Emerging multidrug resistance mechanisms in Campylobacter

弯曲杆菌中新兴的多药耐药机制

• 批准号: 10569586
• 负责人: EDWARD W YU
• 金额: $ 62.91万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-17 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569586
• 关键词: Affect; Amino Acids; Animal Model; Animals; Antibiotic Resistance; Antibiotics; Bile fluid; Binding; Biological Models; CRISPR/Cas technology; Campylobacter; Campylobacter infection; Campylobacter jejuni; Carbapenems; Centers for Disease Control and Prevention (U.S.); Chimeric Proteins; Clinical; Clinical Treatment; Coculture Techniques; Colistin; Crystallization; Culture Media; Data; Databases; Development; Diarrhea; Drug Efflux; Drug-resistant Campylobacter; Genes; Genetic; Genetic Polymorphism; Goals; Homologous Gene; Human; Image; In Vitro; Individual; Intervention; Intestines; Ion Channel; Knowledge; Ligands; Maps; Mediating; Membrane; Membrane Fusion; Membrane Transport Proteins; Multi-Drug Resistance; Mutation; Pathogen detection; Pharmaceutical Preparations; Physiology; Play; Positioning Attribute; Prevalence; Proteins; Protomer; Public Health; Pump; Research Personnel; Resistance; Solid; Structure; Structure-Activity Relationship; System; Techniques; Treatment Failure; Variant; Work; World Health Organization; X-Ray Crystallography; antibiotic efflux; antimicrobial; bacterial fitness; clinically relevant; co-infection; conformational conversion; coping; efflux pump; enteric pathogen; experimental study; fitness; fluoroquinolone resistance; genome sequencing; gut colonization; human pathogen; in vivo; insight; novel; pathogenic bacteria; periplasm; pressure; resistance mechanism; single-molecule FRET; therapy development; transmission process; whole genome

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.

项目摘要 细菌多药外排转运蛋白赋予对结构上不同的抗菌剂的抗性，这是细菌多药外排转运蛋白的一种。 临床治疗失败的主要原因。空肠弯曲菌是一种主要的肠道病原菌， 抗生素耐药性的各种机制。最近，世界卫生组织和 疾病预防和控制中心已将弯曲杆菌列为“严重的抗生素耐药性威胁”。在 弯曲杆菌，多药外排泵CmeABC，一种RND型外排系统，在耐药中起关键作用 通过介导胆汁抗性而在肠内定植。在CmeABC中， 蛋白质组装形成一个强大的三方机制，允许底物直接流出， 革兰氏阴性细胞包膜的膜。CmeABC是C.空肠，因为它有一个重要的 胆汁抗性的天然功能，这是弯曲杆菌在动物肠道中定殖所需的。典型地， CmeABC需要与其他抗性机制（如靶向突变）协同发挥作用， 赋予临床相关的抗生素抗性。然而，CmeABC的“超级”抗性增强变体（RE-1000）是一种新的抗性增强变体。 CmeABC）最近在临床分离的C.空肠。该变体泵具有独特的CmeB序列 并且在赋予多药耐药性方面更有效。此外，我们发现RE-CmeABC是 在临床分离株中越来越普遍，并介导对氟喹诺酮类药物的极高水平耐药性， 临床上重要的治疗弯曲杆菌病的抗生素。我们的初步数据进一步表明， RE-CmeABC的外排功能是由于RE-CmeB转运蛋白的序列变异。开始理解 CmeABC是如何挤出抗菌剂的，我们已经开始了破译CmeABC结构基础的工作， 介导的外排。我们的初步结晶数据表明，CmeB形成同源三聚体，其中单个 原聚体独立地结合并输出底物。基于坚实的初步数据，我们建议在此 应用程序追求三个具体目标：1）识别导致外排增强的特定突变 2）定义CmeB介导的抗生素外排的结构基础以及如何序列化CmeB的结构基础。 多态性影响结构-功能关系; 3）决定RE-cmeABC的水平分布 及其对C.空肠健身的存在和不存在抗生素选择压力。我们将使用一个 高通量作图策略，一种基于CRISPR-Cas9的高效基因编辑和替换技术， 体外和动物模型系统，X射线晶体学，和单分子FRET，以实现的目标， 三个具体目标。调查人员团队拥有强大的互补专业知识， 定位进行拟议的工作，预计将揭示新的机制所使用的RND型 抗生素外排转运蛋白和增强的多药耐药性。这些知识应该是 可转移到其他细菌外排泵，这一发现可能有助于开发新的策略 以控制多药耐药弯曲杆菌的出现和传播。