Mycobacterium tuberculosis environmental signal integration: single cell in vivo understanding of its influence on infection heterogeneity and treatment efficacy

项目摘要/摘要 离子环境是结核分枝杆菌(Mtb)信号的重要来源，是其能力的关键 适应当地环境。它进一步代表了一个方面，其固有的不一致性可以用来驱动 在感染期间观察到的结核分枝杆菌应答和病变结局的显著异质性，这是一个关键 有效治疗的障碍。事实上，新的荧光报告Mtb菌株已经发现了标记 离子环境(pH和氯化物)的异质性和结核分枝杆菌在体内的复制状态。然而，如何 MTB整合了来自多个环境线索的信息，人们对此知之甚少，在 了解药物治疗如何影响当地环境，并受到当地环境的影响 体内单个结核分枝杆菌。为了解决知识上的这一关键差距，本项目的目标1将定义 Mtb整合对不同离子信号的反应的机制。这将包括(I) 最近发现的影响细菌反应的主要Mtb离子信号调节因子的转录研究 PH、氯化物和钾，以及(Ii)使用新型诱导剂筛选新的主调节器 氯化物和pH响应荧光背景下的转录因子过表达文库 记者Mtb菌株。目标2将描述对离子线索的综合结核分枝杆菌反应如何影响感染。 使用关键主离子的缺失和诱导过表达的Mtb菌株的异质性和结果 信号调节器和小鼠结核分枝杆菌感染模型，概括了在人类感染中观察到的损伤类型。 这些研究是通过利用独特的荧光环境和复制来实现的 报告Mtb菌株，以及一种创新的成像方法，使单细胞体内可视化和 信号量化。最后，目标3试图了解局部离子环境和 体内对结核分枝杆菌复制和损伤特性的治疗调节。这将集中在两种当前的药物上 受到离子环境(吡嗪酰胺和氯法齐明)的影响，以及最近 确定了调节结核分枝杆菌对氯化物反应的新化合物。该项目在概念上具有创新性 它的重点是了解MTB整合来自多个信号的信息的机制，特别是 在未被充分研究的离子线索的背景下。在使用新的集成成像方面也有创新 用单菌水平揭示结核分枝杆菌对环境线索和药物治疗反应的方法 体内分辨率，同时保留完整病变和组织结构的空间信息。这些研究 将阐明代表新的治疗靶点的关键环境响应整合节点。 所取得的发现将进一步建立一个模型，推动该领域超越Mtb的总读数 环境适应和感染/治疗结果，对于实现机制和分子上的 了解结核分枝杆菌-宿主在体内的相互作用是改进治疗方法发展所必需的。

PROJECT SUMMARY/ABSTRACT The ionic milieu is an important source of signals for Mycobacterium tuberculosis (Mtb), key to its ability to adapt to the local environment. It further represents a facet whose inherent non-uniformity can serve to drive the marked heterogeneity in Mtb response and lesion outcome observed during infection, which is a critical impediment to efficient therapy. Indeed, novel fluorescent reporter Mtb strains have uncovered marked heterogeneity in the ionic environment (pH and chloride) and replication status of Mtb in vivo. However, how Mtb integrates information from multiple environmental cues is poorly understood, and there is a gap in knowledge of how drug treatment both affects, and is impacted by, the local environment experienced by individual Mtb in vivo. To address this critical gap in knowledge, Aim 1 of this project will define the mechanisms by which Mtb integrates response to disparate ionic signals. This will encompass (i) transcriptional studies of a recently identified master Mtb ionic signal regulator that affects bacterial response to pH, chloride, and potassium, and (ii) a screen for new master regulators, using a novel inducible transcription factor over-expression library in the background of a chloride and pH-responsive fluorescent reporter Mtb strain. Aim 2 will delineate how an integrated Mtb response to ionic cues affects infection heterogeneity and outcome, using deletion and inducible over-expression Mtb strains of critical master ionic signal regulators and a murine Mtb infection model that recapitulates lesion types observed in human infection. These studies are made possible through exploitation of unique fluorescent environmental and replication reporter Mtb strains, and an innovative imaging approach that enables single cell in vivo visualization and signal quantification. Finally, Aim 3 seeks to understand the relationship between local ionic environment and therapeutic modulation on Mtb replication and lesion properties in vivo. This will focus on two current drugs that are affected by, or influence, the ionic environment (pyrazinamide and clofazimine), as well as recently identified novel compounds that modulate Mtb response to chloride. This project is conceptually innovative in its focus on understanding mechanisms by which Mtb integrates information from multiple signals, particularly in the context of under-studied ionic cues. There is also innovation in the use of a novel integrated imaging approach to reveal Mtb response to environmental cues and drug treatment with single bacterium level resolution in vivo, while retaining spatial information from intact lesion and tissue architecture. These studies will illuminate critical environmental response integration nodes that represent novel therapeutic targets. Discoveries made will further build a model that drives the field beyond aggregate readouts of Mtb environmental adaptation and infection/treatment outcome, vital for achieving the mechanistic and molecular understanding of Mtb-host interactions in vivo required for the development of improved therapeutics.