Our approach is a molecular and cellular dissection of the mechanisms of rupture, invasion, and inflammatory destruction of the intestinal epithelium by Shigella to decipher the pathogenesis of bacillary dysentery, an infectious disease that is particularly prevalent in infants and young children in impoverished areas of the planet. Beyond the medical relevance of this work, Shigella is a remarkable probe we use to discover and analyze basic mechanisms of immune homeostasis of the intestinal mucosa, because it has developed an array of dedicated toxins that "surgically strike" key regulators and effectors of the host immune system.
Expression of a type III secretory system (TTSS) is essential for Shigella to express its pathogenic properties. Interaction of Shigella flexneri with epithelial cells entails contact with membrane rafts through engagement of CD44 and secretion of Ipa proteins through its TTSS. GTPases of the Rho family and c-src elicit a cascade of signals that causes rearrangements of the cytoskeleton, allowing invasion of cells. 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 family that activate the NF-kappaB and MAPKinases pathways 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, 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. Inflammation also responds to the emission of "danger signals" by cells that are engaged by pathogens. A typical example is extracellular release of ATP, which appears to be a potent proinflammatory signal in addition to regulating the cell cytoskeleton. A Shigella effector, IpgD, was identified as a phosphatidyl-inositol phosphatase hydrolyzing PI(4,5)P2 into PI5P, thereby inducing the closing of hemiconnexons, which blocks further release of ATP in the extracellular milieu. This is the first example of a bona fide inhibitor of danger signaling by infected cells.
Finally, plasmid-encoded enzymes (Osps and IpaHs), whose corresponding genes are transcribed only when the TTSS is activated, appear to act in concert, following intracellular injection, to downregulate several pathways of the innate response. They essentially proceed to post-translational modification of key signaling proteins. 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 anti-inflammatory I-kappaB from degradation. 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 a family of 10 IpaH proteins gathers isoforms of a new type of E3 ligases for which cellular targets are actively investigated. These recent data confirm that Shigella has evolved amazing strategies to dampen innate immune responses, including expression of antimicrobial peptides that are key molecules controlling the growth of bacteria colonizing the epithelial surface. In addition, IpgD blocks T cell migration and 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.
We more recently realized that mucosal homeostasis itself results from cross-talk between innate sensors of the host intestinal mucosa and the commensal bacterial flora called the microbiota. We therefore decided to apply the successful cellular microbiology approach that allowed us to decipher mechanisms of Shigella pathogenesis to identify key mechanisms of the gut-microbiota cross-talks. Our focus is on the identification of the bacterial factors that support survival and colonization of the gut mucosa by commensal microorganisms, with Lactobacillus casei as our model organism. We are also working on deciphering microbiota-cell cross-talk in the intestinal crypt, where stem cells support epithelial regeneration.
As of September 26, 2012