Identifying the formate-sensory mechanism in Shigella flexneri

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Abstract The enteric bacteria and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis (dysentery) per year worldwide and as many as one million deaths. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance amongst Shigella spp. is on the rise. To cause disease, Shigella flexneri is ingested into a human host, and then migrates through the digestive tract and invades colonic epithelial cells where it replicates and causes acute inflammation; this pathogenesis requires a coordinated program of virulence gene expression for S. flexneri to adapt to different host environments and states. Even within a host epithelial cell, S. flexneri virulence gene expression is dynamic, and one signal that S. flexneri uses to regulate this expression is its own production of formate. As S. flexneri replicates within a host cell, it produces and secretes formate, a byproduct of mixed acid fermentation, and this formate accumulates in the host cell. S. flexneri then senses this formate accumulation to positively regulate the expression of virulence genes associated with intercellular spread and dampening the host immune response; however, the mechanism by which S. flexneri recognizes formate accumulation within a host cell is unknown. The goal of this study is to identify the S. flexneri system used to sense extracellular formate accumulation in the host cell cytoplasm during pathogenesis. The first aim of this proposal is to use the differential radial capillary action of ligand assay (DRaCALA) to identify S. flexneri formate binding proteins, and then characterize their role in promoting S. flexneri plaque formation. The second aim of this proposal is to perform a genetic screen of a S. flexneri transposon mutant library to identify genes associated with the S. flexneri response to formate. Identifying this important virulence regulatory system will enable future studies to characterize the dynamics of formate signaling in infected host cells, and potentially provide novel targets for antimicrobial therapeutics and vaccine development.