Mycobacterium turberculosis metal transport P-type ATPases

描述(由申请人提供)： 结核病是世界范围内的主要健康问题。结核分枝杆菌的毒力是基于其居住在巨噬细胞内的能力。寄主防御包括活性氧物种、活性氮物种、去除金属微量营养素和降低pH。微生物的部分生存方式是分泌各种金属酶(超氧化物歧化酶、过氧化氢酶等)。以克服巨噬细胞的防御。其致病策略的核心是处理所涉及的重金属(铜、锌、铁、锰等)、质子，可能还有碱金属，如K+和Mg2+。结核分枝杆菌基因组揭示了大量金属转运蛋白的存在。其中比较突出的是P型ATPase。结核分枝杆菌有12个编码这些蛋白(命名为CTP，阳离子转运蛋白)的基因：3个铜+-ATPase，1个K+-ATPase，4个底物未知的重金属ATPase和4个可能运输H+或碱金属的ATPase。我们假设这些转运蛋白的病理生理作用主要是由它们的底物特异性决定的。这些P型ATPase的一个子集的金属特异性将通过在大肠杆菌中表达这些酶，检查细菌的功能获得，并测量酶和运输活性来确定。以前的研究表明，CTPC和CtpD与体内(巨噬细胞)结核分枝杆菌的生长特别相关。为了测试这一点，结核分枝杆菌突变株将与过度表达这些蛋白的菌株一起构建。将评估它们的毒力(在小鼠和培养的巨噬细胞中)和在各种介质中生长的适合性。突变和过度表达这些蛋白对结核分枝杆菌金属动态平衡及其对宿主氧化还原攻击的反应能力的影响将被确定。这些研究的结果将提供对这些蛋白质重要性的初步认识，并使关于这些转运蛋白的生物学功能的有意义的可验证假说得以产生。公共卫生相关性：世界上大约三分之一的人口感染了结核分枝杆菌。尽管有可用的疫苗和有效的治疗方法，但每年仍有200万人死于结核病。该项目将表征一组似乎对结核病毒力至关重要的蛋白质。

DESCRIPTION (provided by applicant): Tuberculosis is a leading health problem worldwide. Mycobacterium tuberculosis virulence is based on its capability to reside inside macrophages. Host defense includes reactive oxygen species, reactive nitrogen species, removal of metal micronutrients and lowered pH. The microorganism survives, in part, by secreting various metalloenzymes (SODs, catalases, etc.) to overcome macrophage defenses. At the center of its pathogenic strategy is the handling of involved heavy metals (Cu+, Zn2+, Fe2+, Mn2+, etc.), protons and perhaps alkali metals such as K+ and Mg2+. M. tuberculosis genome reveals the presence of numerous metal transporters. Among these, the more prominent are P-type ATPases. M. tuberculosis has 12 genes coding for these proteins (named Ctp, cation transport protein): 3 Cu+-ATPases, a K+-ATPase, 4 heavy metal ATPases of unknown substrate and 4 ATPases that likely transport H+ or alkali metals. We hypothesize that the pathophysiological role of these transporters is primarily determined by their substrate specificity. Metal specificities of a subset of these P-type ATPases will be determined by expressing these in E. coli, examining bacterial gain of function, and measuring the enzymatic and transport activity. Previous studies suggest that CtpC and CtpD are particularly relevant for in vivo (macrophage) M. tuberculosis growth. Testing this, M. tuberculosis mutants will be constructed along with strains overexpressing these proteins. Their virulence (in mice and cultured macrophages) and fitness to growth in various media will be assessed. The impact of mutating and overexpressing these proteins on M. tuberculosis metal homeostasis and its ability to respond to the host redox attack will be determined. Results form these studies will provide an initial appreciation of these proteins importance and enable the generation meaningful testable hypothesis on the biological function of these transporters. PUBLIC HEALTH RELEVANCE: Approximately one-third of the world's population is infected with Mycobacterium tuberculosis. In spite of an available vaccine and effective treatments, there are 2 million tuberculosis-related deaths per year. This project will characterize a group of proteins that appear essential for tuberculosis virulence.