Illuminating the essential role of the outer membrane component and drug target, lipopolysaccharide

阐明外膜成分和药物靶点脂多糖的重要作用

• 批准号: 10668249
• 负责人: Ryan Adam Valdez
• 金额: $ 3.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668249
• 关键词: Acinetobacter baumannii; Address; Animals; Antibiotics; Applied Genetics; Atomic Force Microscopy; Bacteria; Biology; Bypass; Cell Separation; Cell Survival; Cell Wall; Cell division; Cell physiology; Cells; Cellular Morphology; Charge; Clinical; Colistin; Collection; Compensation; Complement; Cytology; Data; Defect; Divalent Cations; Drug Targeting; Escherichia coli; Fellowship; Filament; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Grant; Growth; Immune Targeting; Immune system; Infection; Innate Immune System; Lead; Lipopolysaccharides; Mediator; Membrane; Mentors; Microscopy; Modeling; Morphology; Mutation; Nature; Organism; Osmolar Concentration; Osmotic Shocks; Peptidoglycan; Permeability; Phenotype; Phospholipids; Physiology; Process; Protein Biochemistry; Research Personnel; Role; Signal Transduction; Signaling Molecule; Structure; Testing; Time; Work; career; cell envelope; cell growth; colistin resistance; conditional mutant; crosslink; experimental study; gain of function; genetic approach; insight; interest; knock-down; mutant; pathogen; pressure; professor; single cell analysis; skills; stem

The gram-negative outer membrane (OM) is asymmetric, with phospholipids on the inner leaflet and lipopolysaccharide (LPS) on the outer leaflet. A defining part of the gram-negative cell envelope, LPS is a major signal for infection in animals and is a target of last resort antibiotics, such as colistin. Normally essential, clinical use of colistin has selected for Acinetobacter baumannii strains that do not produce LPS. Although LPS has been implicated in diverse functions, its precise role in gram-negative biology is unclear. Recent work in E. coli suggests LPS is capable of contributing as much rigidity to the cell envelope as the cell wall, a function that requires polyionic interactions between negative moieties on LPS and divalent cations. Analysis of conditional E. coli mutants defective in LPS synthesis and transport revealed formation of filaments and cell chains, suggesting an additional role for LPS in cell division and separation. The essentiality of LPS in either process is unclear. To evaluate the essential role(s) of LPS in gram-negative biology, I will leverage a set of E. coli mutants to conditionally knockdown LPS synthesis and transport, manipulate LPS charge (and thereby its interactions with divalent cations to provide rigidity), and produce a minimal LPS structure. Using this collection in Aim 1, I will systematically characterize the effect(s) of LPS defects on cell growth and morphology to understand the contribution of LPS to both phenomena. In Aim 2, I will test my hypothesis that providing cell envelope rigidity is a primary, essential function of LPS. Aim 2.1 will evaluate the ability of hyperosmotic conditions (which reduce the force exerted by turgor pressure on the cell envelope) to compensate for LPS defects. Because the force of turgor pressure is spread between the cell wall and the OM, Aim 2.2 will test whether increasing rigidity of the cell envelope via cell wall crosslinking can compensate for LPS defects. In Aim 3, I will identify the steps in cell division and separation impacted by defects in LPS synthesis and transport, respectively. This effort will illuminate the mechanistic basis of filamentation and chaining in LPS mutants. Examination of A. baumannii LPS deletion mutants identified a correlation between division and cell survival. I am thus particularly interested in testing whether enhancing division using complementary genetic strategies promotes growth of LPS deficient E. coli. If enhancing cell envelope rigidity is an essential role of LPS, additional septa may offset lethality associated with LPS defects through a positive impact on the structural integrity of the cell as a whole. A major signaling molecule for the immune system and target for last resort antibiotics, a better understanding of the essential role of LPS in gram-negative biology will provide insights into mitigation and treatment of gram- negative pathogens. Through this F31 fellowship, I will develop expertise in microscopy, protein biochemistry, and single cell analysis. Further, it will grant me the time and support necessary to hone my skills as a scientific mentor and communicator to prepare me for a career as a professor and independent investigator.

革兰氏阴性外膜 (OM) 是不对称的，内层有磷脂， 外层有脂多糖（LPS）。 LPS 是革兰氏阴性细胞包膜的定义部分 动物感染的主要信号，也是粘菌素等抗生素最后手段的目标。通常必不可少， 粘菌素的临床使用选择了不产生LPS的鲍曼不动杆菌菌株。虽然脂多糖 涉及多种功能，但其在革兰氏阴性生物学中的确切作用尚不清楚。最近在 E 的工作。 大肠杆菌表明 LPS 能够为细胞膜提供与细胞壁一样多的刚性，这一功能 需要 LPS 上的负基团和二价阳离子之间的聚离子相互作用。条件分析 LPS 合成和运输缺陷的大肠杆菌突变体揭示了细丝和细胞链的形成， 表明 LPS 在细胞分裂和分离中发挥着额外的作用。 LPS 在这两个过程中的重要性是 不清楚。 为了评估 LPS 在革兰氏阴性生物学中的重要作用，我将利用一组大肠杆菌突变体来 有条件地抑制 LPS 合成和运输，操纵 LPS 电荷（及其相互作用） 与二价阳离子以提供刚性），并产生最小的 LPS 结构。在目标 1 中使用这个集合，我 将系统地表征 LPS 缺陷对细胞生长和形态的影响，以了解 LPS 对这两种现象的贡献。在目标 2 中，我将检验我的假设：提供细胞包膜刚性 是 LPS 的一个主要的、必要的功能。目标 2.1 将评估高渗条件的能力（其中 减少膨胀压力对细胞膜施加的力）以补偿 LPS 缺陷。因为 膨胀压力的力在细胞壁和 OM 之间传播，目标 2.2 将测试是否增加刚性 通过细胞壁交联改变细胞膜可以弥补 LPS 的缺陷。在目标 3 中，我将确定步骤 细胞分裂和分离分别受到 LPS 合成和运输缺陷的影响。这项努力将 阐明 LPS 突变体丝化和链化的机制基础。鲍曼不动杆菌的检查 LPS 缺失突变体确定了分裂和细胞存活之间的相关性。因此我特别感兴趣 测试使用互补遗传策略增强分裂是否促进 LPS 的生长 缺乏大肠杆菌。如果增强细胞包膜刚性是 LPS 的重要作用，则额外的隔膜可能会抵消 通过对整个细胞的结构完整性产生积极影响，与 LPS 缺陷相关的致死率。 免疫系统的主要信号分子和最后手段抗生素的目标，更好地理解 LPS 在革兰氏阴性生物学中的重要作用将为缓解和治疗革兰氏阴性提供见解。 阴性病原体。通过这个 F31 奖学金，我将发展显微镜、蛋白质生物化学、 和单细胞分析。此外，它将给予我必要的时间和支持来磨练我作为一名 科学导师和沟通者，帮助我为教授和独立研究者的职业生涯做好准备。