The role of cardiolipin in the biogenesis of the Gram-negative bacterial cell envelope

心磷脂在革兰氏阴性细菌细胞包膜生物发生中的作用

• 批准号: 10731444
• 负责人: Michael Stephen Trent
• 金额: $ 67.71万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10731444
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Affinity; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Automobile Driving; Bacteria; Bacterial Infections; Binding Sites; Biochemical; Biogenesis; Biology; Cardiolipins; Cell Membrane Permeability; Cell Survival; Cells; Cessation of life; Clinical; Cytoplasm; Data; Defect; Development; Drug Efflux; Encapsulated; Enzymes; Equilibrium; Escherichia coli; Face; Feedback; Future; Genes; Genetic; Glycerophospholipids; Gram-Negative Bacteria; Growth; Healthcare; Heterogeneity; Infection; Libraries; Lipid A; Lipids; Lipopolysaccharides; Maintenance; Membrane; Membrane Lipids; Modeling; Molecular; Multi-Drug Resistance; Organism; Pathway interactions; Peptidoglycan; Pump; Regulation; Resistance; Role; Side; Surface; System; Transmembrane Transport; antimicrobial; cardiolipin synthase; cell envelope; combinatorial; density; design; economic cost; fighting; fitness; global health; insight; lipidome; lipophilicity; membrane biogenesis; mutant; new therapeutic target; novel; novel therapeutics; pathogen; prevent; protein transport; stem

Abstract The increasing rise in antibiotic resistance and the diminished discovery of new antimicrobials threatens global healthcare. Of particular concern are Gram-negative pathogens, as these organisms are intrinsically resistant to multiple classes of antibiotics and the discovery of novel drugs targeting these bacteria has remained challenging. The innate resistance of these organisms is provided primarily by their outer membrane (OM), a defining feature of Gram negatives that encapsulates their peptidoglycan layer. Unlike the inner membrane (IM) that is composed solely of glycerophospholipids (GPLs), the OM is asymmetrical with GPLs found in the inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane organization affords protection from large polar molecules, as well as lipophilic compounds, creating an impervious barrier. Since the OM is essential, pathways required for its assembly are key targets for antimicrobial design. Currently, there are no antibiotics that directly target OM biogenesis in clinical use and first attempts have proven difficult. Thus, it remains critical to investigate cell envelope biology for future and current antimicrobial design. Recently, we discovered a connection between the GPL cardiolipin (CL) and the synthesis and transport of LPS. E. coli harbors three distinct enzymes that synthesize CL, yet CL is not required for cell viability and is the least abundant of the three major GPLs in Gram negatives. We found LpxM, the enzyme that adds the last acyl chain to the lipid anchor of LPS, to be critical for viability in the absence of clsA. Suppressors of clsA and lpxM synthetic lethality were identified in msbA, a gene that encodes the essential, homodimeric ABC transporter that “flips” LPS across the IM. Multiple pieces of genetic and biochemical data supported a model in which CL enhances MsbA activity driving LPS transport. Also, we observed that single mutants lacking either ClsA, the primary CL synthase, or LpxM have reduced LPS levels. This suggests the cell can “sense” defects in LPS transport at the cytoplasmic face of the IM and slow LPS synthesis to balance OM lipid content. In the current application we will define (i) the functional role of CL in MsbA-dependent LPS transport, (ii) characterize specific MsbA-CL interactions and determine how they impact MsbA activity, (iii) determine if ClsA and MsbA are co-localized in the bacterial cell envelope, and (iv) determine how defects in LPS transport results in feedback inhibition of LPS synthesis. Completion of these Aims will provide novel insights into cell envelope biogenesis and promote the development of novel therapeutics targeting Gram-negative pathogens.

摘要 抗生素耐药性的增加和新抗菌素发现的减少威胁着全球 医疗保健。特别令人担忧的是革兰氏阴性病原体，因为这些微生物具有内在的抵抗力 对多种抗生素和针对这些细菌的新药的发现 很有挑战性。这些生物的先天抵抗力主要是由它们的外膜(OM)提供的 革兰氏底片的定义特征，它包裹着它们的肽聚糖层。与内膜不同 (IM)，只由甘油磷脂(GPL)组成，OM与在 内叶和脂多糖(LPS)定位于外叶。这种独特的膜结构 提供保护，不受大极性分子和亲脂化合物的影响，形成不透水屏障。 由于OM是必不可少的，其组装所需的途径是抗菌设计的关键目标。 目前，临床上还没有直接针对OM生物发生的抗生素，第一次尝试已经 事实证明这很难。因此，研究细胞被膜生物学对于未来和当前的抗菌剂仍然是至关重要的。 设计。 最近，我们发现甘油三酯(GPL)与心磷脂(CL)的合成和转运之间存在联系。 LP。大肠杆菌含有合成CL的三种不同的酶，但CL不是细胞存活所必需的，而是 在革兰氏阴性的三种主要GPL中最不丰富的。我们发现了LpxM，一种添加最后一个酰基的酶 在没有里昂证券的情况下，脂链与脂锚的结合对生存至关重要。里昂证券和LpxM的抑制者 在编码基本的同源二聚体ABC转运蛋白的基因msba中发现了合成致死性 这让IM中的内毒素“翻转”过来。多条遗传和生化数据支持CL 增强MSBA活性，推动内毒素转运。此外，我们还观察到，缺乏CLSA的单个突变体 初级CL合成酶或LpxM降低了内毒素水平。这表明细胞可以“感觉到”内毒素的缺陷。 在IM的细胞质表面运输，并减缓内毒素的合成，以平衡OM的脂质含量。在当前 应用我们将定义(I)CL在依赖MSBA的内毒素转运中的功能作用，(Ii)表征 具体的MSBA-CL相互作用并确定它们如何影响MSBA活动，(Iii)确定里昂证券和MSBA 共定位于细菌细胞被膜中，以及(Iv)确定内毒素转运缺陷如何导致 反馈抑制内毒素合成。这些目标的完成将为细胞包膜提供新的见解 生物发生和促进针对革兰氏阴性病原体的新疗法的开发。