Characterizing CaeA-mediated rifampin tolerance in MTB

摘要 结核病（TB）是全球十大死亡原因之一。只有两类新的结核病药物 （二芳基喹啉类和硝基咪唑类）自从引入 目前的多药疗法。在确定新药靶点方面缺乏成功，这表明 重新研究现有药物的作用机制和耐药性可能是一个更有成效的方法。 approach.利福平是一种以分枝杆菌RNA聚合酶为靶点的杀菌药物 允许现代的“短期”6个月疗程。预防结核病所需的长期结核病治疗 复发反映了结核分枝杆菌（Mtb）在宿主中以改变的生理活性持续存在的能力。 对一线抗结核药物具有可逆耐受性的状态。了几种机制 据报道，有助于结核分枝杆菌抗生素耐受性，包括诱导药物外排泵和减少 药物渗透到坏死的结核病损中并穿过蜡质的分枝杆菌细胞壁。目前， 然而，为了消除结核分枝杆菌的“持续存在者”，最大限度地暴露于利福平是很有意义的。 对利福平和其他抗结核药物的耐受机制在很大程度上仍不明确。为了 为了解决这一重大的知识缺口，我们进行了高通量转座子（Tn）突变体筛选， 鉴定在营养丰富肉汤中和在生理相关应激中对利福平耐受性所需的Mtb基因 条件在rv 2224 c/caeA基因中含有Tn插入的突变体对 利福平比野生型Mtb.这种差异性抗生素敏感性表型是使用 靶向caeA缺失突变体（ΔcaeA）及其互补菌株。我们还发现，caeA缺乏是 与利福平蓄积增加和极性分子乙锭渗透性增加相关 溴，结核分枝杆菌。以前的研究表明，CaeA，一种细胞壁相关的羧酸酯酶和丝氨酸 蛋白酶是Mtb毒力的关键。破坏caeA导致感染小鼠的存活时间延长， 减少肺部病变。进一步支持靶向CaeA在TB治疗中的潜在效用， 这种蛋白质与结核分枝杆菌对一线药物乙胺丁醇和抗结核药物的敏感性增加有关。 二线药物链霉素采用有针对性和不带偏见的办法， 将检验CaeA通过修饰Mtb细胞壁组成介导Mtb对利福平耐受性的假设， 从而降低药物渗透性。在使用最近生成的诱导型CaeA的概念验证研究中， 降解菌株，我们还将评估靶向CaeA作为抗结核病的治疗缩短潜力 在临床相关的动物模型中进行治疗。我们的研究结果预计将对 了解结核分枝杆菌和其他持续性细菌感染的抗生素耐受性。

ABSTRACT Tuberculosis (TB) is one of the top ten causes of death worldwide. Only two new classes of TB drugs (diarylquinolines and nitroimidazoles) have been introduced into clinical practice since the introduction of the current multidrug regimen in the 1970s. The lack of success in identifying novel drug targets suggests that the reinvestigation of mechanisms of action and resistance of currently available drugs may be a more fruitful approach. Rifampin, which targets the mycobacterial RNA polymerase, is one of the key sterilizing drugs permitting the modern “short-course” 6-month regimen. The lengthy duration of TB treatment required to prevent relapse reflects the ability of Mycobacterium tuberculosis (Mtb) to persist in the host in an altered physiological state characterized by reversible tolerance to first-line anti-tubercular drugs. Several mechanisms have been reported to contribute to Mtb antibiotic tolerance, including the induction of drug efflux pumps and reduced penetration of drugs into necrotic tuberculous lesions and across the waxy mycobacterial cell wall. Currently, there is significant interest in maximizing exposures to rifampin in order to eliminate Mtb “persisters”, however the mechanisms of tolerance to rifampin and other anti-tubercular drugs remain largely undefined. In order to address this significant knowledge gap, we conducted a high-throughput transposon (Tn) mutant screen to identify Mtb genes required for tolerance to rifampin in nutrient-rich broth and in physiologically relevant stress conditions. Mutants containing a Tn insertion in the rv2224c/caeA gene were 294-fold more susceptible to rifampin than was wild-type Mtb. This differential antibiotic susceptibility phenotype was confirmed using a targeted caeA deletion mutant (ΔcaeA) and its complemented strain. We also found that caeA deficiency is associated with increased rifampin accumulation and increased permeability to the polar molecule ethidium bromide in Mtb. Previous studies have shown that CaeA, a cell wall-associated carboxylesterase and serine protease, is critical for Mtb virulence. Disruption of caeA led to prolonged survival of infected mice and highly reduced lung pathology. Further supporting the potential utility of targeting CaeA in TB treatment, deficiency of this protein has been associated with increased Mtb susceptibility to the first-line drug ethambutol and the second-line drug streptomycin. Using a combination of targeted and unbiased approaches, the current proposal will test the hypothesis that CaeA mediates Mtb tolerance to rifampin by modifying Mtb cell wall composition, thereby reducing drug permeability. In proof-of-concept studies using a recently generated inducible CaeA degradation strain, we also will evaluate the treatment-shortening potential of targeting CaeA as adjunctive TB therapy in a clinically relevant animal model. Our findings are expected to have far-reaching implications for understanding antibiotic tolerance in Mtb and other persistent bacterial infections.