Role of WhiB3 in M. tuberculosis virulence

描述(申请人提供)：结核分枝杆菌(结核分枝杆菌)是全球主要死亡原因之一，每年夺走数百万人的生命。全世界约有17亿人无症状地感染结核杆菌，构成了全球公共卫生控制措施的主要障碍。以前的工作已经表明，牛分枝杆菌的主要西格玛因子RpoV 4.2区域的一个点突变(Arg515-&gt；His)正在减弱。利用酵母双杂交系统，我们已经建立了毒力Mtb的4.2结构域与调节蛋白WhiB3特异性相互作用的机制。相反，含有单点突变的弱化RpoV等位基因不能与WhiB3相互作用。我们构建了一个Mtb WhiB3突变体(DeltawhiB3)，并表明它在小鼠和豚鼠体内的复制能力方面与野生型菌株相同。感染AwhiB3的小鼠的存活时间明显长于感染野生型Mtb的小鼠。此外，感染AwhiB3的小鼠的肺似乎受到的不利影响要小得多。值得注意的是，这种毒力基因不会使用传统的筛查方法检测到，例如签名标记突变，它筛选主要生长缺陷的突变株，而不是毒力。此外，我们已经证明，与AwhiB3相比，强毒牛分枝杆菌的WhiB3突变体在豚鼠中的生长完全减弱。MTB包含7个WhiB同源物，它们与链霉菌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WhiB3调节调节宿主免疫系统的分枝杆菌成分的表达。为了更好地了解WhiB3在结核分枝杆菌毒力中的作用机制，我们将利用电子顺磁共振波谱技术对WhiB3Fe-S簇基因进行生化表征，鉴定WhiB3调控基因，并鉴定与WhiB家族相互作用的蛋白质。我们还将证明WhiB3是一种DNA结合蛋白，能够激活特定靶基因的转录。我们将研究WhiB家族在体内的表达及其在毒力中的作用。这些研究将把WhiB家族定性为干预措施的潜在目标，这些干预措施可能会消除毒力，但不会消除增长。这些研究还将有助于理解结核病是否是对持久细菌的异常免疫反应，是细菌本身导致致命的免疫病理，还是两者的组合。

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.