In vivo Reporters of Mycobacterium tuberculosis ESX-5 Secretion

结核分枝杆菌 ESX-5 分泌的体内报告基因

• 批准号: 10493351
• 负责人: Anna DeGraff Tischler
• 金额: $ 19.38万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10493351
• 关键词: Acute; Aerosols; Alveolar; Cues; Data; Development; Drug Targeting; Drug resistance; ELF3 gene; Environment; Family; Flow Cytometry; Future; Gene Expression; Genes; Genetic Transcription; Goals; Growth; Hand; In Vitro; Infection; Iron; Knowledge; Lung; Mission; Modeling; Monitor; Mus; Mycobacterium tuberculosis; Nitrogen; Nutrient; Nutritional; Oxidative Stress; Oxygen; Pathogenesis; Phagocytes; Production; Protein Export Pathway; Protein Secretion; Proteins; Quantitative Reverse Transcriptase PCR; Regulation; Reporter; Reporting; Research; Role; Signal Transduction; Stress; System; Testing; Tissues; Tuberculosis; United States National Institutes of Health; activating transcription factor; acute infection; antimicrobial; burden of illness; cell type; chronic infection; combat; drug development; environmental stressor; genome-wide; in vitro activity; in vivo; innovation; inorganic phosphate; interstitial; macrophage; monocyte; mutant; new therapeutic target; nitrosative stress; novel; novel therapeutics; overexpression; pathogen; response; small molecule; small molecule inhibitor; transcription factor; transcriptomics; tuberculosis drugs; tuberculosis treatment

PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb) requires the specialized Type VII protein secretion system ESX-5 for growth in vitro, but little is known about the expression or activity of ESX-5 during infection. A recent report suggested ESX-5 genes are transcriptionally induced at the acute stage of infection in mice. However, the regulatory networks and host signals that stimulate ESX-5 expression as well as the stage(s) of infection at which ESX-5 is induced remain to be comprehensively identified. The overall objective of this proposal is to characterize how Mtb regulates ESX-5 secretion during infection. The proposed research will test the central hypothesis that ESX-5 is activated in response to specific nutritional cues or environmental stressors to counteract growth restriction in host interstitial macrophages. Prior studies implicated phosphate limitation as one signal that induces ESX-5 activity in vitro, but disrupting phosphate-dependent ESX-5 regulation did not impact Mtb growth or persistence in mice, suggesting that other uncharacterized regulators control ESX-5 in the host. Preliminary data show that over-expression of two different transcriptional regulators can induce ESX-5 secretion in vitro. Specific Aim 1 will test the hypothesis that specific transcription factors activate ESX-5 in response to nutritional cues and stress conditions that Mtb encounters in host macrophages. In Specific Aim 2, fluorescent ESX-5 transcriptional reporters will be developed and tested in macrophage and mouse infection models using flow cytometry to determine when and in what cell types ESX-5 expression is induced during infection. Fluorescent reporters of ESX-5 substrate export will also be developed for analysis of ESX-5 activity during macrophage infection. The innovative fluorescent reporters developed will enable future screens for small molecules that disrupt ESX-5 expression and/or activity. The proposed research is significant because it will fill important gaps in knowledge of ESX-5 function by identifying the host cues and regulatory factors that control ESX-5 expression and establishing the stage(s) of infection when Mtb will be most vulnerable to drugs targeting ESX-5 or its regulation.

项目摘要/摘要 结核分枝杆菌(Mtb)生长需要专门的VII型蛋白分泌系统ESX-5 在体外，但对ESX-5在感染过程中的表达或活性知之甚少。最近的一份报告显示 ESX-5基因在小鼠感染的急性阶段被转录诱导。然而，监管机构 刺激ESX-5表达的网络和宿主信号以及ESX-5感染的阶段(S) 其诱因仍有待全面鉴定。这项提案的总体目标是 结核分枝杆菌在感染过程中如何调节ESX-5分泌。这项拟议的研究将检验中心假设 ESX-5被激活，以响应特定的营养线索或环境应激源来抵消生长 限制宿主间质巨噬细胞。先前的研究表明，磷酸盐限制是一个信号 体外诱导ESX-5活性，但干扰磷酸依赖的ESX-5调节不影响Mtb 在小鼠体内的生长或持久性，表明其他未确定的调节因子控制宿主中的ESX-5。 初步数据显示，两种不同转录调控因子的过度表达可以诱导ESX-5 体外分泌。特定目标1将测试特定转录因子激活ESX-5在 对结核分枝杆菌在宿主巨噬细胞中遇到的营养线索和应激条件的反应。在具体目标2中， 将开发荧光ESX-5转录报告，并在巨噬细胞和小鼠感染中进行测试 使用流式细胞术确定ESX-5在何时以及在什么细胞类型中表达的模型 感染。还将开发ESX-5底物出口的荧光记者，用于分析ESX-5的活性 在巨噬细胞感染期间。开发的创新荧光记者将使未来的屏幕 干扰ESX-5表达和/或活性的小分子。这项拟议的研究意义重大，因为它 将通过确定宿主提示和监管因素来填补ESX-5功能知识中的重要空白 控制ESX-5的表达，确立结核分枝杆菌对药物最敏感的感染阶段(S) 目标是ESX-5或其监管。