Minimizing in vivo Drug Tolerance induction in tuberculosis.

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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.