Molecular basis for substrate discrimination by transporter protein MexY of the MexXY-OprM efflux pump in Pseudomonas aeruginosa

铜绿假单胞菌中 MexXY-OprM 外排泵转运蛋白 MexY 区分底物的分子基础

• 批准号: 10469329
• 负责人: Logan Kavanaugh
• 金额: $ 4.68万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10469329
• 关键词: Adaptor Signaling Protein; Affinity; Aminoglycoside resistance; Aminoglycosides; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biological Assay; Calorimetry; Carrier Proteins; Cells; Centers for Disease Control and Prevention (U.S.); Charge; Childbirth; Clinical; Complement; Complex; Computer Models; Coupled; Cryoelectron Microscopy; Crystallization; Dangerousness; Development; Discrimination; Distal; Drug Efflux; Electron Microscopy; Escherichia coli; Family; Health; In Vitro; Infection; Knock-out; Lead; Membrane; Membrane Proteins; Membrane Transport Proteins; Minimum Inhibitory Concentration measurement; Modeling; Molecular; Mutagenesis; Nodule; Operative Surgical Procedures; Pathway interactions; Pharmaceutical Preparations; Pneumonia; Procedures; Process; Property; Proteins; Pseudomonas aeruginosa; Public Health; Publishing; Pump; Reporting; Research; Research Personnel; Resistance; Resolution; Roentgen Rays; Sepsis; Shapes; Site-Directed Mutagenesis; Structure; Substrate Specificity; System; Testing; Therapeutic; Titrations; Training; Variant; Vestibule; antibiotic efflux; base; beta-Lactams; cancer care; career; combat; cryogenics; design; efflux pump; in silico; in vivo; inhibitor; insight; mutant; novel; novel strategies; pathogen; pathogenic bacteria; periplasm; preference; protein folding; resistance mechanism; skills; uptake

PROJECT SUMMARY/ ABSTRACT Antibiotic resistance is a major global public health threat, with approximately three million resistant infections reported each year in the US alone. Many distinct mechanisms of antibiotic resistance have been observed in bacteria, but pervasive among Gram-negative pathogens is the ability to actively efflux drugs out of the cell. Efflux pumps in the Resistance-Nodulation-Division (RND) family contribute extensively to intrinsic, clinical antibiotic resistance. RND pumps are tripartite complexes composed of an inner membrane transporter protein, a periplasmic adaptor protein, and an outer membrane factor protein. Many Gram-negative pathogens encode multiple RND systems; for example, the serious threat pathogen Pseudomonas aeruginosa contains four RND efflux systems that contribute to antibiotic resistance. Here, two of these pumps, MexAB-OprM and MexXY- OprM, will serve as an ideal model to define the basis of substrate selectivity due to their overlapping but distinct preferences for b-lactams and aminoglycosides, respectively. Although preferred substrates are known, the molecular determinants behind substrate recognition are not currently understood. Guided by the published structure of MexAB-OprM, our lab generated a model for MexXY-OprM. Using this structural framework for comparison of the transporter proteins MexB and MexY, specific regions and residues within them were identified that could underpin substrate selectivity. In particular, the distal binding pocket (DBP) is predicted to be critical for substrate selection and translocation within the transporter protein. Based on these findings, I hypothesize that critical residues within the distal binding pocket (DBP) of MexY define its physicochemical properties (shape, charge, distribution, and volume) that control aminoglycoside substrate recognition and translocation. In this project, I will test this hypothesis and elucidate the molecular basis of substrate selectivity and translocation through the transporter MexY of the P. aeruginosa RND efflux pump MexXY-OprM. In Aim 1, I will determine the preferred aminoglycoside entry channel(s) from the cell periplasm into the transporter MexY using mutagenesis coupled with in vivo functional assays in both lab and pan-aminoglycoside resistant clinical isolates and high-resolution cryogenic electron microscopy structural studies. In Aim 2, I will define the residues within the DBP of MexY that control selectivity for substrates over non-substrates (e.g. aminoglycosides over b-lactams) by using in vitro binding affinity and high-resolution X-ray crystallographic structural studies, complemented with in vivo functional assays. Understanding what defines uptake, binding, and selection for substrates versus non-substrates by RND transporters can provide critical insight into antibiotic resistance mechanisms and influence the redesign of current therapeutics or design of novel efflux pump inhibitors. Because 11 of the 14 bacterial pathogens currently identified by the Centers for Disease Control and Prevention as “urgent” or “serious” contain at least one RND efflux pump, these alternative therapeutic strategies are urgently needed to combat the growing threat of antibiotic resistance.

项目概要/摘要 抗生素耐药性是全球主要的公共卫生威胁，约有 300 万耐药感染者 仅在美国每年都有报道。已观察到许多不同的抗生素耐药机制 细菌，但革兰氏阴性病原体普遍具有将药物主动排出细胞的能力。 阻力结节分裂 (RND) 系列中的外排泵对内在、临床做出了广泛贡献 抗生素耐药性。 RND 泵是由内膜转运蛋白组成的三联复合物， 周质衔接蛋白和外膜因子蛋白。许多革兰氏阴性病原体编码 多个 RND 系统；例如，严重威胁病原体铜绿假单胞菌含有四种RND 导致抗生素耐药性的外排系统。这里有两个泵，MexAB-OprM 和 MexXY- OprM 将作为定义底物选择性基础的理想模型，因为它们重叠但又不同 分别偏好β-内酰胺类和氨基糖苷类。尽管优选的基材是已知的，但是 目前尚不清楚底物识别背后的分子决定因素。以已发布的 根据 MexAB-OprM 的结构，我们的实验室生成了 MexXY-OprM 的模型。使用这个结构框架 比较转运蛋白 MexB 和 MexY，确定其中的特定区域和残基 这可以支撑底物选择性。特别是，预计远端结合袋 (DBP) 至关重要 用于转运蛋白内的底物选择和易位。根据这些发现，我假设 MexY 远端结合袋 (DBP) 内的关键残基决定了其理化性质 控制氨基糖苷类底物识别的特性（形状、电荷、分布和体积） 和易位。在这个项目中，我将测试这个假设并阐明底物的分子基础 通过铜绿假单胞菌 RND 外排泵 MexXY-OprM 的转运蛋白 MexY 进行选择性和易位。 在目标 1 中，我将确定从细胞周质进入细胞的首选氨基糖苷类进入通道 在实验室和泛氨基糖苷中使用诱变结合体内功能测定的转运蛋白 MexY 耐药临床分离株和高分辨率低温电子显微镜结构研究。在目标 2 中，我将 定义 MexY 的 DBP 内控制底物相对于非底物的选择性的残基（例如 通过使用体外结合亲和力和高分辨率 X 射线晶体学，确定氨基糖苷类药物（氨基糖苷类药物优于 β-内酰胺类药物） 结构研究，辅以体内功能测定。了解什么定义了摄取、结合、 RND 转运蛋白对底物与非底物的选择可以为抗生素提供重要的见解 耐药机制并影响当前疗法的重新设计或新型外排泵的设计 抑制剂。因为疾病控制中心目前确定的 14 种细菌病原体中，有 11 种是 “紧急”或“严重”的预防至少包含一个 RND 外排泵，这些替代治疗策略 迫切需要应对日益严重的抗生素耐药性威胁。