Brucella Stationary Phase Gene Expression and Virulence

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DESCRIPTION (provided by applicant): The host factor (HF-I) proteins of Escherichia coil and Salmonella RNA binding proteins that are required for optimal translation of the mRNA encoding the stationary phase specific alternative c factor RpoS. The hfq gene encodes HF-I, and E. coil and S. typhimurium hfq mutants display essentially the same generalized stationary phase defective phenotype as E. coil and S. typhimurium rpoS mutants. Previous studies in our laboratory have shown that the B. abortus hfq mutant Hfq3 displays a generalized stationary phase defect in vitro, but more importantly, mutational studies have clearly established that HF-I is required for the wild-type virulence of B. abortus 2308 in the mouse model. In studies funded by our current award from NIAID, we have identified 18 genetic loci in B. abortus 2308 that are regulated by HF-I, 6 of which are required for wild-type virulence in mice. Three (ahpCD, sodC and cydAB) encode stationary phase antioxidants that protect the intracellular brucellae from oxidative damage in the phagosomal compartment. Interestingly, the virB operon (which encodes the Type IV secretion system) and the bvrRS two-component regulatory system (which regulates genes involved in maintaining cell envelope integrity) also require HF-I for normal expression in B. abortus 2308. The Brucella spp. lack a typical RpoS homolog, so the nature of the regulatory link between HF-I and stationary phase gene expression in not yet clear. Consequently, the objectives of the project described in this competing renewal application are a) to investigate the individual contributions of the B. abortus HF-I regulated genes dps, cfa, znuA, oppD, and bolA to stationary phase physiology in vitro and virulence in mice; b) to better define the role of HF-I in the regulation of the virB and bvrRS operons, and c) to test the hypothesis that the alternative a factor RpoE2 links HF-I and stationary phase gene expression in B. abortus 2308, and thus serves as a "functional RpoS homolog in this bacterium. Defining the basis for HF-I mediated stationary phase gene expression in Brucella and elucidating the contributions of individual stationary phase gene products to successful survival and replication in host macrophages will provide us with important basic information that will be useful for the design of novel vaccine candidates and improved chemotherapeutic approaches.