Role of WhiB3 in M. tuberculosis virulence

描述（由申请人提供）：M。结核病（Mtb）是全世界主要的死亡原因之一，每年夺去数百万人的生命。全世界约有17亿人无症状地感染结核杆菌，这是全世界公共卫生控制措施的主要障碍。先前的工作已经表明，牛分枝杆菌中主要的σ因子RpoV的4.2结构域中的点突变（Arg 515->His）是减毒的。使用酵母双杂交系统，我们已经建立了4.2结构域的毒性Mtb特异性相互作用的调节蛋白WhiB 3。相反，含有单点突变的减毒RpoV等位基因不能与WhiB 3相互作用。我们构建了一个Mtb whiB 3突变体（deltawiB 3），并表明它的行为相同的野生型菌株就其在小鼠和豚鼠体内复制的能力。感染AwhiB 3的小鼠显示出比感染野生型Mtb的小鼠显著更长的存活时间。此外，AwhiB 3感染小鼠的肺部似乎受到的不利影响要小得多。值得注意的是，使用传统筛选（例如标记突变）无法检测到该毒力基因，该筛选主要是生长缺陷而不是毒力缺陷的突变体。此外，我们已经证明，一个毒力M.与AwhiB 3相反，Bovis在豚鼠中的生长完全减弱。Mtb含有七个WhiB同源物，其与链霉菌属中对孢子形成至关重要的蛋白质具有很强的同源性。我们假设WhiB 3调节分枝杆菌组分的表达，从而调节宿主免疫系统。为了更好地了解WhiB 3在结核分枝杆菌毒力中的机制，我们将使用电子顺磁共振光谱（EPR）对WhiB 3 Fe-S簇基因进行生物化学表征，鉴定WhiB 3控制下的基因，并表征与WhiB家族相互作用的蛋白质。我们还将证明WhiB 3是一种能够激活特定靶基因转录的DNA结合蛋白。我们将研究whiB家族的体内表达及其在毒力中的作用。这些研究将描述WhiB家族作为干预措施的潜在目标，这些干预措施可能会消除毒力，但不会抑制生长。这些研究还将提供深入了解结核病是否是一种异常的免疫反应，在持续的杆菌，是否杆菌本身诱导致命的免疫病理学，或者如果它是两者的组合。

DESCRIPTION (provided by applicant): M. tuberculosis (Mtb) is one of the leading causes of death worldwide and claims millions of lives annually. Approximately 1.7 billion people worldwide are asymptomatically infected with the tubercle bacillus and constitute a major impediment to worldwide public health control measures. Previous work had shown that a point mutation (Arg515->His) in the 4.2 domain of RpoV, the principal sigma factor in Mycobacterium bovis, is attenuating. Using the yeast two-hybrid system, we have established that the 4.2 domain of virulent Mtb specifically interacts with a regulatory protein WhiB3. In contrast, the attenuated RpoV allele containing the single point mutation was unable to interact with WhiB3. We constructed a Mtb whiB3 mutant (deltawhiB3) and showed that it behaved identical to the wild-type strain with respect to its ability to replicate in mice and guinea pigs in vivo. Mice infected with AwhiB3 showed significantly longer survival times than mice infected with the wild type Mtb. In addition, the lungs of AwhiB3-infected mice appeared much less adversely affected. It is notable that this virulence gene would not have been detected using conventional screens such as signature tagged mutagenesis, which screens for mutants primarily defective in growth, and not virulence. Furthermore, we have shown that a whiB3 mutant of virulent M. bovis, in contrast to AwhiB3, was completely attenuated for growth in guinea pigs. Mtb contain seven WhiB homologues that show strong homology to proteins that are critical for sporulation in Streptomyces spp. We hypothesize that WhiB3 regulates the expression of mycobacterial components that modulate the host immune system. To better understand the mechanism of whiB3 in Mtb virulence, we will use electron paramagnetic resonance spectroscopy (EPR) to biochemically characterize the WhiB3 Fe-S cluster genes, identify genes under WhiB3 control, and characterize proteins that interact with the WhiB family. We will also demonstrate that WhiB3 is a DNA binding protein capable of activating transcription of specific target genes. We will study the in vivo expression of the whiB family and their role in virulence. These studies will characterize the WhiB family as potential targets for interventions that may abolish virulence, but not growth. These studies will also provide insight into understanding whether TB is an anomalous immunological reaction in response to the persistent bacilli, whether the bacilli themselves induce lethal immunopathology, or if it is a combination of both.