Our group is interested in the pathogenesis of bacillary dysentery; prevalent in the developing world, the disease is particularly lethal in infants and young children in endemic zones. Our approach is a molecular and cellular dissection of the mechanisms of rupture, invasion, and inflammatory destruction of the intestinal epithelium by Shigella. Beyond this objective, we use Shigella as a probe to study the signaling processes that achieve homeostasis of intestinal epithelium in the presence of the gut microbiota.
Interaction of Shigella flexneri with epithelial cells entails contact with membrane rafts through engagement of CD44 and release of Ipa proteins through an activatable, type III secretory apparatus—Mxi/Spa. GTPases of the Rho family and c-src elicit a cascade of signals that causes rearrangements of the cytoskeleton, allowing bacterial entry by macropinocytosis. Then, the bacterium initiates intracytoplasmic movement as a result of the assembly of actin filaments caused by IcsA, a surface protein that recruits N-WASP and Arp2/3. This allows passage to adjacent cells via protrusions that are engulfed by a cadherin-dependent process. A paracrine pathway involving secretion of ATP by hemiconnexons and calcium fluxes facilitates cytoskeletal rearrangements, thus boosting entry and cell-to-cell spread of bacteria.
In addition, epithelial cells are able to sense intracellular bacteria through their peptidoglycan (PGN) by proteins of the NOD/CARD family that are able to activate the NF-kappaB pathway in invaded cells, thus reprogramming them to produce proinflammatory cytokines. Global transcriptional analysis of infected cells reveals a typical pattern of increased transcription of proinflammatory cytokines and cytokines dominated by massive expression of IL-8 mRNAs and consequent production of this potent chemoattractant for polymorphonuclear cells. This is particularly the case for epithelial Nod1/CARD4, which was shown to specifically recognize PGN from Gram-negative microorganisms. As Shigella invades an increasing number of epithelial cells, the colonic epithelium becomes a major provider of IL-8, thereby inducing massive recruitment of polymorphonuclear leukocytes, which account for the destructive inflammatory process so characteristic of shigellosis.
Finally, a pool of plasmid-encoded proteins (Osps and IpaHs), whose corresponding genes are transcribed only when the type III secretory system is activated, appear to act in concert to downregulate several key pathways involved in establishing the innate response upon their injection into cells. We have characterized the function of several of them: OspG is a kinase that binds to the ubiquitinated E2 (ubiquitin transfer enzyme) isoforms involved in ubiquitination of the I-kappaB molecule and blocks this ubiquitination process, thereby preserving I-kappaB from degradation. I-kappaB is a potent anti-inflammatory protein. OspF is a dual phosphatase that translocates into the nucleus of infected cells and dephosphorylates the MAP kinases ERK and P38, leading to the dephosphorylation of serine-10 on the tail of histone H3. This leads to the closing of a restricted number of promoters of proinflammatory genes, particularly IL-8. OspF regulates migration of polymorphonuclear cells through the epithelium during Shigella invasion. Finally, we have shown that the family of 10 IpaH proteins corresponds to a series of isoforms of a new type of E3 ligases for which cellular targets are actively investigated. These recent data confirm that Shigella has evolved an efficient strategy to dampen the innate immune response, including the expression of antimicrobial peptides that are key molecules controlling the growth of bacteria colonizing the epithelial surface. In addition, OspF controls the adaptive immune response by suppressing the Th1 response of the host, thereby creating an immunosuppressive environment that is undoubtedly favorable to bacterial survival. These data should have a major impact on the development of second-generation live attenuated vaccine candidates. Vaccine projects encompass live attenuated candidates as well as a novel approach based on the conjugation of chemically synthesized polysaccharides to a protein carrier. Clinical trials are ongoing.
In addition to the Shigella project, we are developing two projects that are more limited in scope—one on infection of the respiratory tract by Klebsiella spp. and one on the cross-talk between commensal bacteria and the intestinal epithelium. This allows us, in the first case, to tackle a model of nosocomial infection that is a particular threat in modern hospitals, and, in the second, to develop a model of mutualism that is characteristic of commensal microorganisms, with the aim of deciphering the cross-talk underlying homeostasis and its possible disruption in the case of inflammatory bowel diseases.
Last updated September 2009
