Structure and mechanism of membrane enzymes responsible for bacterial lipid modification and polymyxin resistance

负责细菌脂质修饰和多粘菌素抗性的膜酶的结构和机制

• 批准号: 10713771
• 负责人: Vasileios I Petrou
• 金额: $ 39.25万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-10 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713771
• 关键词: Anabolism; Antibiotic Resistance; Antibiotics; Antimicrobial Cationic Peptides; Antimicrobial Resistance; Bacteria; Binding; Biochemical Reaction; Biological Assay; Catalysis; Chemicals; Cryoelectron Microscopy; Development; Drug Design; Electrostatics; Endotoxins; Enzymes; Escherichia coli; Future; Goals; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Growth; Health; Human; Incidence; Infection; Innate Immune System; Lipid A; Lipids; Lipopolysaccharides; Membrane; Membrane Proteins; Metals; Modification; Molecular; Multi-Drug Resistance; Mutagenesis; Pathway interactions; Polymyxin Resistance; Polymyxins; Predisposition; Protein Biochemistry; Proteins; Pseudomonas aeruginosa; Research; Resistance; Resistance development; Salmonella enterica; Structure; Techniques; Therapeutic; Training; antimicrobial drug; antimicrobial peptide; bacterial genetics; cofactor; design; enzyme pathway; enzyme structure; experience; glycosyltransferase; inorganic phosphate; insight; multidisciplinary; novel therapeutics; prevent; programs; structural biology

Project Summary/Abstract Antibiotic resistance is a rapidly growing threat to human health, further exacerbated by the limited development of new antibiotics. Thus, there is a dire need for research informing the design of new therapeutic options to counter the rise of antibiotic resistance. Polymyxins are cationic antimicrobial peptides that associate with the outer membrane of Gram-negative (GN) bacteria through electrostatic interactions and are considered the last line of defense against multi-drug resistant GN bacterial infections. Yet, resistance to polymyxins develops often and with relative ease, due to modifications that bacteria have developed as defenses against antimicrobial peptides (AMPs) produced by the innate immune system or secreted by other bacterial species. Modification of Lipid A, the lipidic anchor of the bacterial lipopolysaccharide (LPS or endotoxin) decorating the outer membrane of GN bacteria, with diverse chemical moieties, is a common mechanism leading to resistance to antimicrobial agents. In E. coli, S. enterica and P. aeruginosa, “capping” of the phosphates of Lipid A with an aminoarabinose moiety (L-Ara4N) is the predominant modification leading to resistance against polymyxins and AMPs. The aminoarabinose “cap” is synthesized by GN bacteria through an enzymatic relay of eight proteins collectively called the aminoarabinose biosynthetic pathway. The mechanistic basis of function for the membrane enzymes of the pathway is poorly understood, in large part due to the technical challenges associated with studying enzymes that function at or near the membrane and utilize lipidic substrates. As part of this research program, we will use a variety of experimental techniques, including cryo-electron microscopy (cryoEM), mutagenesis, bacterial growth assays, bacterial genetics, and microscale thermophoresis (MST), to achieve the following core goals: (1) Structure determination and substrate-binding characterization for the three bona fide membrane enzymes that operate in the aminoarabinose biosynthetic pathway (the polyprenol phosphate glycosyltransferase ArnC, the deformylase ArnD and the lipid-to-lipid glycosyltransferase ArnT), and (2) Investigating the mechanistic basis of enzymatic function, metal cofactor coordination, and catalysis, in each of the three membrane enzymes under study. The research program will leverage our multidisciplinary training in membrane protein biochemistry and structural biology, and experience gained from having successfully solved several structures of the enzyme ArnT bound to different lipidic substrates. The impact of the program lies within its potential to: i) Provide detailed mechanistic insights into the structural basis of a diverse set of enzymatic functions responsible for aminoarabinose biosynthesis and polymyxin resistance in GN bacteria, ii) Advance our understanding of protein-lipid interactions with undecaprenyl phosphate, as all three enzymes under study utilize undecaprenyl phosphate as either a donor or acceptor substrate, and iii) Inform structure-based drug design of compounds capable of restoring susceptibility to polymyxins by targeting enzymes of the aminoarabinose pathway.

项目总结/摘要 抗生素耐药性是对人类健康的一个迅速增长的威胁， 开发新的抗生素。因此，迫切需要进行研究，为新的治疗方法的设计提供信息。 应对抗生素耐药性上升的选择。多粘菌素是阳离子抗微生物肽， 通过静电相互作用与革兰氏阴性（GN）细菌的外膜结合， 被认为是抵抗多重耐药GN细菌感染的最后一道防线。然而， 多粘菌素的发展往往和相对容易，由于修改，细菌已发展为 抗先天性免疫系统产生的或其他免疫系统分泌的抗菌肽（AMP）的防御 细菌种类脂质A的修饰，细菌脂多糖（LPS或LPS）的粘附锚， 内毒素）装饰GN细菌的外膜，具有不同的化学部分，是常见的 导致抗微生物剂耐药性的机制。在大肠coli、S.肠杆菌和铜绿假单胞菌，“加帽” 具有氨基阿拉伯糖部分的脂质A的磷酸盐（L-Ara 4 N）是导致脂质A的主要修饰。 对多粘菌素和抗菌肽的抗性。氨基阿拉伯糖“帽”由GN细菌通过以下途径合成： 这是一个由八种蛋白质组成的酶促中继，统称为氨基阿拉伯糖生物合成途径。的 在很大程度上，对该途径的膜酶的功能的机械基础知之甚少。 由于与研究在膜处或膜附近起作用的酶相关的技术挑战， 利用无水基质。作为这项研究计划的一部分，我们将使用各种实验技术， 包括冷冻电子显微镜（cryoEM）、诱变、细菌生长测定、细菌遗传学，以及 微尺度热泳（MST），以实现以下核心目标：（1）结构测定和 三个真正的膜酶，在工作的底物结合特性， 氨基阿拉伯糖生物合成途径（磷酸聚戊烯醇糖基转移酶ArnC、脱甲酰基酶 ArnD和脂-脂糖基转移酶ArnT），以及（2）研究酶促反应的机理基础。 功能，金属辅因子协调，和催化，在每一个研究中的三个膜酶。 该研究计划将利用我们在膜蛋白生物化学的多学科培训， 结构生物学，以及从成功解决酶的几种结构中获得的经验 ArnT与不同的底物结合。该计划的影响在于其潜力：i）提供 详细的机械见解的结构基础的一组不同的酶功能负责 氨基阿拉伯糖生物合成和多粘菌素抗性，ii）推进我们对 与磷酸十一异戊二烯酯的蛋白质-脂质相互作用，因为研究中的所有三种酶都利用十一异戊二烯酯 磷酸盐作为供体或受体底物，和iii）告知化合物的基于结构的药物设计 能够通过靶向氨基阿拉伯糖途径的酶来恢复对多粘菌素的敏感性。