Minimizing in vivo Drug Tolerance induction in tuberculosis.

摘要项目 3：最大限度地减少结核病的体内耐药性诱导。 结核分枝杆菌的表型耐药性是一种最重要的表达 正是这些特性使结核菌成为全球健康的主要威胁。耐受性促进细菌在体内的持久存在 面对药物治疗并扩大了基因编码耐药性出现的窗口。的目标 该项目是合理设计一种新的治疗方法，通过以下方式最大限度地减少药物耐受性的诱导： 操纵宿主细胞或免疫环境导致药物耐受。这样的结果将会 增强当前结核病药物治疗方案的功效并减少遗传性耐药性的出现。 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。拉塞尔实验室有 开发了在直接分离的细胞中同时分析宿主和病原体转录组的能力 来自经过药物治疗的动物。萨塞蒂实验室开发了一个补充遗传数据库 全面量化感染过程中细菌突变对耐药性的影响，并利用 CRISPR-Cas9 用于改造原代巨噬细胞系的基因组。 指导该项目的总体假设是 Mtb 的表型耐药性是由 宿主免疫环境，可以对其进行专门调节以提高药物疗效。 目标 1：确定影响 Mtb 药物耐受性的巨噬细胞免疫或代谢途径。 我们将应用不同的 RNA 测序模式来检测抗生素治疗小鼠肺部受感染的巨噬细胞， 识别与耐受相关细菌基因表达相关的宿主途径。融合多元 结核分枝杆菌分离株将这些机制观察结果与临床相关表型联系起来。 目标 2：表征巨噬细胞代谢状态与 Mtb 药物耐受性之间的机制联系。 我们将利用新兴的遗传工具来探索细菌和宿主巨噬细胞的生物学功能 验证导致 Mtb 耐药性诱导的候选途径。我们将使用以下组合 培养物和宿主细胞模型系统将特定的免疫途径与细菌耐药性的诱导联系起来。 目标 3：维持/增强一线药物疗效的治疗干预措施的概念验证。 我们将使用合成 mRNA 和 siRNA 方法来探索体内药物耐受性的途径 最小化，并且可以有效维持一线药物的疗效。还原论动物的组合 模型和人类临床标本将用于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.