Role of Rpf Homologues in M tuberculosis Reactivation

描述（由申请人提供）：此应用程序概述了职业生涯 结合密集的研究经验的发展计划 和细菌致病机理的教学培训。 长期目标是 准备候选人的职业生涯作为一个独立的调查员在一个 学术机构。 结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是一种人类结核病病原体， 每年造成约300万人死亡 并潜在地感染了世界三分之一的人口。 尽管密集 许多实验室致力于阐明结核分枝杆菌的分子机制 发病机制，很少了解在分子水平上关于结核分枝杆菌 休眠和复活 这个问题至关重要，因为 临床上明显的结核病通常是再活化的结果， 而目前的化疗药物对潜在形式的 疾病 最近人们已经了解了许多关于适应不利环境的知识。 不形成经典的细菌属之间的环境条件， 形态分化的孢子，其中一些适应可能具有 与结核杆菌的体内持久性直接相关。藤黄微球菌， 像结核分枝杆菌一样，一种具有富含GC基因组的细菌， 培养物不受搅动地孵化，进入休眠状态。 对数相位M。 黄体分泌 16 kDa蛋白，复苏促进因子（Rpf）， 能够唤醒休眠的M黄体，并具有促进生长的作用， 分枝杆菌培养也是如此。 结核分枝杆菌基因组编码五个同源物， 黄枝藓 该提案概述了一系列旨在 探讨该基因家族在结核分枝杆菌中的功能。 实验 方法包括纯化重组His标记的Rpf同源物， 为了检测它们对分枝杆菌生长动力学的影响， 产生用于中和和表达测定的抗血清。 表达 Rpf同源物将在多种生长条件下在体外Mtb中进行研究。 条件下，包括厌氧模式的休眠，在蛋白质和mRNA 程度.将进行Rpf同源物的基因破坏，并且生长 在持续感染的鼠模型中研究的突变体的动力学。的 将使用微阵列探索对Rpf应答的基因的鉴定 技术和互补/启动子捕获测定。 预计各国 对这个假定的细菌信息素家族的研究将提高我们的 了解结核分枝杆菌在不同环境条件下的生长规律 条件，并可能提供深入了解休眠的重要现象 和重新激活。

DESCRIPTION (provided by applicant): This application outlines a career development plan consisting of combination of intensive research experience and didactic training in bacterial pathogenesis. The long-term goal is to prepare the candidate for a career as an independent investigator at an academic institution. Mycobacterium tuberculosis (Mtb) is a human pathogen of tremendous importance, responsible for an estimated 3 million deaths per year and latently infecting one third of the world s population. Despite intensive effort by numerous laboratories to elucidate the molecular mechanisms of Mtb pathogenesis, little is understood at the molecular level regarding Mtb dormancy and reactivation. This issue is of critical importance, since clinically apparent tuberculous disease is often the result of reactivation, and current chemotherapies have little activity against the latent form of disease. Much has been learned recently about adaptation to adverse environmental conditions among genera of bacteria which do not form classical, morphologically differentiated spores, and some of these adaptations may have direct relevance for the in vivo persistence of Mtb. Micrococcus luteus is, like Mtb, a bacterium with a GC-rich genome, and is able, upon extended and unagitated incubation of cultures, to enter a state of dormancy. Log phase M. luteus secrete an 16 kDa protein, resuscitation-promoting factor (Rpf), which is able to revive dormant M. luteus, and has growth-promoting effects on mycobacterial cultures as well. The Mtb genome encodes five homologues of the M luteus Rpf. This proposal outlines a series of experiments designed to investigate the functions of this gene family in Mtb. The experimental approaches include purification of recombinant his-tagged Rpf homologues, in order to examine their effects on mycobacterial growth kinetics, and to generate antisera for neutralization and expression assays. Expression of the Rpf homologues will be studied in Mtb in vitro under a variety of growth conditions, including anaerobic models of dormancy, at the protein and mRNA levels. Gene disruption of the Rpf homologues will be performed and the growth kinetics of the mutants studied in a murine model of persistent infection. The identification of genes responsive to Rpf will be explored using microarray technology and a complementation/promoter trap assay. It is anticipated that study of this family of putative bacterial pheromones will improve our understanding of the growth regulation of Mtb under various environmental conditions, and may provide insight into the important phenomena of dormancy and reactivation.