Characterizing CaeA-mediated rifampin tolerance in MTB

摘要 结核病是全球十大死因之一。只有两类新的结核病药物 (二芳基喹啉类和硝基咪唑类)自推出以来已进入临床实践 20世纪70年代目前的多药疗法。在识别新的药物靶点方面缺乏成功表明 重新研究现有药物的作用机制和耐药性可能会更有成效 接近。针对分枝杆菌rna聚合酶的利福平是主要的杀菌药物之一。 允许现代的“短程”6个月的养生法。预防结核病所需的长期治疗 复发反映了结核分枝杆菌(Mtb)在改变的生理状态下在宿主体内持续存在的能力。 对一线抗结核药物具有可逆耐受性的状态。有几种机制已经被 据报道，结核分枝杆菌对抗生素的耐受性，包括诱导药物外排泵和减少 药物穿透坏死性结核病变并穿过蜡质分枝杆菌细胞壁。目前， 然而，人们对最大限度地暴露于利福平以消除结核分枝杆菌“顽固者”有很大兴趣。 利福平和其他抗结核药物的耐受机制在很大程度上仍不清楚。为了 为了解决这一重大的知识差距，我们进行了高通量转座子(TN)突变筛选 确定营养丰富的肉汤和生理相关应激中耐受利福平所需的mtb基因 条件。在rv2224c/caeA基因中含有TN插入的突变体，其易感性增加294倍 利福平为野生型Mtb。这种不同的抗生素敏感性表型是用 靶向caeA缺失突变体(ΔcaeA)及其互补株。我们还发现，CaeA缺乏是 与利福平蓄积增加和对极性分子乙锭的渗透性增加有关 结核分枝杆菌中的溴。先前的研究表明，CaeA，一种与细胞壁相关的羧酸酯酶和丝氨酸 蛋白酶对结核分枝杆菌的毒力是至关重要的。破坏caeA导致感染小鼠存活时间延长，并高度 减少肺部病理改变。进一步支持靶向CaeA在结核病治疗中的潜在效用，不足 这种蛋白与结核分枝杆菌对一线药物乙胺丁醇的敏感性增加有关 二线药物链霉素。使用有针对性和不偏不倚的方法相结合，当前的提案 将验证CaeA通过改变结核分枝杆菌细胞壁成分来介导结核分枝杆菌对利福平耐受性的假设， 从而降低药物的渗透性。在使用最近产生的诱导性CAEA的概念验证研究中 降解菌株，我们还将评估靶向CaeA作为辅助结核病的治疗缩短潜力 临床相关动物模型的治疗。我们的发现预计将对 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.