MOLECULAR ANALYSES OF VIRULENCE DETERMINANTS OF MYCOBACTERIA

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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，我们使用M。marinum（Mm）作为模式生物，为研究提供便利。Mm是一种鱼类、两栖动物和机会性人类病原体，生长速度比Mtb快四倍，在实验室操作中提供了安全性和便利性。 我们利用基于mariner的转座子构建了Mm转座子突变体库，并筛选了约1000个突变体。鉴定了表现出如下缺陷表型的突变体：i）在静息或INF-g活化的巨噬细胞中的细胞内生长; 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）对防御素的抗性;以及v）不产生或产生红色素的色素沉着变体。 转座子连接的测序已经确定了与毒力有关的Mtb同源基因内的插入。其中几个在与M的RD 1基因座同源的基因组区域中。与毒力因子分泌有关的结核病。 我们有些变种人的分泌模式很不正常。我们有兴趣与质谱设备合作，以确定I）野生型和突变生物体之间差异分泌的蛋白质;和II）RD 1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架中发现了突变体，这些突变体影响受感染的巨噬细胞分泌细胞因子。我们想用质谱来确定这些突变体中蛋白质和脂质改变的性质。

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.