Protein Secretion Pathways of Mycobacterium tuberculosis

描述(申请人提供)：结核病仍然是一个严重的全球健康问题。更好地了解导致这种疾病的结核分枝杆菌，将有助于制定新的抗结核战略。与其他细菌病原体一样，结核分枝杆菌的分泌和表面定位蛋白在毒力和宿主免疫发展中具有重要作用。这项研究的长期目标是确定负责将这些蛋白质输出到适当位置的系统，并确定它们在结核分枝杆菌发病机制中所起的作用。我们的研究有助于表明分枝杆菌具有保守的蛋白质输出途径：一般分泌(SEC)途径和双精氨酸转位(TAT)途径。我们还确定了一个专门的系统，专门用于选择分泌和表面蛋白的子集。这种依赖SecA2的输出系统对毒力是重要的，正如结核分枝杆菌secA2突变体在巨噬细胞和小鼠中的减弱表型所表明的那样。感染secA2突变体的巨噬细胞产生更高水平的促炎细胞因子和效应器。我们推测，SecA2在毒力中的作用是限制宿主免疫反应，可能是通过调节MyD88依赖的途径。这项建议的具体目标如下。1)通过对由SecA2改变的巨噬细胞反应进行全球分析，并测试其在巨噬细胞和小鼠体内的免疫调节意义，来研究SecA2在限制宿主反应和促进巨噬细胞生长中的作用。2)用定量蛋白质组学方法鉴定SecA2依赖的蛋白质，并研究SecA2与新发现的Dos调控的细胞膜蛋白之间的关系。3)通过鉴定SecA2出口蛋白中的靶向结构域和鉴定出口中与SecA2一起工作的蛋白质来表征SecA2出口的机制。本方案的研究将加深我们对SecA2输出系统在结核分枝杆菌致病机制中作用的理解，并将阐明这一新型专业化输出途径的机制基础。这一结果可能揭示疾病干预的新方法，并有助于构建具有更强的输出和呈递保护性抗原的能力的分枝杆菌活疫苗。公共卫生相关性：结核病是一个严重的世界卫生问题。结核分枝杆菌是这种疾病的病原体，其分泌和表面定位的蛋白对毒力很重要。这项拟议的研究将定义将这些蛋白质输出到必要位置的系统，并确定它们在结核分枝杆菌发病机制中所起的作用。

DESCRIPTION (provided by applicant): Tuberculosis remains a severe global health problem. A better understanding of Mycobacterium tuberculosis, the bacterium responsible for this disease, will facilitate development of new anti-tuberculosis strategies. As in other bacterial pathogens, the secreted and surface-localized proteins of M. tuberculosis have important roles in virulence and in the development of host immunity. The long-term objective of this research is to define the systems responsible for exporting these proteins to their proper location and determine the role they play in M. tuberculosis pathogenesis. Our research has helped show mycobacteria to possess conserved protein export pathways: the general secretion (Sec) pathway and the twin-arginine translocation (Tat) pathway. We also identified a specialized system that is dedicated to a select subset of secreted and surface proteins. This SecA2-dependent export system is important to virulence as shown by the attenuated phenotype of a M. tuberculosis secA2 mutant in macrophages and mice. Macrophages infected with the secA2 mutant produce higher levels of pro-inflammatory cytokines and effectors. We hypothesize that the role of SecA2 in virulence is to limit host immune responses, possibly through modulation of MyD88-dependent pathways. The specific aims of this proposal are the following. 1) Investigate the role of SecA2 in limiting host responses and promoting growth in macrophages by performing a global analysis of macrophage responses altered by SecA2 and testing the significance of the immunomodulation in macrophages and mice. 2) Identify SecA2-dependent proteins by quantitative proteomics and study the relationship between SecA2 and newly identified Dos regulated cell envelope proteins. 3) Characterize the mechanism of SecA2 export by identifying targeting domains in SecA2-exported proteins and by identifying proteins that work with SecA2 in export. The research in this proposal will improve our understanding of the role played by the SecA2 export system in M. tuberculosis pathogenesis and it will clarify the mechanistic basis of this new type of specialized export pathway. The results may reveal new approaches for disease intervention and facilitate construction of live mycobacterial vaccines with enhanced ability to export and present protective antigens. PUBLIC HEALTH RELEVANCE: Tuberculosis is a severe world health problem. Secreted and surface-localized proteins of Mycobacterium tuberculosis, the causative agent of this disease, are important to virulence. The proposed research will define the systems that export these proteins to their necessary locations and determine the role they play in M. tuberculosis pathogenesis.