MOLECULAR ANALYSES OF VIRULENCE DETERMINANTS OF MYCOBACTERIA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。本项目旨在了解分枝杆菌感染发病的分子和生化机制。为了阐明分枝杆菌致病的分子机制，我们以海洋分枝杆菌(Mm)为模式生物进行研究。MM是一种鱼、两栖动物，也是一种有机会的人类病原体，它的生长速度是结核杆菌的四倍，在实验室操作中提供了安全性和简便性。我们利用Mariner转座子构建了一个mM转座子突变体文库，并筛选了大约1000个突变体。突变株表现出缺陷的表型如下：i)在静息或INF-g激活的巨噬细胞中的细胞内生长；ii)对巨噬细胞的细胞毒性；iii)接触依赖性的红细胞溶血和巨噬细胞的细胞溶解；iv)对防御素的抗性；v)产生NO或红色色素的色素变异体。对转座子连接的测序已经确定了与毒力有关的Mtb同源基因中的插入。其中几个基因位于与结核分枝杆菌Rd1基因座同源的基因组区域，该基因座与毒力因子的分泌有关。我们的一些突变体有非常不正常的分泌模式。我们有兴趣与质谱仪合作，以确定i)野生型和突变型生物之间差异分泌的蛋白质；以及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。我们希望使用质谱仪来确定这些突变体中蛋白质和脂肪变化的性质。

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.