New Opportunities: The Nitrosative Stress Response of Burkolderia thailandensis

激活的吞噬细胞产生活性氧(ROS)和氮(RNS)物种，它们抑制 通过靶向必需的蛋白质硫醇和金属中心在细胞内的微生物。我们的实验室已经研究了 十多年来与ROS/RNS相关的抗菌行动，并帮助确立了核心作用 这些介体在宿主对细胞内病原体的抗性中起作用。拟议的研究将具体地 寻求确定伯克霍尔德氏菌和弗朗西斯氏菌是否存在。通过适应直接抵抗吞噬细胞杀伤 氧化和亚硝化应激，或者通过干扰ROS/RNS来避免巨噬细胞的杀伤 制作。相对非致病的泰兰伯克霍尔德氏菌和图拉氏方格氏菌 SSP。Novicida将与它们的高毒力同行假鼻疽杆菌(NIAID B类)进行比较 和图拉氏针茅F.tularensis SSP.Holarctica(NIAID A类)。具体目标是： 1.确定Burkholderia和Francisella spp.的氧化和亚硝化应激反应 转录微阵列和实时RT-PCR。 2.伯克霍尔德氏菌和弗朗西斯氏菌的遗传决定因素鉴定对ROS/RNS的抗性 NWRCE生成的综合转座子文库和AIM One的数据，以及 地理上不同的环境和临床菌株的集合。 3.检测巨噬细胞抗Bur/r/io/der/a和抗Franca/Se//a活性中的ROS/RNS。 A.测量ROS/RNS的产生和细胞因子/TLR依赖的产生 Burkholderia和Francisella spp. 确定ROS/RNS对伯克霍尔德氏菌和弗朗西斯氏菌的影响。细胞内存活率 巨噬细胞。 4.评估ROS/RNS在小鼠对伯克霍尔德氏菌和弗朗西斯杆菌抗性中的作用。 感染-使用目标二中确定的同源KO小鼠和突变体。

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.