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

项目摘要/摘要 抗生素耐药性是一个主要的全球公共卫生威胁，大约有300万人感染了抗生素 每年仅在美国就有报道。已经观察到许多不同的抗生素耐药机制 细菌，但在革兰氏阴性病原体中普遍存在的是主动将药物排出细胞的能力。 阻力结节分部(RND)家族的外排泵广泛应用于临床 抗生素耐药性。RND泵是由内膜转运蛋白组成的三元复合体， 一个是周质接头蛋白，一个是外膜因子蛋白。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包含四个RND 导致抗生素耐药性的外排系统。在这里，两个这样的泵，MexAB-OprM和Mexxy- OPRM，由于它们重叠但不同，将成为定义底物选择性基础的理想模型 分别对β-内酰胺类和氨基糖苷类的偏好。虽然首选底物是已知的，但 底物识别背后的分子决定因素目前还不清楚。以出版商为指导 MexAB-OprM的结构，我们实验室为Mexxy-OprM建立了一个模型。使用此结构框架可 对转运蛋白MexB和MexY进行比较，确定了MexB和MexY的特定区域和残基 这可能会加强底物的选择性。特别是，预测远端结合口袋(DBP)是关键的 用于底物选择和转运蛋白内的转位。基于这些发现，我假设 MexY末端结合口袋(DBP)中的关键残基定义了它的物理化学 控制氨基糖苷类底物识别的性质(形状、电荷、分布和体积) 和易位。在这个项目中，我将检验这一假设，并阐明底物的分子基础。 铜绿假单胞菌RND外排泵Mexxy-OprM转运子MexY的选择性和转运。 在目标1中，我将确定首选的氨基糖苷类进入通道(S)从细胞质进入 转运蛋白MexY在实验室和泛氨基糖苷类中的诱变结合体内功能检测 耐药临床分离株及高分辨低温电子显微镜结构研究。在《目标2》中，我会 定义在MexY的DBP内控制底物对非底物的选择性的残基(例如 用体外结合亲和力和高分辨X射线结晶学研究 结构研究，辅之以体内功能分析。了解什么定义了摄取、结合、 RND转运蛋白对底物和非底物的选择可以提供对抗生素的关键洞察 阻力机制及其对现有疗法重新设计或新型外排泵设计的影响 抑制剂。因为美国疾病控制和预防中心目前确认的14种细菌中有11种 预防“紧急”或“严重”至少包含一个RND外排泵，这些替代治疗策略 迫切需要与日益增长的抗生素耐药性威胁作斗争。

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.