanding how Shigella causes the invasion and inflammatory destruction of the intestinal barrier, we have demonstrated that the key role played by the cytosolic protein Nod1 (Card4) that recognizes the peptidoglycan of gram-negative microorganisms and initiates activation of the NF-κB and JNK pathways, thereby leading to a strong inflammatory reaction, demonstrated by the expression of IL-8, the transcriptomal profile of which has been characterized by Affymetirx microarrays. This inflammatory reaction, however, appears to be efficiently down regulated by some secreted proteins such as the already mentioned OpsG and OspF. It seems that Shigella "sculpts" a particular transcriptome that is adapted to the development of the infectious process. We have also demonstrated that the lack of IL-8 expression largely accounts for the unability of mice to develop significant intestinal inflammation (thus the lack of a murine model for bacillary dysentery) in the presence of invasive Shigella.
Under the selective pressure of the innate immune response, it appears that Shigella has developed a shortening process of LPS O-side chains responding to a mechasim that does not involve deletion (a process that leads to extreme susceptibilty to killing by anti-microbial peptides and activated complement), but rather a transition from a linear to a helicoidal structure that involves glycosylation of the basic Rha-Rha-Rha-NAcGlu motif. This glycosylation process is encoded by a lysogenic bacteriophage, the process allowing better access of the tip of the type III secretroy system of Shigella to the surface of eukaryotic cells while maintaining resistance to the innate response.
We have also demonstrated the protective role of commensal/probiotic bacterial such as Lactobacillus casei against inflammation caused by invasive Shigella.
We are now developing methods for real time analysis of infectious processes based on magnetic resonance imaging and intravital microscopy. We are also developing murine models of intestinal inflammation, including transgenic mice and moels of inflammation of the respiratory tract. These approaches have allowed us to introduce a new research theme aimed at studying the molecular, cellular and tissue mechansims of infection of the tracheo-bronchial and pulmonary tissues by Klebsiella pneumoniae.
Photo 3: Reconstruction 3D of mice lungs.
Innate and adaptive immunity in bacillary dysentery.
Group leader: Armelle Phalipon. Post-doctoral scientists: Gernot Sellge. PhD Student: Joao Gamelas-Magalhaes. Technician: Myriam Tanguy.
We increasingly 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. Shigella, at the very early stages of primary infection, modulate INF-γ production. This may constitute a key step in the induction and orientation of the protective humoral response, as well as a way to inhibit an efficient Th1-oriented cell response.
The humoral response specific for the polysaccharidic part of LPS (O-Ag) is crucial for protection against re-infection. It is likely that the secreted effector proteins such as Osps play a major role in this regulatory process.
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 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-κ B with slower kinetics than those observed with the invasive bacterium. Interestingly, LPS-mediated NF-κ B activation is inhibited by a monoclonal IgA antibody 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.
Scientists : Armelle Phalipon, Philippe Sansonetti.
With the support of DGA, we have completed a phase I study and are conducting a phase II 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. In addition, we are developping a sub-unit vaccine approach based on the chemical synthesis of polysaccharidic antigens. Our preliminary immunogenicity results in mice are extremely encouraging.
Keywords: Shigella, intestinal epithelium, invasion, inflammation, immune response, vaccine