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

项目摘要/摘要 结核分枝杆菌(Mtb)感染的一个显著特征是异质性，它包括几个 在一些方面，包括细菌复制状况和当地微环境。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，正如在单个细菌水平上所揭示的那样 结合使用荧光报告Mtb菌株的集成成像系统，建立小鼠感染模型 这概括了干酪性坏死性病变的特点，以及共焦成像。而环境信号，如 由于已知一氧化氮(NO)能够使结核分枝杆菌生长停滞，因此结核分枝杆菌如何协调其复制 对环境线索的反应在很大程度上仍是未知的。此外，这两个关键系统之间的相互作用 它们在MTB信号转导中起着核心作用，即丝氨酸/苏氨酸蛋白激酶(STPKs)和两个- 组件系统(TCS)，也是知之甚少。为此，我们最近发现了 转录因子PrrA，PrrAB TCS的一部分，作为(I)MTB对多种环境反应的调节因子 信号，包括NO，和(Ii)其功能受STPK磷酸化显著调节的TCS， NO对结核分枝杆菌复制状态的后续影响。因此，本提案的目标1试图澄清 PrrA的STPK调控对结核分枝杆菌适应进入非复制型的全球转录影响 使用PrrA STPK磷酸化突变体，在延长不暴露时处于状态。细菌双杂交种 将进一步采取方法来发现负责PrrA磷酸化的STPKs。目标2 重点了解PrrA的STPK调控对MTB复制状态的功能影响 体内感染过程中，随单个细菌的分解。这将利用复制报告程序- 用我们的集成成像系统表达STPK磷酸化PrrA突变体，以描绘STPK是如何 PrrA的调节可能不同地影响不同病变亚组的结核分枝杆菌生长，并揭示其 关系到当地没有条件。这个项目在概念上是创新的，它关注的是 Mtb中的STPKs和TCSs，以及Mtb环境反应和复制调控之间的关系。通过铺设 这些研究将为揭示这些未被研究的概念中的关键连接节点奠定基础 洞察结核分枝杆菌感染生物学对细菌定植成功的关键方面，并开辟新的途径 旨在了解和利用这些重要方面以达到治疗目的的研究。

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.