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

阐明结核分枝杆菌钾稳态与其脂质代谢和体内生长之间的联系

• 批准号: 10509286
• 负责人: Shumin Tan
• 金额: $ 24.75万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-25 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10509286
• 关键词: Affect; Architecture; Bacteria; Biology; C3HeB/FeJ Mouse; Carbon; Chlorides; Cholesterol; Complement; Comprehension; Cues; Data; Development; Ensure; Environment; Environmental Impact; Equilibrium; Foamy Macrophage; Foundations; Genetic Transcription; Goals; Growth; Heterogeneity; Homeostasis; Human; Individual; Infection; Lesion; Light; Link; Lipids; Maintenance; Metabolism; Methodology; Microscopy; Modeling; Mus; Mycobacterium tuberculosis; Necrotic Lesion; Nutrient; Pathway interactions; Physiology; Potassium; Reporter; Reporting; Resolution; Role; SS DNA BP; Signal Transduction; Source; Structure; System; Testing; Therapeutic; Tissues; Tracer; Tuberculosis; Variant; environmental disparity; experience; experimental study; extracellular; host colonization; in vivo; innovation; insight; lipid metabolism; mutant; novel; overexpression; response; success; transcription factor; transcriptome sequencing; uptake

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.

项目总结/摘要 结核分枝杆菌（Mtb）的成功宿主定殖需要细菌感测和 在感染过程中协调地对不同的环境线索做出反应，并相应地调整其生理机能。 这包括响应酸性pH和维持细菌内离子稳态的能力，以及 在感染期间利用脂质碳源。然而，如何结核杆菌离子线索的反应和稳态， 其脂质代谢在功能上的联系仍然知之甚少。有趣的是，我们发现结核分枝杆菌 脂质代谢和其钾（K+）反应/体内平衡是相关联的。例如，细菌对 胆固醇在细菌内K+稳态破坏时受到抑制， Mtb突变体在K+稳态中被破坏，在pH 7下受到阻碍，但在pH 6下不受阻碍。因此，本提案的目标1力求 确定结核分枝杆菌K+稳态破坏对细菌利用胆固醇的能力的全球影响， 检查转录反应并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+稳态和Mtb复制状态，单个细菌分辨率。它将 利用一种新的荧光报告基因，报告细菌内K+水平，并对野生型进行测试 几种关键脂质利用调节因子的结核分枝杆菌及其突变体。体内单个细菌水平分析特别适用于 关键是由于在当地的pH值和营养来源的显着异质性经历的结核分枝杆菌在 感染，其在典型干酪样坏死病变内扩展到空间上的不均匀性。因此，Aim 2 研究将进一步开发荧光复制报告基因与被破坏的Mtb突变体的组合， 在小鼠感染模型中再现高度结构化干酪性坏死的细菌内K+稳态 在人类感染期间观察到的病变。这些体内实验将能够阐明差异如何 在当地环境中（pH、碳源等）在感染期间可能会改变对活性物质的相对需求， 通过Mtb维持细菌内K+稳态以获得复制成功。这个项目在概念上 在研究结核分枝杆菌K+稳态如何以pH依赖性方式影响其脂质代谢方面具有创新性， Mtb感染生物学重要方面的交叉点尚未探索。还有一种方法 创新开发了一种新的细菌内[K+]报告基因，以描述局部环境的影响 体内对Mtb K+稳态的影响。这些研究将为理解一个概念的基础， 结核分枝杆菌代谢与离子提示反应和稳态之间内在联系，结核分枝杆菌感染生物学的一个方面 有可能被用于治疗目的。