Characterization of a novel family of Small Regulatory Proteins modulating virulence in Enterobacteriaceae

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PROJECT SUMMARY/ABSTRACT In the United States, the Center for Disease Control and Prevention (CDC) has estimated that more than 200 million episodes of acute gastroenteritis occur annually, resulting in nearly 1 million hospitalizations and over 5000 deceases. Vaccines against enteric pathogens are not available due, in part, to limited knowledge of the many virulence mechanisms that enteropathogens have evolved to colonize, multiply and escape host defenses. We have recently discovered the ANR (AraC Negative Regulators) family, comprising a large number of small regulatory proteins produced by diverse clinically important enteric pathogens including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC), enteropathogenic EPEC, enterohaemorragic (EHEC) and enteroaggregative E. coli (EAEC). Members of the ANR family modulate the expression of virulence factors such as fimbriae, toxins, type-3 and type-6 secretion systems, and genes associated with metabolism, stress-response and fitness, by protein-protein interactions with positive transcriptional regulators of the AraC/XylS family and negative regulators of the HNS family. Furthermore, the absence of this regulatory system affects bacterial pathogenesis in a natural model of infection. Our overall hypothesis is that the ANR family plays a critical role in the concerted regulation of fitness and virulence in pathogens of the Enterobacteriaceae family by taking concomitant control of AraC and HNS global regulators. Here, we propose to obtain mechanistic insights into ANR molecular interactions (AIM-1), regulation (AIM 2) and pathogenesis (AIM-3) using EAEC, EPEC and C. rodentium as pathogen models, human stem cell technology and a natural animal model of disease, coupled with genetic, biochemical and molecular approaches such as Surface Plasmon Resonance (SPR), Time-lapse fluorescence microscopy (TLFM) and ChIP-seq. Our proposed studies will advance our knowledge of global gene regulatory mechanisms that govern fitness and virulence in pathogenic bacteria, and will likely open new avenues for therapeutic intervention.