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

项目概要/摘要 结核分枝杆菌 (Mtb) 感染的一个显着特征是异质性，包括多种因素 方面，包括细菌复制状态和局部微环境。这种异质性是存在的 不仅在时间上，而且在空间上，甚至在单个病变内，正如在单个细菌水平上所揭示的那样 集成成像系统，结合使用荧光报告 Mtb 菌株（一种小鼠感染模型） 概括了标志性的干酪样坏死病变和共聚焦成像。虽然环境信号如 由于已知一氧化氮 (NO) 能够促使 Mtb 生长停滞，Mtb 如何协调其复制 与环境线索的反应仍然很大程度上未知。此外，两个关键系统之间的相互作用 在 Mtb 信号转导中发挥核心作用，即丝氨酸/苏氨酸蛋白激酶 (STPK) 和二 组件系统（TCS）也知之甚少。为此，我们最近发现了关键的 转录因子 PrrA，PrrAB TCS 的一部分，作为 (i) Mtb 对多种环境反应的调节剂 线索，包括 NO，和 (ii) 其功能受到 STPK 磷酸化显着调节的 TCS， NO 响应对 Mtb 复制状态的后续影响。因此，该提案的目标 1 旨在阐明 STPK 调节 PrrA 对 Mtb 适应性进入非复制状态的全局转录影响 利用 PrrA STPK 磷酸消融突变体在延长 NO 暴露后状态。细菌二杂交体 将进一步采取方法来揭示负责 PrrA 磷酸化的 STPK。目标2 重点了解 STPK 调节 PrrA 对 Mtb 复制状态的功能影响 在体内感染期间，具有单一细菌分辨率。这将利用复制记者的使用 - 使用我们的集成成像系统表达 STPK 磷酸消融 PrrA 突变体，以描述 STPK 如何 PrrA 的调节可能对不同病变亚位的 Mtb 生长产生不同的影响，并揭示其 与当地 NO 条件有关。该项目在概念上具有创新性，重点关注两者之间的联系 Mtb 中的 STPKs 和 TCS，以及 Mtb 环境响应和复制调节之间的关系。通过铺设 这些研究将为揭示这些待研究概念中的关键连接节点奠定基础 深入了解结核分枝杆菌感染生物学对细菌定植成功至关重要的各个方面，并开辟新的途径 研究旨在理解和利用这些重要方面以达到治疗目的。

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.