Role of Rpf Homologues in M tuberculosis Reactivation

描述(由申请者提供)：这份申请表概述了你的职业生涯 由密集的研究经验组成的发展计划 以及细菌发病机制的说教训练。长期目标是 为候选人成为一名独立调查员做好准备 学术机构。结核分枝杆菌(Mtb)是一种人类致病菌。 非常重要，据估计每年造成300万人死亡 并潜伏着感染了世界三分之一的S人口。尽管强度很大 众多实验室为阐明结核分枝杆菌的分子机制所做的努力 发病机制，在分子水平上对结核分枝杆菌知之甚少。 休眠和重新激活。这个问题至关重要，因为 临床上明显的结核性疾病通常是重新激活的结果， 而目前的化疗方法对潜伏期的癌症几乎没有活性。 疾病。最近关于适应逆境的研究已经有了很多。 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 与结核分枝杆菌体内持久性直接相关。黄微球菌是， 像结核分枝杆菌一样，一种具有丰富GC基因组的细菌，并且能够在扩展和 未经发酵培养，进入休眠状态。对数阶段M。 黄体分泌一个16 kDa的蛋白质，即复苏促进因子(RPF)， 能使休眠的黄体分枝杆菌苏醒，并有促进生长的作用。 还有分枝杆菌培养。结核分枝杆菌基因组编码5个同源基因 M luteus rpf.这份提案概述了一系列旨在 研究该基因家族在结核分枝杆菌中的功能。实验性的 方法包括纯化重组的组氨酸标记的RPF同系物，在 为了检测它们对分枝杆菌生长动力学的影响，并 制备中和和表达检测用抗血清。的表达 RPF同系物将在Mtb的体外各种生长条件下进行研究 条件，包括无氧休眠模式，在蛋白质和mRNA 级别。RPF同源物的基因破坏将被执行，并生长 在持续感染的小鼠模型中研究了突变的动力学。这个 将利用微阵列探索识别对RPF有反应的基因 技术和互补/启动子诱捕试验。预计 对这一可能的细菌信息素家族的研究将改善我们的 对不同环境下Mtb生长规律的认识 条件，并可能提供对休眠的重要现象的洞察 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.