Unraveling the link between serine/threonine protein kinase and two-component system regulation of environment-mediated Mycobacterium tuberculosis growth arrest

项目总结/摘要 结核分枝杆菌（Mtb）感染的一个显著特征是异质性，包括几个 这些方面包括细菌复制状态和局部微环境。这种异质性存在 不仅在时间上，而且在空间上，甚至在单个病变内，如在单个细菌水平上通过 整合成像系统，其结合使用荧光报告Mtb菌株、鼠感染模型 它概括了标志性干酪样坏死病变和共聚焦成像。虽然环境信号， 由于已知一氧化氮（NO）能够驱动Mtb进入生长停滞，Mtb如何协调其复制 与环境线索反应之间的关系仍然是未知的。此外，两个关键系统之间的相互作用 在Mtb信号转导中起核心作用的蛋白激酶，即丝氨酸/苏氨酸蛋白激酶（STPKs）和两种- 组件系统（TCS）也知之甚少。为此，我们最近发现了 转录因子PrrA，PrrAB TCS的一部分，作为（i）Mtb对多种环境应答的调节剂 包括NO在内的信号，和（ii）其功能受STPK磷酸化显著调节的TCS， 因此，本提案的目的1旨在阐明 STPK调节PrrA对Mtb适应性进入非复制型细胞的全局转录影响 状态下延长NO暴露，利用PrrA STPK磷酸化突变体。细菌双杂交 将进一步采取方法来揭示负责PrrA磷酸化的STPKs。目的2 重点是了解STPK调节PrrA对Mtb复制状态的功能后果 在体内感染期间，具有单个细菌分辨率。它会利用复制报告器- 表达STPK磷酸消融PrrA突变体与我们的集成成像系统，以描绘如何STPK PrrA的调节可能会不同程度地影响不同病变亚部位的结核分枝杆菌生长，并揭示其 与当地的NO条件有关。这个项目在概念上是创新的，重点是 STPKs和TCS在结核分枝杆菌，结核分枝杆菌的环境反应和复制调节之间。通过铺设 为揭示这些未充分研究的概念中的关键连接节点奠定基础，这些研究将提供 深入了解对细菌定植成功至关重要的结核分枝杆菌感染生物学方面，并开辟新的途径， 研究旨在了解和利用这些重要方面的治疗目的。

PROJECT SUMMARY/ABSTRACT A marked feature of Mycobacterium tuberculosis (Mtb) infection is heterogeneity that encompasses several aspects, including in bacterial replication status and in the local microenvironment. This heterogeneity exists not just temporally, but also spatially even within a single lesion, as revealed at the single bacterium level by an integrated imaging system that combines the use of fluorescent reporter Mtb strains, a murine infection model that recapitulates hallmark caseous necrotic lesions, and confocal imaging. While environmental signals such as nitric oxide (NO) are known to be able to drive Mtb into growth arrest, how Mtb coordinates its replication with environmental cue response remains largely unknown. Further, the interplay between the two key systems that play central roles in Mtb signal transduction, namely serine/threonine protein kinases (STPKs) and two- component systems (TCSs), is also poorly understood. To this end, we recently uncovered the essential transcription factor PrrA, part of the PrrAB TCS, as (i) a regulator of Mtb response to multiple environmental cues, including NO, and (ii) a TCS whose function is significantly modulated by STPK phosphorylation, with consequent effects on Mtb replication status in response to NO. Aim 1 of this proposal thus seeks to elucidate the global transcriptional impact of STPK regulation of PrrA on the adaptive entry of Mtb into a non-replicating state upon extended NO exposure, utilizing a PrrA STPK phosphoablative mutant. A bacterial-two-hybrid approach will further be undertaken to uncover the STPKs responsible for phosphorylation of PrrA. Aim 2 focuses on understanding the functional consequences of STPK regulation of PrrA on Mtb replication status during infection in vivo, with single bacterium resolution. This will exploit the use of a replication reporter- expressing STPK phosphoablative PrrA mutant with our integrated imaging system, to delineate how STPK regulation of PrrA may differentially influence Mtb growth in disparate lesion sublocations, and reveal its relation to local NO conditions. This project is conceptually innovative in its focus on the connection between STPKs and TCSs in Mtb, and between Mtb environmental response and replication regulation. By laying the groundwork for revealing key connecting nodes in these understudied concepts, these studies will provide insight into facets of Mtb infection biology critical for bacterial colonization success, and open new avenues of study targeted at understanding and exploiting these vital aspects for therapeutic purposes.