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

项目总结/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300万耐药感染 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性机制， 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-分裂（RND）家族中的外排泵广泛地促进了内在的、临床的、 抗生素耐药性RND泵是由内膜转运蛋白， 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包含四个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，将作为一个理想的模型，以确定基础的底物选择性，由于他们的重叠，但不同的 分别优选β-内酰胺类和氨基糖苷类。尽管优选的基底是已知的， 底物识别背后的分子决定因素目前还不清楚。以出版的《 MexAB-OprM的结构，我们的实验室生成了MexXY-OprM的模型。利用这种结构框架， 通过对转运蛋白MexB和MexY的比较，确定了它们的特定区域和残基 这可能是底物选择性的基础。特别是，远端结合口袋（DBP）被预测为关键 用于底物选择和转运蛋白内的转运。基于这些发现，我假设 MexY的远端结合口袋（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性质（形状、电荷、分布和体积） 和易位。在这个项目中，我将验证这一假设，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的选择性和易位。 在目标1中，我将确定氨基糖苷类药物从细胞周质进入细胞的首选通道。 在实验室和泛氨基糖苷类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， 氨基糖苷类抗生素优于β-内酰胺类抗生素）的体外结合亲和性和高分辨率X射线晶体学研究 结构研究，辅以体内功能测定。理解什么定义了吸收、结合、 RND转运蛋白对底物和非底物的选择可以为抗生素 耐药机制并影响当前治疗方法的重新设计或新型外排泵的设计 抑制剂的因为疾病控制中心目前确定的14种细菌病原体中有11种， 预防为“紧急”或“严重”包含至少一个RND外排泵，这些替代治疗策略 迫切需要对抗日益增长的抗生素耐药性威胁。

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.