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.

项目概要/摘要 抗生素耐药性对人类健康构成了迅速增长的威胁，而有限的抗生素耐药性进一步加剧了这一威胁。 因此，迫切需要为新疗法的设计提供研究信息。 对抗抗生素耐药性上升的选择 多粘菌素是阳离子抗菌肽。 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。 由于细菌已发展为多粘菌素，多粘菌素经常且相对容易地发展 防御由先天免疫系统产生或由其他系统分泌的抗菌肽（AMP） 脂质 A 的修饰，细菌脂多糖（LPS 或）的脂质锚。 内毒素）装饰 GN 细菌的外膜，具有多种化学基团，是一种常见的细菌 导致大肠杆菌、肠沙门氏菌和铜绿假单胞菌产生耐药性的机制。 脂质 A 的磷酸酯与氨基阿拉伯糖部分 (L-Ara4N) 的修饰是主要的修饰 GN 细菌通过合成氨基阿拉伯糖“帽”来抵抗多粘菌素和 AMP。 八种蛋白质的酶促中继，统称为氨基阿拉伯糖生物合成途径。 该途径的膜酶的功能机制基础在很大程度上知之甚少 由于研究在膜上或膜附近起作用的酶相关的技术挑战， 作为该研究计划的一部分，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、细菌遗传学和 微尺度热泳（MST），实现以下核心目标：（1）结构测定和 三种真正的膜酶的底物结合表征 氨基阿拉伯糖生物合成途径（聚戊烯醇磷酸糖基转移酶 ArnC、去甲酰基酶 ArnD 和脂质间糖基转移酶 ArnT)，以及 (2) 研究酶促作用的机制基础 所研究的三种膜酶中的每一种的功能、金属辅因子配位和催化作用。 该研究计划将利用我们在膜蛋白生物化学和 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 与不同的脂质底物结合，该计划的影响在于其潜力： i) 提供 对一组不同酶功能的结构基础的详细机制见解 GN 细菌中的氨基阿拉伯糖生物合成和多粘菌素抗性，ii) 增进我们对 蛋白质-脂质与十一异戊二烯基磷酸盐相互作用，因为所研究的所有三种酶都利用十一异戊二烯基 磷酸盐作为供体或受体底物，以及 iii) 为化合物的基于结构的药物设计提供信息 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。