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.

抽象的 抗生素耐药性的日益增加和新抗菌药物发现的减少威胁着全球 卫生保健。特别值得关注的是革兰氏阴性病原体，因为这些生物体具有本质抵抗力 多种类型的抗生素和针对这些细菌的新药的发现仍然存在 具有挑战性的。这些生物体的先天抵抗力主要由其外膜 (OM) 提供，外膜是 封装其肽聚糖层的革兰氏阴性菌的定义特征。与内膜不同 (IM) 仅由甘油磷脂 (GPL) 组成，OM 与中发现的 GPL 不对称 内叶和脂多糖（LPS）定位于外叶。这种独特的膜组织 提供大极性分子和亲脂性化合物的保护，形成不渗透的屏障。 由于 OM 至关重要，因此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，首次尝试已 事实证明很困难。因此，研究细胞包膜生物学对于未来和当前的抗菌药物仍然至关重要 设计。 最近，我们发现了 GPL 心磷脂 (CL) 与合成和转运之间的联系 脂多糖。大肠杆菌具有三种合成 CL 的不同酶，但 CL 并不是细胞活力所必需的，而是 革兰氏阴性菌中三个主要 GPL 中含量最少的。我们发现了 LpxM，添加最后一个酰基的酶 与 LPS 脂质锚的链，在缺乏 clsA 的情况下对于活力至关重要。 clsA 和 lpxM 抑制剂 在 msbA 中鉴定出合成致死性，msbA 是编码必需的同二聚体 ABC 转运蛋白的基因 将 LPS“翻转”到 IM 上。多项遗传和生化数据支持 CL 模型 增强驱动 LPS 运输的 MsbA 活性。此外，我们观察到缺乏 ClsA 的单一突变体 初级 CL 合酶 (LpxM) 降低了 LPS 水平。这表明细胞可以“感知”LPS 的缺陷 在 IM 的细胞质表面运输并减缓 LPS 合成以平衡 OM 脂质含量。在当前 应用中，我们将定义 (i) CL 在 MsbA 依赖性 LPS 运输中的功能作用，(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 ClsA 和 MsbA 是否 共定位于细菌细胞包膜中，并且 (iv) 确定 LPS 运输缺陷如何导致 LPS 合成的反馈抑制。完成这些目标将为细胞包膜提供新的见解 生物发生并促进针对革兰氏阴性病原体的新型疗法的开发。