M.tuberculosis Rpfs: Modulators of Reactivation

描述(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死灰复燃，因为艾滋病毒感染者每年有10%的重新激活风险，而具有免疫能力的人的终身风险为-10%。由于世界上三分之一的人口感染了结核分枝杆菌(Mtb)并面临重新激活的风险，如果要控制结核病流行的努力取得成功，解决潜伏感染的负担至关重要。尽管再激活在结核分枝杆菌发病机制中起着中心作用，但人们对维持潜伏期和允许疾病稍后复发的机制知之甚少。特别是，在从休眠中苏醒的过程中发挥作用的细菌因素仍然难以捉摸。MTB编码5个与黄微球菌复苏促进因子(RPF)同源的基因。RPF是一种16 kDa的分泌性蛋白质，当加入“休眠”的黄体分枝杆菌时，它能刺激1000倍的菌落数量增加。MTB rPFS在体外也能刺激BCG静止期的生长，但该基因家族在体内的作用尚不完全清楚。我观察到，在体外或体内，在急性感染期间，rPFS可以从结核分枝杆菌染色体上单独删除，而没有明显的生长缺陷。我现在使用小鼠结核病潜伏期模型发现，在给予一氧化氮合酶抑制剂后，Mtb Rv1009(Rpf)缺失突变体的重新激活动力学明显延迟。据我们所知，这是第一个在体内存在重新激活缺陷的结核分枝杆菌突变体。对这种表型的更充分的理解将有助于阐明分枝杆菌休眠和重新激活的机制，并可能提供新的治疗靶点。该建议解决了Rv1009是一个刺激“休眠”杆菌重新激活的分泌因子的假设，并从三个角度检查了Rv1009突变体的延迟重新激活表型：使用小鼠重新激活模型表征潜在的免疫机制，探索体外无氧休眠模型，以及确定多个RPF基因敲除是否显示延迟重新激活。相关性：结核病每年导致200万人死亡。结核分枝杆菌(Mtb)可在感染者体内潜伏数十年，然后重新激活而致病。由于更好地了解重新激活过程对控制结核病至关重要，该建议侧重于Mtb基因家族，据信该家族在调节休眠状态的重新激活方面发挥作用。

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.