UNIVERSITY OF MASSACHUSETTS
Shigellae are a group of Gram-negative enteric bacilli that cause acute bacillary dysentery in humans. The signature feature of this disease is exhibited by an intense inflammatory reaction manifested clinically as passage of bloody stools. Humans and some non-human primates, namely Old World monkeys in Africa and Asia serve as the only hosts that are naturally susceptible to Shigella infection. The relationship between Shigella and the human intestinal epithelium and the subsequent inflammatory response, which determines clinical virulence, is undoubtedly complicated and relatively little information is known pertaining to this process. We have identified key components of the Shigella-host relationship that may explain some of the host adaptation exhibited by this organism, in addition to strategic mechanisms underlying Shigella flexneri pathogenicity. First, we have demonstrated that S. flexneri has evolved the ability to regulate functional components of the epithelial tight junctional complex following early interactions at the apical pole of model intestinal epithelia. Second, we have demonstrated a critical role for the eicosanoid hepoxilin A3 (HXA3) in the transepithelial migration of neutrophils (PMN) induced by S. flexneri. HXA3 is a derivative of arachidonic acid formed from the enzymatic action of the 12- lipoxygenase (12-LO) pathway. We have acquired evidence to indicate that HXA3 is secreted through an efflux transport system located at the apical surface of the intestinal epithelia that involves the ABC transporter MRP2. In pilot studies we also show that infection of polarized monolayers with S. flexneri results in a profound up-regulation of MRP2 functional activity. Moreover, blocking MRP2 function corresponds to a marked decrease in the ability of S. flexneri to induce PMN migration. Together, such findings reveal a novel mechanism whereby HXA3 is released apically from the intestinal epithelium. Consequently, the long-term goal of this project is to understand the molecular mechanisms underlying S. flexneri-intestinal epithelial interactions that lead to acute infectious colitis so that novel therapeutic strategies for the treatment of this disease can be developed. The persistence and prevalence of shigellosis worldwide necessitates the study of Shigella pathogenesis, especially given the increase in antibiotic resistance, its potential use as a bio-weapon, and the lack of an efficacious vaccine. We hypothesize that specific host-Shigella interactions that lead to the release of HXA3 will provide novel therapeutic intervention strategies for the treatment of shigellosis. At the same time much information will be learned about the immunology of mucosal surfaces as well as the underlying mechanisms of S. flexneri pathogenesis. The following related but independent Specific Aims are logically aligned to test this hypothesis, as we will examine important aspects underlying Shigella's unique invasion strategy (Specific Aim 1), its ensuing pathogenesis (Specific Aim 2), and in addition, we will explore the development of new therapeutic targets for the treatment of shigellosis (Specific Aim 3). Specific Aim 1: Dissect the molecular mechanisms by which S. flexneri regulates the tight junction proteins. Our evidence suggests that S. flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical pole of model intestinal epithelia, although the details are largely unknown. Such data support a model in which S. flexneri can interact with the tight junctions of the intestinal epithelium and functionally modulate components of the tight junction in order to facilitate paracellular passage to the basolateral surface and foster invasion. Specific Aim 2: Determine the specifics of the molecular cross-talk (between the host and S. flexneri) that lead to induction of mucosal inflammation. We have recently shown that S. flexneri-induced neutrophil movement across mode