Molecular Analysis of the Rip1 Virulence Pathway of M. tuberculosis

M.结核病感染是一个持续的全球健康危机，需要新的药物或疫苗进行有效控制。虽然许多个别基因已被证明是重要的M。结核菌在小鼠体内的致病机制，目前对M.结核病在分子水平上对不同的宿主环境有反应。Rip 1（Rv 2869 c）膜内蛋白酶是位点2蛋白酶（S2 P）类膜内金属蛋白酶的成员，其在跨膜结构域内切割底物蛋白。我们以前已经证明了Rip 1是M的一个临界行列式。结核病的生长和持久性的小鼠和控制细胞包膜组成通过转录调节脂质生物合成基因。本文提供的进一步初步数据确定了三种抗sigma因子作为Rip 1底物：抗SigK（RskA），抗SigL（RslA）和抗SigM（RsmA）。因此，我们表明SigK、SigL和SigM靶基因在'rip 1菌株中没有被激活，这表明Rip 1的缺失使三个下游转录程序失活。此外，我们表明，Rip 1是必需的铁响应基因的调节，响应铁限制，也需要SigL和SigM的反应。基于这些数据，我们提出并检验了主要假设，即Rip 1空菌株的严重减毒是由于SigK、SigL、SigM调节子的同时失活。为了验证这一假设，我们提出了一个为期两年的项目，以确定Rip 1途径的遗传和生化特征，具体目标如下。我们将确定SigK/anti-sigK，SigL/anti-sigL和SigM/anti-sigM的贡献Rip 1依赖的转录调控的转录谱的定义突变株在高和低铁。我们将在相同条件下使用抗σ因子抗体在野生型和Rip 1缺陷菌株中使用蛋白质印迹法分析抗σ因子切割。最后，我们将通过基因切除候选位点1蛋白酶并在这些突变株中测试抗σ因子裂解来定义抗σ因子位点1裂解的分子基础。 .

M. tuberculosis infection is an ongoing global health crisis that requires new drugs or vaccines for effective control. Although many individual genes have been shown to be important for M. tuberculosis pathogenesis in the mouse, we still understand relatively little about how M. tuberculosis responds to varied host environments at the molecular level. The Rip1 (Rv2869c) intramembrane protease is a member of the Site two protease (S2P) class of intramembrane metalloproteases which cleave substrate proteins within transmembrane domains. We have previously shown that Rip1 is a critical determinant of M. tuberculosis growth and persistence in mice and controls cell envelope composition through transcriptional regulation of lipid biosynthetic genes. Further preliminary data presented here identifies three anti-sigma factors as Rip1 substrates: anti-SigK (RskA), anti-SigL (RslA), and anti-SigM (RsmA). Accordingly, we show that SigK, SigL, and SigM target genes are not activated in the 'rip1 strain, demonstrating that loss of Rip1 inactivates three downstream transcriptional programs. In addition, we show that Rip1 is required for the regulation of iron responsive genes in response to iron limitation, a response that also requires SigL and SigM. Based on this data, we advance and test the major hypothesis that the severe attenuation of the Rip1 null strain is due to simultaneous inactivation of SigK, SigL, SigM regulons. To test this hypothesis, we propose a two year project to define the genetic and biochemical characteristics of the Rip1 pathway outlined in the specific aims below. We will determine the contribution of SigK/anti-sigK, SigL/anti-sigL, and SigM/anti-sigM to Rip1 dependent transcriptional regulation by transcriptional profiling of defined mutant strains in high and low iron. We will analyze anti-sigma factor cleavage in the same conditions using western blotting using anti-sigma factor antibodies in wild type and Rip1 deficient strains. Finally, we will define the molecular basis for site one cleavage of anti-sigma factors by genetically ablating candidate site one proteases and testing anti-sigma factor cleavage in these mutant strains. .