LptA-mediated transport of LPS

LptA 介导的 LPS 转运

• 批准号: 9275484
• 负责人: CANDICE S KLUG
• 金额: $ 29.07万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9275484
• 关键词: Affinity; Alanine; Amino Acids; Antibiotics; Assessment tool; Bacteria; Binding; Binding Sites; Biological Assay; C-terminal; Calorimetry; Carrier Proteins; Cell surface; Cells; Cessation of life; Characteristics; Crystallization; Data; Development; Disease; Drug Design; Electron Spin Resonance Spectroscopy; Employee Strikes; Endotoxins; Environment; Escherichia coli; Foundations; Future; Genes; Genetic Screening; Genetic study; Gram-Negative Bacteria; Growth; Human; Hydrophobicity; In Vitro; Infection; Inflammatory; Knowledge; Lasers; Lead; Libraries; Lipid Binding; Lipopolysaccharides; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Modeling; Molecular Conformation; N-terminal; Names; Periplasmic Proteins; Physiologic pulse; Plasmids; Process; Proteins; Pseudomonas aeruginosa; Role; Salmonella typhimurium; Septic Shock; Site; Spectrum Analysis; Structure; Techniques; Temperature; Time; Titrations; Transport Process; biophysical techniques; cell growth; experimental study; in vivo; innovation; insight; invention; light scattering; mutant; new therapeutic target; novel; pathogen; pathogenic bacteria; periplasm; pressure; protein protein interaction; protein transport; public health relevance

DESCRIPTION (provided by applicant): Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane (OM) of Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium and many other important pathogens. LPS, also referred to as endotoxin, is essential for survival in this large class of bacteria and serves as a first line of defense against hostile environments encountered during host infection. Given the essential role of LPS in the survival of Gram-negative bacteria - i.e., the bacterial cells die if any step o LPS transport does not occur - and the unique cell surface it creates, a detailed understanding of the proteins and mechanisms involved in LPS synthesis and transport will be the foundation on which to develop novel antibiotics against these promising new drug targets. Many of the proteins involved in LPS transport have been identified through recent genetics studies, suggesting that a set of seven inner membrane (IM), periplasmic, and OM proteins (named LptA, LptB, LptC, LptD, LptE, LptF, and LptG) are directly involved in moving LPS from the IM to the OM. However, the mechanism of how this group of proteins transports LPS to the OM is yet unknown. One of the most striking questions about this process is how the hydrophobic domain of LPS crosses the periplasm. Therefore, the proposed studies will focus on how the periplasmic protein LptA receives LPS from the IM-associated protein LptC, how LptA protects the hydrophobic acyl chains of LPS as it crosses the periplasm, and how LptA delivers LPS to LptDE at the OM. The successful completion of the proposed studies will include the development of a novel functional assessment tool for LptA, the creation of a comprehensive library of in vivo growth assay results to identify LptA amino acids critical for its structure or function, the identification of the specific LptA sites and conformational changes involved in LPS binding, and the characterization of the interactions between LptA and its binding partners LptC, LptDE, and LPS. The results of the novel genetic screenings, the laser light scattering analyses, the innovative electron paramagnetic resonance (EPR) spectroscopy studies, and the isothermal titration calorimetry measurements will provide detailed insights into the mechanism of LPS transport across the periplasm of Gram-negative bacteria. This unique knowledge will greatly enhance our growing understanding of LPS transport in bacteria and set the stage for future studies on the other Lpt proteins of unknown structure and function.

描述（申请人提供）： 脂多糖（LPS）是革兰氏阴性菌（如大肠杆菌、鼠伤寒沙门氏菌和许多其他重要病原体）外膜（OM）外叶的主要成分。 LPS，也称为内毒素，对于这一大类细菌的生存至关重要，并且是宿主感染期间遇到的恶劣环境的第一道防线。鉴于 LPS 在革兰氏阴性细菌生存中的重要作用（即，如果 LPS 运输的任何步骤不发生，细菌细胞就会死亡）以及它所产生的独特的细胞表面，对 LPS 合成和运输所涉及的蛋白质和机制的详细了解将成为开发针对这些有前景的新药物靶点的新型抗生素的基础。 最近的遗传学研究已经鉴定了许多参与 LPS 运输的蛋白质，表明一组七个内膜 (IM)、周质和 OM 蛋白质（名为 LptA、LptB、LptC、LptD、LptE、LptF 和 LptG）直接参与 LPS 从 IM 到 OM 的运输。然而，这组蛋白质如何将 LPS 转运至 OM 的机制尚不清楚。关于这一过程最引人注目的问题之一是 LPS 的疏水域如何穿过周质。因此，拟议的研究将重点关注周质蛋白LptA如何从IM相关蛋白LptC接收LPS，LptA如何在LPS穿过周质时保护LPS的疏水酰基链，以及LptA如何将LPS传递到OM处的LptDE。拟议研究的成功完成将包括开发一种新型的 LptA 功能评估工具，创建一个综合的体内生长测定结果库，以确定对其结构或功能至关重要的 LptA 氨基酸，识别 LptA 特定位点和参与 LPS 结合的构象变化，以及表征 LptA 与其结合伙伴 LptC、LptDE 和 LPS 之间的相互作用。新颖的遗传筛查、激光散射分析、创新的电子顺磁共振（EPR）光谱研究和等温滴定量热测量的结果将为LPS跨革兰氏阴性菌周质运输的机制提供详细的见解。这一独特的知识将极大地增强我们对细菌中脂多糖转运的了解，并为未来研究其他结构和功能未知的脂多糖蛋白奠定基础。