Minimizing in vivo Drug Tolerance induction in tuberculosis.

摘要项目3：最大限度地减少结核病体内药物耐受诱导。 表型耐药是结核分枝杆菌表达的一个最重要的单 这些特性使结核病成为全球健康的主要威胁。耐受性促进细菌在环境中的持久存在。 面对药物治疗，并扩大了出现遗传编码耐药性的窗口。的目标 该项目是一种新的治疗方法的合理设计，以尽量减少药物耐受性的诱导， 操纵宿主细胞或免疫环境，导致药物耐受性。这样的结果将 提高目前结核病药物治疗方案的疗效，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。罗素实验室 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 药物治疗的动物。Sassetti实验室开发了一个互补的基因数据库， 全面量化了细菌突变对感染期间耐药性的影响，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的表型药物耐受性是由以下因素诱导的： 宿主免疫环境，其可以被特异性地调节以增加药物功效。 目的1：鉴定影响结核分枝杆菌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-seq模式从经抗肿瘤治疗的小鼠的肺中感染巨噬细胞， 鉴定与耐受性相关细菌基因表达相关的宿主途径。融合多样 结核分枝杆菌分离株将这些机制的观察临床相关的表型。 目的2：表征巨噬细胞代谢状态与结核分枝杆菌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测细菌和宿主巨噬细胞的生物学功能， 验证导致Mtb中药物耐受性诱导的候选途径。我们将结合使用 培养和宿主细胞模型系统，以将特异性免疫途径与细菌药物耐受性的诱导联系起来。 目标3：维持/增强一线药物疗效的治疗干预的概念验证。 我们将使用合成的mRNA和siRNA方法来探索体内药物耐受性的途径， 并能有效地维持前线药物的疗效。一种还原主义动物 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.