Mycobacterium tuberculosis (Mtb) infection is notoriously difficult to treat. Treatment efficacy is limited by Mtb’s intrinsic drug resistance, as well as its ability to evolve acquired resistance to all antituberculars in clinical use. A deeper understanding of the bacterial pathways that influence drug efficacy could facilitate the development of more effective therapies, identify new mechanisms of acquired resistance, and reveal overlooked therapeutic opportunities. Here we developed a CRISPR interference chemical-genetics platform to titrate the expression of Mtb genes and quantify bacterial fitness in the presence of different drugs. We discovered diverse mechanisms of intrinsic drug resistance, unveiling hundreds of potential targets for synergistic drug combinations. Combining chemical genetics with comparative genomics of Mtb clinical isolates, we further identified several previously unknown mechanisms of acquired drug resistance, one of which is associated with a multidrug-resistant tuberculosis outbreak in South America. Lastly, we found that the intrinsic resistance factor whiB7 was inactivated in an entire Mtb sublineage endemic to Southeast Asia, presenting an opportunity to potentially repurpose the macrolide antibiotic clarithromycin to treat tuberculosis. This chemical-genetic map provides a rich resource to understand drug efficacy in Mtb and guide future tuberculosis drug development and treatment.

众所周知，结核分枝杆菌(Mtb) 感染很难治疗。治疗效果受到结核分枝杆菌固有耐药性及其对临床使用的所有抗结核药物产生获得性耐药性的能力的限制。更深入地了解影响药物疗效的细菌途径可以促进更有效疗法的开发，确定获得性耐药的新机制，并揭示被忽视的治疗机会。在这里，我们开发了一个 CRISPR 干扰化学遗传学平台，用于滴定 Mtb 基因的表达并量化不同药物存在下的细菌适应性。我们发现了内在耐药性的多种机制，揭示了数百个协同药物组合的潜在靶点。将化学遗传学与结核分枝杆菌临床分离株的比较基因组学相结合，我们进一步确定了几种以前未知的获得性耐药机制，其中之一与南美洲的耐多药结核病爆发有关。最后，我们发现内在耐药因子whiB7在东南亚流行的整个结核分枝杆菌亚系中失活，这为重新利用大环内酯类抗生素克拉霉素来治疗结核病提供了机会。该化学遗传图谱为了解 Mtb 药物疗效并指导未来结核病药物开发和治疗提供了丰富的资源。