Molecular Analysis of the Rip1 Virulence Pathway of M. tuberculosis

结核分枝杆菌感染是一种持续的全球健康危机，需要新药或疫苗才能有效控制。虽然许多单独的基因已经被证明在小鼠中对结核分枝杆菌的致病作用很重要，但我们对结核分枝杆菌在分子水平上如何响应不同的宿主环境的了解仍然相对较少。RIP1(Rv2869c)膜内蛋白水解酶是S2P类膜内金属蛋白水解酶中的一员，其作用是在跨膜区内切割底物蛋白。我们先前已经证明，RIP1是小鼠中结核分枝杆菌生长和持续存在的关键决定因素，并通过脂类生物合成基因的转录调控来控制细胞膜成分。进一步的初步数据确定了三种抗Sigma因子为RIP1底物：抗SigK(RskA)、抗Sigl(RSLA)和抗Sigm(RSMA)。因此，我们证明了SigK、Sig1和Sigm靶基因在‘RIP1菌株中没有激活，表明RIP1的缺失会使三个下游转录程序失活。此外，我们还发现RIP1是调节铁反应基因对铁限制作出反应所必需的，这一反应也需要Sig1和Sigm。基于这一数据，我们提出并检验了主要假设，即RIP1缺失株的严重衰减是由于SigK、Sig1、Sigm调节子同时失活所致。为了验证这一假设，我们提出了一个为期两年的项目，以定义下面具体目标中概述的RIP1途径的遗传和生化特征。我们将通过定义的高铁和低铁突变株的转录图谱来确定SigK/anti-SigK、Sigl/anti-Sigl和Sigm/anti-Sigm对RIP1依赖的转录调控的贡献。我们将使用抗Sigma因子抗体在相同条件下用免疫印迹法分析野生型和RIP1缺陷株中抗Sigma因子的裂解。最后，我们将通过在这些突变株中去除候选的第一位点蛋白酶并测试抗西格玛因子的切割来确定第一位点裂解抗西格玛因子的分子基础。 。

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. .