Project 3: Defining and defeating the mechanisms of outer membrane biogenesis in Gram-negative bacteria

项目 3：定义并破解革兰氏阴性菌外膜生物发生机制

• 批准号: 10699956
• 负责人: Thomas G Bernhardt
• 金额: $ 87.6万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-07 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10699956
• 关键词: Acinetobacter baumannii; Adopted; Affect; Affinity; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biochemical; Biochemistry; Bioinformatics; Cell Membrane Permeability; Cell Wall; Cell membrane; Cell surface; Cells; Chemosensitization; Clinical; Complex; Cryoelectron Microscopy; Cytoplasm; Development; Disease; Drug Targeting; ESKAPE pathogens; Educational process of instructing; Enterobacter; Epitopes; Equilibrium; Escherichia coli; Exclusion; Genetic; Glycolipids; Gram-Negative Bacteria; Gram-Positive Bacteria; Growth; Homeostasis; Infection; Klebsiella pneumoniae; Learning; Lipid A; Lipids; Lipopolysaccharides; Medical; Membrane; Membrane Lipids; Membrane Proteins; Methods; Modification; Molecular Conformation; Multi-Drug Resistance; Natural regeneration; New Agents; O Antigens; Organism; Pathway interactions; Penetration; Peptide Hydrolases; Peptidoglycan; Permeability; Pharmaceutical Preparations; Phenotype; Phospholipids; Play; Polymers; Polysaccharides; Predisposition; Process; Protein Family; Proteins; Proteolysis; Pseudomonas aeruginosa; Pump; Reaction; Recycling; Regulation; Resistance; Role; Signaling Molecule; Staphylococcus aureus; Structure; Surface; System; TRAP Complex; Testing; Therapeutic; United States; beta barrel; cell envelope; cell killing; inorganic phosphate; insight; member; membrane assembly; membrane biogenesis; model organism; nanobodies; novel; pathogen; prevent; resistance mechanism; screening; transposon sequencing; uptake; virtual

PROJECT SUMMARY Project 3: Defining and defeating the mechanisms of outer membrane biogenesis in Gram-negative bacteria Gram-negative bacteria are surrounded by an outer membrane (OM) composed of lipopolysaccharide (LPS) that creates a formidable permeability barrier preventing the uptake of many drugs. The spread of acquired antibiotic resistance mechanisms among Gram-negative bacteria combined with the intrinsic resistance conferred by the OM has severely limited treatment options for infections with these organisms. Therefore, the development of new antibiotics effective against Gram-negative bacteria is an urgent medical need. To enable the discovery of these treatments, our CARBIRU team will uncover new vulnerabilities and targets required for OM assembly using E. coli as a model organism. Our approach will be three-pronged. Aim 1 will focus on the essential Bam machine that assembles beta-barrel proteins in the OM. We have purified and characterized different states of the six-member complex, including an intermediate state engaged with a substrate. To determine which states of the machine are the most effective to target with drugs that disrupt the OM permeability barrier, we will use our nanobody screening platform to identify nanobodies that selectively bind purified complexes locked in different conformations. The ability of the nanobodies to engage with surface exposed Bam epitopes on cells and promote cell killing or permeability will then be assessed. Structures of the nanobody-bound complexes will also be determined using cryo-EM methods that successfully elucidated the structure of the stalled Bam machine. The results will provide insights into the mechanism of Bam function and define susceptible domains within the machine. The second aim will investigate the role of the essential membrane protein YejM in regulating LPS synthesis. LpxC is the committed step in the pathway, and it has long been known to be subjected to proteolysis by the FtsH protease. However, it has remained unclear how LpxC proteolysis is regulated to coordinate LPS synthesis with OM assembly. We will test the hypothesis that YejM is responsible for this coordination and elucidate the regulatory mechanism. The results will teach us how the important drug target LpxC is regulated in enterobacterial pathogens and identify new ways to disrupt LPS synthesis for antibiotic development. Finally, we will investigate the mechanism by which undecaprenyl-phosphate (Und-P) is recycled. Und-P is the lipid carrier used for the synthesis of most cell surface polysaccharides, including peptidoglycan and the O-antigen of LPS. Our genetic and bioinformatic analyses identified two conserved protein families as candidates for the long-sought flippases that recycle Und-P. We will investigate their role in Und-P transport and how their inactivation affects OM assembly and OM modifications that promote antibiotic resistance. Because Und-P recycling is central to cell envelope assembly, the results will define an attractive new class of targets for antibiotics or antibiotic potentiators. Overall, our results with these conserved systems will be highly relevant to the development of novel treatments for infections with a broad-spectrum of Gram-negative pathogens.

项目总结 项目3：革兰氏阴性细菌外膜生物发生机制的确定和破解 革兰氏阴性细菌被由脂多糖(LPS)组成的外膜(OM)所包围 制造了一道强大的渗透屏障，阻止了许多药物的吸收。获得性抗生素的传播 革兰氏阴性杆菌的耐药机制与其内在耐药性相结合 OM对感染这些微生物的治疗选择严重有限。因此，中国的发展 新的有效对抗革兰氏阴性细菌的抗生素是医学上的迫切需要。为了能够发现 通过这些处理，我们的CARBIRU团队将发现OM组装所需的新漏洞和目标 以大肠杆菌作为模式生物。我们的方法将是三管齐下的。目标1将专注于Essential Bam 在有机质中组装贝塔桶蛋白质的机器。我们已经提纯并表征了不同状态的 该六元络合物包括与底物接合的中间态。要确定哪些州 是以破坏OM通透性屏障的药物为靶点最有效的，我们将使用 我们的纳米体筛选平台可以识别选择性结合锁定的纯化络合物的纳米体 不同的构象。纳米小体与细胞表面暴露的Bam表位结合的能力 并促进细胞杀伤或渗透性，然后将进行评估。纳米体结合络合物的结构将 也是使用冷冻-EM方法成功地阐明了失速的Bam的结构 机器。这些结果将为深入了解BAM的功能机制并确定易感结构域提供依据 在机器里。第二个目的是研究必需的膜蛋白YejM在调节 内毒素合成。LpxC是该途径中的承诺步骤，很早就知道它会受到 FtsH蛋白水解酶的蛋白水解酶。然而，目前尚不清楚LpxC蛋白水解酶是如何调节的。 协调内毒素的合成和OM的组装。我们将检验耶伊姆对此负有责任的假设 协调和阐明监管机制。这一结果将告诉我们重要的药物靶点是如何 LpxC在肠道细菌中受到调控，并发现了扰乱细菌合成抗生素的新方法 发展。最后，我们将研究十一碳烯基磷酸(UND-P)的循环利用机理。 AND-P是用于合成包括肽聚糖在内的大多数细胞表面多糖的脂质载体 和内毒素的O抗原。我们的遗传和生物信息学分析确定了两个保守的蛋白质家族 人们长期以来一直在寻找的、循环使用UD-P的翻转方案的候选人。我们将调查它们在Und-P运输中的作用 它们的失活如何影响OM组装和OM修饰，从而促进抗生素耐药性。因为 AND-P回收是细胞被膜组装的核心，结果将定义一类有吸引力的新靶点 抗生素或抗生素增效剂。总体而言，我们在这些保守系统上的结果将与 开发治疗广谱革兰氏阴性病原体感染的新疗法。