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 （二芳基喹啉和硝基咪唑）自引入以来已被引入临床实践 目前的多药治疗方案是 20 世纪 70 年代的。在确定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 方法。利福平以分枝杆菌RNA聚合酶为靶点，是关键的灭菌药物之一 允许现代“短期”6 个月的治疗方案。为预防结核病的发生，需要进行长期的结核病治疗 复发反映了结核分枝杆菌（Mtb）以改变的生理状态在宿主体内持续存在的能力。 其特征是对一线抗结核药物可逆耐受。已经建立了多种机制 据报道有助于 Mtb 抗生素耐受，包括诱导药物流出泵并减少 药物渗透到坏死的结核病灶中并穿过蜡质分枝杆菌细胞壁。目前， 然而，为了消除结核分枝杆菌“持续存在”，人们对最大限度地增加利福平的暴露量非常感兴趣 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 为了解决这一重大知识差距，我们进行了高通量转座子 (Tn) 突变体筛选 鉴定在营养丰富的肉汤和生理相关应激中对利福平耐受所需的 Mtb 基因 条件。 rv2224c/caeA 基因中含有 Tn 插入的突变体对 利福平比野生型结核分枝杆菌更有效。这种不同的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 缺乏症 与利福平累积增加和极性分子乙锭渗透性增加相关 Mtb 中的溴化物。先前的研究表明 CaeA，一种细胞壁相关的羧酸酯酶和丝氨酸 蛋白酶对于 Mtb 毒力至关重要。 caeA 的破坏导致受感染小鼠的存活时间延长，并且高度 肺部病理减少。进一步支持靶向 CaeA 在结核病治疗中的潜在效用， 该蛋白与 Mtb 对一线药物乙胺丁醇和 二线药物链霉素。目前的提案结合了有针对性和公正的方法 将检验 CaeA 通过改变 Mtb 细胞壁组成来介导 Mtb 对利福平的耐受性的假设， 从而降低药物的渗透性。在使用最近生成的诱导型 CaeA 的概念验证研究中 降解菌株，我们还将评估以 CaeA 作为辅助结核病治疗缩短治疗的潜力 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.