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: 10372365
• 负责人: Anna DeGraff Tischler
• 金额: $ 23.25万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372365
• 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.

项目概要/摘要 结核分枝杆菌 (Mtb) 需要专门的 VII 型蛋白分泌系统 ESX-5 才能生长 在体外，但对感染期间 ESX-5 的表达或活性知之甚少。最近的一份报告建议 ESX-5 基因在小鼠感染急性期被转录诱导。然而，监管 刺激 ESX-5 表达的网络和主机信号以及 ESX-5 的感染阶段 是否诱发还有待全面鉴定。该提案的总体目标是描述 Mtb 在感染过程中如何调节 ESX-5 分泌。拟议的研究将检验中心假设 ESX-5 会根据特定的营养提示或环境压力因素而被激活，以抑制生长 宿主间质巨噬细胞的限制。先前的研究表明磷酸盐限制是一个信号 在体外诱导 ESX-5 活性，但破坏磷酸盐依赖性 ESX-5 调节不会影响 Mtb 小鼠体内的生长或持续存在，表明其他未表征的调节因子控制宿主体内的 ESX-5。 初步数据显示两种不同转录调节因子的过表达可以诱导ESX-5 体外分泌。具体目标 1 将测试特定转录因子在以下情况下激活 ESX-5 的假设： Mtb 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 转录报告基因并在巨噬细胞和小鼠感染中进行测试 使用流式细胞术的模型来确定 ESX-5 表达在何时以及在何种细胞类型中被诱导 感染。还将开发 ESX-5 底物导出的荧光报告基因用于分析 ESX-5 活动 巨噬细胞感染期间。开发的创新荧光报告基因将使未来的屏幕成为可能 破坏 ESX-5 表达和/或活性的小分子。拟议的研究意义重大，因为它 将通过识别主机线索和监管因素来填补 ESX-5 功能知识的重要空白 控制 ESX-5 表达并确定 Mtb 最容易受到药物影响的感染阶段 针对 ESX-5 或其法规。