Emerging multidrug resistance mechanisms in Campylobacter

项目总结 细菌多药外排转运体对结构多样的抗菌素具有耐药性，这是 临床治疗失败的主要原因。空肠弯曲杆菌是一种主要的肠道病原体，已经形成 抗生素耐药性的各种机制。最近，世界卫生组织和疾病控制与预防中心 疾病预防和控制组织已将弯曲杆菌列为“严重的抗生素耐药性威胁”。在……里面 弯曲杆菌，多药外排泵CmeABC，是一种RND型外排系统，在耐药中起关键作用 对各种抗菌剂和通过介导胆汁耐药而在肠道定植。在CmeABC中，三位 蛋白质组装成一个强大的三方机制，允许底物直接流出两端 革兰氏阴性细胞膜。CmeABC对空肠弯曲菌是必不可少的，因为它有一个重要的 自然的胆汁抵抗功能，这是弯曲杆菌在动物肠道定植所必需的。通常， CmeABC要求与其他耐药机制(如靶点突变)协同作用 授予临床相关的抗生素耐药性。然而，CmeABC(RE-1)的一种“超级”抗药性增强型变异体。 最近在空肠弯曲菌临床分离株中出现了CmeABC)。这个变种泵有一个独特的CmeB序列 而且在导致多药耐药性方面要有效得多。此外，我们还发现RE-CmeABC是 在临床分离株中日益普遍，并导致对氟喹诺酮类药物的高度耐药， 治疗弯曲杆菌病的临床重要抗生素。我们的初步数据进一步表明， RE-CmeABC的外流功能是由于RE-CmeB转运体的序列变异所致。开始理解 CmeABC是如何分泌抗菌剂的，我们已经开始破译CmeABC的结构基础-- 中介外排。我们的初步结晶数据表明，CmeB形成了同源三聚体，其中单个 原核糖体独立结合和输出底物。基于坚实的初步数据，我们在此提出 应用程序追求三个特定目标：1)确定导致外排增加的特定突变 在RE-CmeABC中的作用，2)确定CmeB介导的抗生素外排的结构基础和如何序列 多态影响结构-功能关系，3)决定RE-cmeABC的水平分布 以及在没有和存在抗生素选择压力的情况下，它对空肠弯曲菌适合度的影响。我们将使用 高通量作图策略，基于CRISPR-CAS9的高效基因编辑和替换技术， 在体外和动物模型系统中，x射线结晶学，和单分子FRET，以实现 三个具体目标。调查团队拥有强大和互补的专业知识，并具有独一无二的 定位于进行拟议的工作，预计将揭示RND类型的 抗生素排泄和增强多药耐药性的转运体。这种获得的知识应该是 可以转移到其他细菌外排泵，研究结果可能有助于开发新的策略 控制耐多药弯曲杆菌的出现和传播。

PROJECT SUMMARY Bacterial multidrug efflux transporters confer resistance to structurally diverse antimicrobials, which is one of the major causes for clinical treatment failure. Campylobacter jejuni is a major enteric pathogen and has developed various mechanisms for antibiotic resistance. Recently, both the World Health Organization and the Centers for Disease Prevention and Control have designated Campylobacter as a “serious antibiotic resistance threat”. In Campylobacter, the multidrug efflux pump CmeABC, a RND-type efflux system, plays a key role in the resistance to various antimicrobials and in intestinal colonization by mediating bile resistance. In CmeABC, the three proteins assemble to form a powerful tripartite machinery, allowing direct efflux of substrates across both membranes of the Gram-negative cellular envelope. CmeABC is essential for C. jejuni as it has an important natural function for bile resistance, which is required for Campylobacter colonization in animal intestine. Typically, CmeABC requires to function cooperatively with other resistance mechanisms (such as target mutations) to confer clinically relevant antibiotic resistance. However, a “super” resistance-enhancing variant of CmeABC (RE- CmeABC) has recently emerged in clinical isolates of C. jejuni. This variant pump has a distinct CmeB sequence and is much more potent in conferring multidrug resistance. Additionally, we found that RE-CmeABC is increasingly prevalent in clinical isolates and mediates exceedingly high-level resistance to fluoroquinolone, a clinically important antibiotic for treating campylobacteriosis. Our preliminary data further suggest the enhanced efflux function of RE-CmeABC is due to sequence variations in the RE-CmeB transporter. To begin to understand how CmeABC extrudes antimicrobials, we have initiated work to decipher the structural basis of CmeABC- mediated efflux. Our preliminary crystallization data indicate that CmeB forms a homotrimer, where individual protomers bind to and export substrates independently. Based on the solid preliminary data, we propose in this application to pursue three specific aims to 1) identify the specific mutations responsible for the enhanced efflux function in RE-CmeABC, 2) define the structural basis of CmeB-mediated antibiotic efflux and how sequence polymorphisms affect the structure-function relationship, and 3) determine the horizontal spread of RE-cmeABC and its impact on C. jejuni fitness in the absence and presence of antibiotic selection pressure. We will use a high throughput mapping strategy, a CRISPR-Cas9 based technique for efficient gene editing and replacement, in vitro and animal model systems, x-ray crystallography, and single-molecule FRET to achieve the goals of the three specific aims. The team of investigators have strong and complementary expertise, and are uniquely positioned to conduct the proposed work, which is expected to reveal novel mechanisms used by an RND-type transporter for antibiotic extrusion and enhanced multidrug resistance. This gained knowledge should be transferrable to other bacterial efflux pumps, and the findings may facilitate the development of new strategies to control the emergence and spread of multidrug resistant Campylobacter.