MOLECULAR ANALYSES OF VIRULENCE DETERMINANTS OF MYCOBACTERIA

分枝杆菌毒力决定因素的分子分析

• 批准号: 7724169
• 负责人: Eric J. Brown
• 金额: $ 0.67万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724169
• 关键词: Affect; Amphibia; Animal Model; Biochemical; Computer Retrieval of Information on Scientific Projects Database; Cytolysis; Defensins; Erythrocytes; Exhibits; Fishes; Funding; Genomics; Genus Mycobacterium; Grant; Growth; Hemolysis; Homologous Gene; Human; Institution; Laboratories; Libraries; Lipids; Molecular; Mycobacterium Infections; Mycobacterium tuberculosis; Nature; Open Reading Frames; Organism; Pathogenesis; Pattern; Phenotype; Pigmentation physiologic function; Pigments; Proteins; Research; Research Personnel; Resistance; Resources; Rest; Safety; Source; System; Time; United States National Institutes of Health; Variant; Virulence; Virulence Factors; Work; base; cytokine; cytotoxicity; interest; macrophage; mutant; mycobacterial; pathogen; protein protein interaction

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project aims at understanding the molecular and biochemical mechanisms of pathogenesis of mycobacterial infections. To elucidate the molecular mechanisms of mycobacterial pathogenesis, we use M. marinum (Mm) as a model organism to facilitate the research. Mm, a fish, amphibian, and opportunistic human pathogen, grows four times faster than Mtb and provides safety and ease in laboratory manipulations. We constructed a Mm transposon mutant library by using a mariner-based transposon and screened approximately 1000 mutants. Mutants were identified exhibiting defective phenotypes as follows: i) intracellular growth in resting or INF-g-activated macrophages; ii) cytotoxicity to macrophage; iii) contact-dependent hemolysis of red blood cell and cytolysis of macrophage; iv) resistance to defensin; and v) pigmentation variants that produced either no or red pigment. Sequencing of the transposon junctions has identified insertions within Mtb homologous genes that are implicated in virulence. Several of these are in a genomic region homologous to the RD1 locus of M. tuberculosis that has been implicated in secretion of virulence factors. Some of our mutants have quite abnormal secretion patterns. We are interested in working with the mass spec facility to determine I) the proteins that are differentially secreted between wildtype and mutant organisms; and II) the protein-protein interactions involved in the RD1 secretion system. Finally, mutants have been found in unknown open reading frames that affect cytokine secretion by infected macrophages. We would like to use mass spec to determine the nature of the protein and lipid alterations in these mutants.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该项目旨在了解分枝杆菌感染发病机制的分子和生化机制。为了阐明分枝杆菌发病机制的分子机制，我们使用海分枝杆菌（Mm）作为模式生物来促进研究。 Mm 是一种鱼类、两栖动物和机会性人类病原体，其生长速度比 Mtb 快四倍，并且在实验室操作中提供安全性和便利性。 我们利用基于mariner的转座子构建了Mm转座子突变体库，并筛选了大约1000个突变体。突变体被鉴定为表现出如下缺陷表型：i)静息或INF-g激活的巨噬细胞的细胞内生长； ii) 对巨噬细胞的细胞毒性； iii) 红细胞的接触依赖性溶血和巨噬细胞的细胞溶解； iv) 对防御素的抵抗； v) 不产生或产生红色色素的色素沉着变体。 转座子连接点的测序已鉴定出与毒力有关的结核分枝杆菌同源基因内的插入。其中一些基因组区域与结核分枝杆菌的 RD1 位点同源，该区域与毒力因子的分泌有关。 我们的一些突变体具有非常异常的分泌模式。我们有兴趣与质谱设施合作以确定 I) 野生型和突变体生物体之间差异分泌的蛋白质； II) RD1 分泌系统中涉及的蛋白质-蛋白质相互作用。 最后，在未知的开放阅读框中发现了影响受感染巨噬细胞分泌细胞因子的突变体。我们希望使用质谱来确定这些突变体中蛋白质和脂质改变的性质。