New Opportunities: The Nitrosative Stress Response of Burkolderia thailandensis

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Activated phagocytes produce reactive oxygen (ROS) and nitrogen (RNS) species, which inhibit intracellular microbes by targeting essential protein thiols and metal centers. Our lab has studied ROS/RNS-related antimicrobial actions for more than a decade and has helped to establish a central role for these mediators in host resistance to intracellular pathogens. The proposed studies will specifically seek to determine whether Burkholderia and Francisella spp. directly resist phagocyte killing by adapting to oxidative and nitrosative stress, or alternatively avoid macrophage killing by interfering with ROS/RNS production. The relatively non-pathogenic species Burkholderia thailandensis and Francisella tularensis ssp. novicida will be compared with their highly virulent counterparts B. pseudomallei (NIAID category B) and F. tularensis ssp. holarctica (NIAID category A). The specific aims are to: 1. Define Oxidative and Nitrosative Stress Responses in Burkholderia and Francisella spp.- by transcriptional microarray and real time RT-PCR. 2. Identify Genetic Determinants of Burkholderia and Francisella spp. Resistance to ROS/RNSusing comprehensive transposon libraries generated by NWRCE and data from aim one, as well as a collection of geographically diverse environmental and clinical strains. 3. Examine ROS/RNS in Anti-Bur/r/io/der/a and Anti-Franc/se//a Activity of Macrophages. a. Measure production and cytokine/Tlr-dependence of ROS/RNS production elicited by Burkholderia and Francisella spp.. b. Determine effects of ROS/RNS on Burkholderia and Francisella spp. intracellular survival in macrophages. 4. Assess Role of ROS/RNS in Resistance of Mice to Burkholderia and Francisella spp. Infection- using congenic ko mice and mutants identified in aim two.