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

项目总结/摘要 离子环境是结核分枝杆菌（Mtb）信号的重要来源，是其能够 适应当地环境。它还代表了一个方面，其固有的不均匀性可以用来驱动 在感染期间观察到的Mtb应答和病变结果的显著异质性，这是一个关键的 阻碍有效治疗。事实上，新的荧光报告Mtb菌株已经发现了显著的 离子环境（pH和氯化物）的异质性和体内Mtb的复制状态。但如何 MTB整合来自多种环境线索的信息知之甚少， 了解药物治疗如何影响当地环境，并受当地环境的影响， 体内单个Mtb。为了解决这一关键的知识差距，本项目的目标1将确定 Mtb整合对不同离子信号的响应的机制。这将包括（一） 最近鉴定的影响细菌反应的主Mtb离子信号调节剂的转录研究 pH值，氯化物和钾，和（ii）新的主调节剂的筛选，使用新的诱导 在氯化物和pH响应荧光背景下的转录因子过表达文库 报道Mtb菌株。目的2将描绘如何综合结核分枝杆菌响应离子线索影响感染 异质性和结果，使用关键主离子的缺失和诱导性过表达Mtb菌株 信号调节子和小鼠Mtb感染模型，其概括了在人类感染中观察到的病变类型。 这些研究通过利用独特的荧光环境和复制成为可能 报告基因Mtb菌株，以及一种创新的成像方法，能够实现单细胞体内可视化， 信号量化。最后，目标3试图了解局部离子环境与 对体内Mtb复制和损伤性质的治疗调节。这将集中在两个目前的药物， 受离子环境影响（吡嗪酰胺和氯法齐明），以及最近 鉴定了调节Mtb对氯化物的反应的新化合物。该项目在概念上具有创新性， 它的重点是了解Mtb整合来自多种信号的信息的机制，特别是 在未被充分研究的离子线索的背景下。在使用一种新颖的集成成像方面也有创新 一种在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 在体内的分辨率，同时保留来自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反应整合节点。 所做的发现将进一步建立一个模型，推动该领域超越结核分枝杆菌的总读数 环境适应和感染/治疗结果，对于实现机制和分子 了解Mtb-宿主在体内的相互作用需要开发改进的治疗。

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.