Minimizing in vivo Drug Tolerance induction in tuberculosis.

项目3：最大限度地减少结核病体内耐药诱导。 结核分枝杆菌耐药表型是单项表达中最显著的之一 使结核分枝杆菌成为全球主要健康威胁的财产。耐受性促进细菌在体内的持久性 面对药物治疗，并扩大了出现基因编码耐药性的窗口。的目标是 项目是合理设计一种新的治疗方法，通过以下方式将药物耐受的诱导降至最低 对宿主细胞或免疫环境的操纵，导致药物耐受。这样的结果将是 提高当前结核病药物治疗方案的疗效，减少可遗传耐药性的出现。 为了实现这一目标，我们将利用我们实验室的一些最新进展。罗素实验室已经 开发了在直接分离的细胞中同时分析寄主和病原体转录本的能力 从接受药物治疗的动物身上。Sassetti实验室开发了一个互补的基因数据库， 全面量化感染期间细菌突变对药物耐受性的影响，并利用 CRISPR-Cas9用于设计原代巨噬细胞系的基因组。 指导该项目的主要假设是结核分枝杆菌的表型药物耐受是由 宿主免疫环境，可以通过特定的调节来提高药物疗效。 目的1：确定影响结核分枝杆菌耐药的巨噬细胞免疫或代谢途径。 我们将把不同的rna-seq模式应用于经抗生素治疗的小鼠肺部感染的巨噬细胞，以 确定与耐受相关细菌基因表达相关的宿主途径。整合多样化的 结核分枝杆菌分离株将把这些机制观察与临床相关的表型联系起来。 目的2：研究巨噬细胞代谢状态与结核分枝杆菌耐药之间的机制联系。 我们将利用新兴的基因工具来探索细菌和宿主巨噬细胞生物学，以实现功能 结核分枝杆菌耐药诱导候选途径的验证。我们将结合使用 培养和宿主细胞模型系统，将特定的免疫途径与细菌耐药诱导联系起来。 目的3：维持/提高一线药物疗效的治疗干预的概念验证。 我们将使用合成的mRNA和siRNA方法来探索体内耐药的途径 最大限度地减少和有效地维持一线药物的疗效。一种简化论动物的组合 模型和人类临床标本将被用来将机制与治疗相关性联系起来。

Abstract Project 3: Minimizing in vivo drug tolerance induction in tuberculosis. Phenotypic drug tolerance in Mycobacterium tuberculosis is an expression of one of the single most significant properties that make Mtb such a major Global Health threat. Tolerance promotes bacterial persistence in the face of drug therapy and expands the window for emergence of genetically-encoded resistance. The goal of the project is the rational design of a novel therapeutic approach to minimize the induction of drug tolerance through manipulation of the host cells or the immune environment that result in drug tolerance. Such an outcome would enhance the efficacy of current TB drug regimens and reduce emergence of heritable drug resistance. To accomplish this goal, we will leverage a number of recent advances from our labs. The Russell lab has developed the capability to simultaneously profile host and pathogen transcriptomes in cells isolated directly from drug-treated animals. The Sassetti lab has developed a complementary genetic database that comprehensively quantifies the effect of bacterial mutations on drug tolerance during infection, and has exploited CRISPR-Cas9 to engineer the genomes of primary macrophage lines. The overarching hypothesis guiding this project is that phenotypic drug tolerance in Mtb is induced by the host immune environment, which can be specifically modulated to increase drug efficacy. Aim 1: Identify macrophage immune or metabolic pathways that influence Mtb drug tolerance. We will apply different RNA-seq modalities to infected macrophages from the lungs of antibiotic-treated mice to identify host pathways that are linked with the expression of tolerance-related bacterial genes. Integrating diverse Mtb isolates will link these mechanistic observations to clinically-relevant phenotypes. Aim 2: Characterize mechanistic links between macrophage metabolic state and Mtb drug tolerance. We will exploit emergent genetic tools to probe bacterial and host macrophage biology for the functional verification of candidate pathways leading to induction of drug tolerance in Mtb. We will use a combination of culture and host cell model systems to link specific immune pathways to induction of bacterial drug tolerance. Aim 3: Proof-of-Concept for therapeutic interventions to maintain/enhance frontline drug efficacy. We will use synthetic mRNA and siRNA approaches to explore avenues whereby in vivo drug tolerance can be minimized and the efficacy of frontline drugs can be effectively sustained. A combination of reductionist animal models and human clinical specimens will be used to link mechanism with therapeutic relevance.