M.tuberculosis Rpfs: Modulators of Reactivation

描述（由申请人提供）：结核病（TB）是由单一细菌病原体引起的主要死亡原因。艾滋病流行影响了结核病的复苏，因为HIV感染者每年有10%的再激活风险，而免疫功能正常者的终身风险为-10%。由于世界上三分之一的人口感染了结核分枝杆菌（Mtb），并有重新激活的风险，如果要成功控制结核病流行，就必须解决潜伏感染的负担。尽管在结核分枝杆菌发病机制中的再激活的中心地位，很少有人知道的机制，保持潜伏期，并允许以后复发的疾病。特别是，在休眠中重新出现的细菌因素仍然难以捉摸。结核分枝杆菌编码与藤黄微球菌复苏促进因子（Rpf）同源的5个基因。Rpf是一种16 kDa的分泌蛋白，当加入到“休眠”M中时，其刺激菌落计数增加1000倍。黄体。Mtb Rpfs还刺激稳定期M的生长。bovis BCG在体外，但在体内的作用，该基因家族仍然不完全确定。我观察到，rpfs可以删除单独从结核分枝杆菌染色体没有明显的生长缺陷，在体外或体内，在急性感染。我现在发现，使用鼠TB潜伏期模型，Mtb Rv 1009（rpf）缺失突变体在施用一氧化氮合酶抑制剂后显示出明显延迟的再活化动力学。据我们所知，这是第一个Mtb突变体在体内具有特定的再活化缺陷。对这种表型的更全面的了解将有助于阐明分枝杆菌休眠和再激活的机制，并可能提供新的治疗靶点。该提议提出了Rv 1009是刺激“休眠”杆菌再活化的分泌因子的假设，并从三个角度检查了Rv 1009突变体的延迟再活化表型：使用小鼠再活化模型表征潜在的免疫机制，探索作为体外相关性的厌氧休眠模型，以及确定多Rpf敲除是否显示延迟再活化。相关性：结核病每年造成200万人死亡。M.结核病（Mtb）在感染者体内可以潜伏数十年，然后重新激活引起疾病。由于更好地了解再激活过程对控制结核病至关重要，因此本提案侧重于Mtb基因家族，该基因家族被认为在调节从休眠状态的再激活中发挥作用。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is the leading cause of death due to a single bacterial pathogen. The AIDS epidemic has impacted the resurgence of TB, as HIV-infected persons have a 10% annual risk of reactivation vs. an -10% lifetime risk for immunocompetent persons. Since one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb) and at risk for reactivation, it is crucial that the burden of latent infection be addressed if attempts to control the TB epidemic are to succeed. Despite the centrality of reactivation in Mtb pathogenesis, little is known about the mechanisms which maintain latency and permit the later recrudescence of disease. In particular, bacterial factors at play in re-emergence from dormancy remain elusive. Mtb encodes five genes with homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. Rpf is a 16 kDa secreted protein which stimulates a 1000-fold increase in colony counts when added to "dormant" M. luteus. Mtb Rpfs also stimulate growth of stationary phase M. bovis BCG in vitro, yet the in vivo role of this gene family remains incompletely defined. I observed that rpfs can be deleted individually from the Mtb chromosome without obvious growth defects in vitro or in vivo, during acute infection. I have now found, using a murine TB latency model, that the Mtb Rv1009 (rpf) deletion mutant shows markedly delayed kinetics of reactivation following administration of a nitric oxide synthase inhibitor. To our knowledge this is the first Mtb mutant with a specific in vivo defect in reactivation. Fuller understanding of this phenotype should shed light on mechanisms of mycobacterial dormancy and reactivation, and may offer novel therapeutic targets. This proposal addresses the hypothesis that Rv1009 is a secreted factor which stimulates reactivation of "dormant" bacilli and examines the delayed reactivation phenotype of the Rv1009 mutant from three points of view: characterizing the underlying immune mechanisms using murine models of reactivation, exploring an anaerobic dormancy model as an in vitro correlate, and determining whether multi- Rpf knockouts display delayed reactivation. Relevance: Tuberculosis (TB) causes 2 million deaths per year. M. tuberculosis (Mtb) can remain dormant within infected individuals for decades before reactivating to cause disease. Because a better understanding of the reactivation process is critical to control of TB, this proposal focuses on an Mtb gene family believed to play a role in regulating reactivation from the dormant state.