Elucidating the link between Mycobacterium tuberculosis potassium homeostasis and its lipid metabolism and growth in vivo

项目摘要/摘要 结核分枝杆菌(Mtb)成功的宿主定植需要细菌感知和 在感染期间协调地对不同的环境线索做出反应，并相应地调整其生理。 这包括对酸性pH的反应和维持细菌内离子稳态的能力，以及 感染期间脂类碳源的利用。然而，Mtb离子线索的反应和动态平衡以及 它的脂代谢在功能上是相关的仍然知之甚少。有趣的是，我们发现结核分枝杆菌 脂代谢与其钾(K+)反应/动态平衡密切相关。例如，细菌对 胆固醇在破坏细菌内K+动态平衡和在胆固醇介质中生长时受到抑制 在pH 7时，破坏K+动态平衡的MTB突变体会受到阻碍，但在pH 6时不会受到阻碍。因此，本提案的目标1旨在 确定Mtb K+动态平衡破坏对细菌利用胆固醇能力的全球影响， 检测转录反应和利用荧光胆固醇示踪剂分析 胆固醇摄取。目标2侧重于了解当地环境差异对 感染期间细菌内K+动态平衡和Mtb复制状态，具有单一细菌分辨率。会的 开发一种新的荧光报告程序，报告细菌内K+水平，并对野生型进行测试 MTB和突变体在几个关键的脂类利用调节剂中。体内单菌水平分析特别是 由于结核分枝杆菌在当地的PH值和营养源的显著异质性 感染，这延伸到典型干酪坏死性皮损内的空间不均匀。因此，目标2 研究将进一步利用荧光复制报告与Mtb突变体的组合 细菌内K+稳态，在重述高度结构干酪坏死的小鼠感染模型中 在人类感染期间观察到的损伤。这些体内实验将有助于阐明差异是如何 在当地环境中(pH、碳源等)在感染过程中可能会改变相对活跃的需要 Mtb维持细菌内K+动态平衡以获得复制成功。这个项目在概念上是 在研究线粒体K+动态平衡如何以pH依赖的方式影响其脂质代谢方面具有创新性， 尚未探索的结核分枝杆菌感染生物学的重要方面的交叉。还有更多的方法论 创新发展中新的细菌内[K+]记者描绘了当地环境的影响 体内对线粒体K+动态平衡的影响。这些研究将为理解网络的概念提供基础 结核分枝杆菌代谢与离子线索反应和动态平衡之间的内在联系，这是结核分枝杆菌感染生物学的一个方面 这就有可能被用于治疗目的。

PROJECT SUMMARY/ABSTRACT Successful host colonization by Mycobacterium tuberculosis (Mtb) requires that the bacteria sense and respond to disparate environmental cues coordinately during infection, and adapt its physiology accordingly. This includes an ability to respond to acidic pH and maintain intrabacterial ionic homeostasis, as well as utilization of lipid carbon sources during infection. However, how Mtb ionic cue response and homeostasis and its lipid metabolism are functionally connected remains poorly understood. Intriguingly, we have found that Mtb lipid metabolism and its potassium (K+) response/homeostasis is linked. For example, the bacterial response to cholesterol is repressed upon disruption of intrabacterial K+ homeostasis, and growth in cholesterol media of a Mtb mutant disrupted in K+ homeostasis is impeded at pH 7, but not pH 6. Aim 1 of this proposal thus seeks to define the global impact of Mtb K+ homeostasis disruption on the bacterium’s ability to utilize cholesterol, examining both transcriptional response and utilizing a fluorescent cholesterol tracer for the analysis of cholesterol uptake. Aim 2 focuses on understanding the functional impact of local environmental differences on intrabacterial K+ homeostasis and Mtb replication status during infection, with single bacterium resolution. It will exploit the use of a novel fluorescent reporter that reports on intrabacterial K+ levels, with tests on wild type Mtb and mutants in several key lipid utilization regulators. Single bacterium level analysis in vivo is particularly critical due to the marked heterogeneity in both local pH and nutrient sources experienced by Mtb during infection, which extends to non-uniformity spatially within canonical caseous necrotic lesions. As such, Aim 2 studies will further exploit combination of a fluorescent replication reporter with a Mtb mutant disrupted for intrabacterial K+ homeostasis, in a murine infection model that recapitulates highly structured caseous necrotic lesions observed during human infection. These in vivo experiments will enable elucidation of how differences in local environment (pH, carbon source etc.) during infection may alter the relative need for active maintenance of intrabacterial K+ homeostasis by Mtb for replication success. This project is conceptually innovative in examining how Mtb K+ homeostasis affects its lipid metabolism in a pH-dependent manner, an intersection of vital aspects of Mtb infection biology that is unexplored. There is further methodological innovation in the development of a new intrabacterial [K+] reporter to delineate the impact of local environment in vivo on Mtb K+ homeostasis. These studies will provide the foundation for understanding the concept of an intrinsic link between Mtb metabolism and ionic cue response and homeostasis, a facet of Mtb infection biology that holds the potential for exploitation for therapeutic purposes.