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

摘要 抗生素耐药性的增加和新抗菌药物发现的减少威胁着全球 健康护理特别值得关注的是革兰氏阴性病原体，因为这些微生物具有固有的耐药性 和发现针对这些细菌的新药仍然存在 挑战性这些生物体的先天抗性主要由它们的外膜（OM）提供， 包封其肽聚糖层的革兰氏阴性菌的定义特征。不像内膜 (IM)仅由甘油磷脂（GPL）组成，OM与在 内叶和脂多糖（LPS）定位于外叶。这种独特的膜组织 提供保护免受大极性分子以及亲脂性化合物的影响，从而形成不可渗透的屏障。 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，在临床使用中没有直接靶向OM生物发生的抗生素， 证明困难。因此，研究细胞被膜生物学对于未来和当前的抗微生物药物仍然至关重要。 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， LPS。E.大肠杆菌含有三种不同的合成CL的酶，但CL不是细胞活力所必需的， 革兰氏阴性菌中三种主要GPL中丰度最低的。我们发现了LpxM，一种将最后一个酰基 链到LPS的脂质锚，在缺乏clsA的情况下对生存力至关重要。clsA和lpxM的抑制因子 msbA是编码必需的同源二聚体ABC转运蛋白的基因， 将LPS“翻转”穿过IM。多个遗传和生化数据支持一个模型，其中CL 增强驱动LPS转运的MsbA活性。此外，我们观察到缺乏ClsA， 初级CL合酶或LpxM具有降低的LPS水平。这表明细胞可以“感知”LPS中的缺陷 在IM的细胞质面的转运和缓慢LPS合成以平衡OM脂质含量。在当前 我们将定义（i）CL在MsbA依赖性LPS转运中的功能作用，（ii）表征 （iii）确定ClsA和MsbA之间是否存在特异性MsbA-CL相互作用，并确定它们如何影响MsbA活性， 共定位于细菌细胞包膜，和（iv）确定LPS转运缺陷如何导致 LPS合成的反馈抑制。这些目标的完成将为细胞包膜提供新的见解 生物发生和促进革兰氏阴性病原体靶向新疗法的发展。

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.