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; 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.

项目摘要 项目3：定义和打败革兰氏阴性细菌中外膜生物发生的机制 革兰氏阴性细菌被外膜（OM）包围，该膜由脂多糖（LPS）组成 产生强大的渗透性屏障，以防止吸收许多药物。获得的抗生素的传播 革兰氏阴性细菌之间的抗性机制，结合由 OM对这些生物的感染具有严重有限的治疗选择。因此，发展 对革兰氏阴性细菌有效的新抗生素是紧迫的医疗需求。为了发现 这些治疗方法，我们的Carbiru团队将发现OM组装所需的新漏洞和目标 将大肠杆菌作为模型生物。我们的方法将是三个统治的。 AIM 1将重点放在基本的BAM上 在OM中组装β-桶蛋白的机器。我们已经纯化和表征了不同状态的不同状态 由六人组成的综合体，包括与基板接合的中间状态。确定哪些状态 机器的最有效靶向破坏OM渗透性障碍的药物，我们将使用 我们的纳米机构筛选平台，以识别有选择地结合被锁定的纯化复合物的纳米生物。 不同的构象。纳米体与细胞上表面暴露的BAM表位接合的能力 然后将评估促进细胞杀伤或渗透率。纳米结合复合物的结构将 还可以使用成功阐明失速BAM结构的冷冻EM方法确定 机器。结果将提供有关BAM功能机制并定义易感域的见解 在机器内。第二个目标将研究必需膜蛋白YEJM在调节中的作用 LPS合成。 LPXC是途径中的致力步骤，长期以来已知它会受到约束 FTSH蛋白酶的蛋白水解。但是，尚不清楚如何调节LPXC蛋白水解 与OM组装协调LPS合成。我们将检验Yejm负责的假设 协调并阐明调节机制。结果将教会我们重要的药物目标 LPXC在肠杆菌病原体中受到调节，并确定破坏LPS合成抗生素的新方法 发展。最后，我们将研究未依赖磷酸（UND-P）的机制。 UND-P是用于合成大多数细胞表面多糖的脂质载体，包括肽聚糖 和LPS的O-Antigen。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 候选长期回收和P的候选者。我们将调查它们在UND-P运输中的作用， 它们的灭活如何影响OM组装和OM修饰，从而促进抗生素抗性。因为 UND-P回收是细胞信封组件的核心，结果将定义一个有吸引力的新目标 抗生素或抗生素增强剂。总体而言，我们使用这些保守系统的结果将与 用革兰氏阴性病原体广谱的感染的新型治疗方法的发展。