Role of Rpf Homologues in M tuberculosis Reactivation

Rpf 同源物在结核分枝杆菌再激活中的作用

• 批准号: 6511347
• 负责人: JOANN M TUFARIELLO
• 金额: $ 12.68万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511347
• 关键词: Mycobacterium smegmatis; Mycobacterium tuberculosis; bacterial proteins; disease /disorder model; genetically modified animals; laboratory mouse; latent bacterial disease; microorganism growth; protein biosynthesis; protein structure function; tuberculosis

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.

描述（由申请人提供）：该申请概述了职业生涯 结合深入研究经验的发展计划 以及细菌发病机制的教学培训。 长期目标是 为候选人作为独立调查员的职业生涯做好准备 学术机构。 结核分枝杆菌 (Mtb) 是一种人类病原体 极其重要，估计每年导致 300 万人死亡 并潜伏感染世界三分之一的人口。 尽管密集 许多实验室努力阐明 Mtb 的分子机制 对于 Mtb 的发病机制，在分子水平上知之甚少 休眠和重新激活。 这个问题至关重要，因为 临床上明显的结核病通常是重新激活的结果， 目前的化疗对潜在的形式几乎没有活性 疾病。 最近在适应不利条件方面学到了很多知识 不形成经典细菌属的环境条件， 形态上不同的孢子，其中一些适应可能具有 与 Mtb 体内持久性直接相关。藤黄微球菌是， 像 Mtb 一样，一种具有富含 GC 基因组的细菌，并且能够在扩展和 培养物不受搅动孵化，进入休眠状态。 对数相 M。 藤黄体分泌一种 16 kDa 的蛋白质，复苏促进因子 (Rpf)， 它能够使休眠的藤黄微球菌复活，并具有促进生长的作用 分枝杆菌培养物也是如此。 Mtb 基因组编码 5 个同源物 M uteus Rpf。 该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。 实验的 方法包括纯化重组组氨酸标签的 Rpf 同源物， 为了检查它们对分枝杆菌生长动力学的影响，并 产生用于中和和表达测定的抗血清。 的表达 Rpf同源物将在Mtb体外在多种生长条件下进行研究 蛋白质和 mRNA 的条件，包括休眠的厌氧模型 水平。将进行 Rpf 同源物的基因破坏并且生长 在持续感染的小鼠模型中研究的突变体动力学。这 将使用微阵列探索对 Rpf 敏感的基因的鉴定 技术和互补/启动子陷阱测定。 预计 对这个假定的细菌信息素家族的研究将改善我们的 了解Mtb在各种环境下的生长调节 条件，并可以深入了解休眠的重要现象 和重新激活。