Molecular Analysis of the Rip1 Virulence Pathway of M. tuberculosis

结核分枝杆菌 Rip1 毒力途径的分子分析

基本信息

批准号：
7895718
负责人：
Michael Stephen Glickman
金额：
$ 47.48万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-17 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7895718
关键词：
Aerosols Anabolism Antibodies Biochemical Candidate Disease Gene Characteristics Cleaved cell Data Drug Delivery Systems Environment Gene Expression Gene Targeting Genes Genetic Genetic Transcription Growth Individual Infection Iron Lipids Metalloproteases Molecular Molecular Analysis Mus Mutation Mycobacterium tuberculosis Pathogenesis Pathway interactions Peptide Hydrolases Pharmaceutical Preparations Phenotype Proteins Proteolysis Regulation Regulon Relative (related person)Role Siderophores Sigma Factor Signal Transduction Site Starvation Testing Transcriptional Activation Transcriptional Regulation Transmembrane Domain Tuberculosis Vaccines Virulence Western Blotting attenuation base cell envelope deprivation global health in vivo insight member mouse model mutant mycobactins pathogen programs promoter research study response

项目摘要

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. .

结核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。尽管已经证明许多单独的基因对于小鼠的结核分枝杆菌发病机理很重要，但我们仍然对结核分枝杆菌对分子水平的各种宿主环境的反应相对较少。 RIP1（RV2869C）内膜内蛋白酶是位点的成员，两种蛋白酶（S2P）类的膜内金属蛋白酶，它们在跨膜结构域内切割底物蛋白。我们先前已经表明，RIP1是小鼠结核分枝杆菌生长和持久性的关键决定因素，并通过脂质生物合成基因的转录调节来控制细胞包膜组成。此处介绍的进一步的初步数据将三个抗sigma因子确定为RIP1底物：抗SIGK（RSKA），抗SIGL（RSLA）和抗SIGM（RSMA）。因此，我们表明SIGK，SIGL和SIGM靶基因未在RIP1菌株中激活，这表明RIP1的丧失会使三个下游转录程序失活。此外，我们表明，对铁限制的铁反应基因调节是必需的，这一响应也需要Sigl和Sigm。基于这些数据，我们提出并检验了重大假设，即RIP1无菌株的严重衰减是由于SIGK，SIGL，SIGM调节子的同时失活。为了检验这一假设，我们提出了一个为期两年的项目，以定义以下特定目的中概述的RIP1途径的遗传和生化特征。我们将确定SIGK/抗SIGK，SIGL/ANTI-SIGL和SIGM/ANTI-SIGM对RIP1依赖性转录调控的贡献，通过对高和低铁中定义突变菌株的转录分析。我们将使用抗sigma因子因子抗体在野生型和RIP1缺乏菌株中使用蛋白质印迹分析相同条件下的抗盐因子裂解。最后，我们将通过遗传烧蚀候选位点的蛋白酶并在这些突变菌株中测试抗sigma因子裂解，从而定义抗sigma因子的位点裂解的分子基础。。