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.

项目摘要 项目3：确定和击败革兰氏阴性菌外膜生物发生的机制 革兰氏阴性菌被由脂多糖（LPS）组成的外膜（OM）包围， 形成了一个强大的渗透屏障，阻止了许多药物的吸收。获得性抗生素的传播 革兰氏阴性菌之间的耐药机制结合了革兰氏阴性菌赋予的内在耐药性， OM严重限制了这些微生物感染的治疗选择。因此发展 对革兰氏阴性菌有效的新抗生素是迫切的医疗需求。为了能够发现 通过这些治疗，我们的CARBIRU团队将发现OM组装所需的新漏洞和目标 使用大肠大肠杆菌作为模式生物。我们将采取三管齐下的办法。目标1将侧重于基本的BAM 在有机体中组装β-桶蛋白的机器我们已经纯化并表征了不同状态的 六元复合物，包括一个与底物结合的中间态。为了确定哪些州 我们将使用机器的最佳目标是破坏OM渗透屏障的药物 我们的nanobody筛选平台，以确定nanobody选择性结合纯化的复合物锁定在 不同的构象。纳米抗体与细胞上表面暴露的Bam表位接合的能力 并促进细胞杀伤或渗透性。纳米抗体结合的复合物的结构将 也可以使用冷冻EM方法来确定，该方法成功地阐明了失速BAM的结构， 机这些结果将为深入了解Bam功能的机制和确定易感域提供依据 在机器里。第二个目的是研究必需的膜蛋白YejM在调节细胞凋亡中的作用。 LPS合成。LpxC是该途径中的关键步骤，长期以来一直被认为是受 通过FtsH蛋白酶的蛋白水解。然而，目前还不清楚LpxC蛋白水解是如何调节的， 协调LPS合成与OM组装。我们将检验YejM对此负责的假设 协调和阐明监管机制。这些结果将告诉我们重要的药物靶点 LpxC在肠道细菌病原体中受到调控，并确定了破坏抗生素LPS合成的新方法 发展最后，我们将研究十一异戊二烯磷酸（Und-P）的回收机制。 Und-P是用于合成大多数细胞表面多糖（包括肽聚糖）的脂质载体 和LPS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， 我们将研究它们在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 Und-P回收是细胞包膜组装的核心，其结果将定义一个有吸引力的新目标， 抗生素或抗生素增效剂。总的来说，我们对这些保守系统的研究结果将与以下方面高度相关： 开发用于广谱革兰氏阴性病原体感染的新型治疗方法。