M. tuberculosis Genes Regulating Persistent Infection

描述(申请人提供)：结核分枝杆菌是结核病(TB)的病原体，每年导致170多万人死亡和800万新感染者。世界卫生组织(世卫组织)最近在非洲宣布进入紧急状态，在所有与获得性免疫缺陷综合征(艾滋病)相关的死亡中，有近三分之一与结核病有关。目前，预计全球将有20亿人持续感染结核分枝杆菌。控制结核分枝杆菌长期感染的过程是复杂和多因素的，人们对它们仍然知之甚少。如果要根除结核病，迫切需要更好地了解管理结核分枝杆菌持久性的建立、维持和重新激活的分子机制。MprAB已被证明在结核分枝杆菌持续感染的建立和维持中发挥作用。MprAB编码一个两组分的信号系统，用于检测、整合和协调特定的生存计划，以响应应激刺激。受MprAB调控的基因包括mprAB本身、应激反应Sigma因子SiGe和SIGB、一种可能的丝氨酸蛋白酶PepD和DosRST调节子。我们假设，在结核分枝杆菌暴露于应激后，细胞表面诱导的错误折叠或改变的蛋白底物的存在激活了MprAB，并启动了下游的生存程序，其中包括诱导丝氨酸蛋白酶降解或重新折叠改变的蛋白底物(S)和组成DosRST调节子的基因，以启动一般的生存反应程序。在这项提案中，提出了两个具体目标。第一个目标将确定PepD的生化功能及其在结核分枝杆菌生理学和发病机制中的重要性。这将通过纯化各种rPepD衍生物并检测它们的酶活性来实现。此外，还将构建PepD突变株，并将其用于各种体外和体内试验，以研究该基因在结核分枝杆菌生理和毒力方面的重要性。最后，将使用蛋白质组学方法鉴定和验证PepD的天然底物靶标。第二个目的是研究MprAB在DosRST调节子调控中的作用。这将通过鉴定和比较MprA和DosR共同调节基因的启动子区域，并在体外巨噬细胞和潜伏性结核病小鼠模型中检查这些基因在各种结核分枝杆菌衍生物中的调节作用来实现。此外，mprA和/或dosR基因缺失对结核分枝杆菌生理和毒力的影响将使用体外存活分析和体外和体内感染模型系统进行评估。这里概述的建议有望增强我们对导致结核分枝杆菌持续存在的因素的全面了解，包括调节这一动态过程的遗传和环境因素。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB) and is responsible for more than 1.7 million deaths and 8 million new infections annually. A state of emergency was recently declared by the World Health Organization (WHO) in Africa where TB has been associated with nearly one-third of all acquired immunodeficiency syndrome (AIDS)-associated deaths. Currently, 2 billion people are predicted to be persistently infected by M. tuberculosis worldwide. The processes that govern long-term infection by M. tuberculosis are complex and multifactorial, and they remain poorly understood. A better understanding of the molecular mechanisms regulating the establishment, maintenance, and reactivation of persistence by M. tuberculosis is urgently needed if TB is to be eradicated. MprAB has been shown to play a role in the establishment and maintenance of persistent infection by M. tuberculosis. mprAB encodes a two-component signaling system that is used to detect, integrate, and coordinate specific survival programs in response to stressful stimuli. Genes regulated by MprAB include mprAB itself, stress-responsive sigma factors sigE and sigB, a putative serine protease pepD, and the DosRST regulon. We hypothesize that the presence of misfolded or altered protein substrates induced in or at the cell surface following exposure of M. tuberculosis to stress activates MprAB and initiates downstream survival programs that include induction of a serine protease to degrade or refold altered protein substrate(s) and genes comprising the DosRST regulon to initiate a general survival response program. In this proposal, two specific aims have been proposed. The first aim will determine the biochemical function of PepD and its importance in M. tuberculosis physiology and pathogenesis. This will be accomplished by purifying various rPepD derivatives and examining their enzymatic activity. In addition, pepD mutant strains will be constructed and utilized in various in vitro and in vivo assays to investigate the importance of this gene in M. tuberculosis physiology and virulence. Finally, the natural substrate targets of PepD will be identified and verified using proteomic approaches. The second aim will investigate the role of MprAB in regulation of the DosRST regulon. This will be accomplished by characterizing and comparing promoter regions from MprA and DosR co- regulated genes, and examining the regulation of these genes in various M. tuberculosis derivatives during growth in macrophages in vitro and in a murine model of latent TB. In addition, consequences of mprA and/or dosR gene deletion on M. tuberculosis physiology and virulence will be assessed using in vitro survival assays and in vitro and in vivo model systems of infection. The proposal outlined here is expected to enhance our overall understanding of the factors contributing to M. tuberculosis persistence, including the genetic and environmental factors that regulate this dynamic process.