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 抗生素耐药性的增加以及新抗菌剂的发现减少威胁着全球 卫生保健。革兰氏阴性病原体特别关注，因为这些生物本质上具有抗性 多种类别的抗生素和针对这些细菌的新药物的发现仍然存在 具有挑战性的。这些生物的先天抵抗是由它们的外膜（OM）提供的主要的 定义革兰氏负面的特征，该特征封装了其辣椒层层。与内膜不同 （IM）仅由甘油磷脂（GPLS）组成，OM是不对称的，在GPL中发现了GPL 内部小叶和脂多糖（LPS）位于外部小叶。这个独特的膜组织 可以保护大型极性分子以及亲脂化合物，从而产生不透水的障碍。 由于OM是必不可少的，因此其组装所需的途径是抗菌设计的关键目标。 当前，没有直接靶向OM生物发生在临床使用中的抗生素，并且首先尝试 被证明困难。这是至关重要的 设计。 最近，我们发现了GPL Cardiolipin（CL）与合成和运输之间的联系 LPS。大肠杆菌包含合成Cl的三种不同的酶，但细胞活力并不需要Cl，这是 革兰氏底片中三个主要的GPL中的最少。我们发现LPXM是添加最后酰基的酶 链条链至LPS的脂质锚，对于在没有CLSA的情况下对于生存力至关重要。 CLSA和LPXM的抑制剂 在MSBA中鉴定了合成致死性，该基因编码了必需的同型ABC转运蛋白 那个“翻转”唱片横跨IM。多个遗传和生化数据支持了一个模型，其中Cl 增强MSBA活动驱动LPS运输。另外，我们观察到缺乏CLSA的单个突变体， 一级CL合酶或LPXM的LPS水平降低。这表明细胞可以在LPS中“感知”缺陷 在IM的细胞质面和LPS合成缓慢的脂质含量。在电流中 应用我们将定义（i）Cl在MSBA依赖性LPS传输中的功能作用，（ii）表征 特定的MSBA-CL相互作用并确定它们如何影响MSBA活动，（iii）确定CLSA和MSBA是否是否 在细菌细胞包膜中共定位，（iv）确定LPS转运中的缺陷如何导致 反馈抑制LPS合成。这些目标的完成将为细胞信封提供新颖的见解 生物发生并促进针对革兰氏阴性病原体的新型治疗的发展。