LptA-mediated transport of LPS

LptA 介导的 LPS 转运

• 批准号: 8756571
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8756571
• 关键词: Affinity; Alanine; Amino Acids; Antibiotics; Bacteria; Binding; Binding Sites; Biological Assay; C-terminal; Calorimetry; Carrier Proteins; Cell surface; Cells; Cessation of life; Characteristics; Data; Development; Disease; Drug Design; Drug Targeting; Electron Spin Resonance Spectroscopy; Employee Strikes; Endotoxins; Environment; Escherichia coli; Foundations; Future; Genes; Genetic; Genetic Screening; Gram-Negative Bacteria; Growth; Human; In Vitro; Infection; Inflammatory; Knowledge; Lasers; Lead; Libraries; Lipid Binding; Lipopolysaccharides; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Modeling; N-terminal; Names; Periplasmic Proteins; Physiologic pulse; Plasmids; Process; Proteins; Pseudomonas aeruginosa; Role; Salmonella typhimurium; Septic Shock; Site; Spectrum Analysis; Staging; Structure; Techniques; Temperature; Time; Titrations; Transport Process; biophysical techniques; cell growth; in vivo; innovation; insight; light scattering; mutant; novel; pathogen; pathogenic bacteria; periplasm; pressure; protein protein interaction; protein transport; public health relevance; research study; tool

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.

说明(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。内毒素，也被称为内毒素，是在这一大类细菌中生存所必需的，是抵御宿主感染期间遇到的恶劣环境的第一道防线。鉴于内毒素在革兰氏阴性细菌生存中的重要作用--即如果没有发生内毒素转运的任何步骤，细菌细胞就会死亡--以及它所创造的独特的细胞表面，对内毒素合成和转运所涉及的蛋白质和机制的详细了解将是开发针对这些有希望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确定了许多参与内毒素转运的蛋白质，表明一组7种内膜(IM)、周质和OM蛋白(命名为LPTA、LptB、LptC、LptD、LptE、LptF和LptG)直接参与将内毒素从IM转移到OM。然而，这组蛋白质如何将内毒素转运到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内毒素的疏水结构域是如何穿过周质的。因此，拟议的研究将集中在周质蛋白LPTA如何从IM相关蛋白LptC接收内毒素，LPTA如何在穿过周质时保护内毒素的疏水酰链，以及LPTA如何将内毒素运送到OM处的LptDE。成功完成的研究将包括开发一种新的LPTA功能评估工具，创建一个全面的体内生长分析结果文库，以确定LPTA对其结构或功能至关重要的氨基酸，确定LPTA的特定位点和涉及内毒素结合的构象变化，以及表征LPTA与其结合伙伴LptC、LptDE和LPS之间的相互作用。新的基因筛选、激光光散射分析、创新的电子顺磁共振(EPR)光谱研究和等温滴定热法测量的结果将为深入了解内毒素在革兰氏阴性细菌周质中的转运机制提供详细的见解。这一独特的知识将极大地提高我们对细菌内毒素转运的了解，并为未来研究其他未知结构和功能的LPT蛋白奠定基础。