LptA-mediated transport of LPS

描述（由申请人提供）：脂多糖（LPS）是革兰氏阴性菌外膜（OM）外小叶的主要成分，如大肠杆菌、鼠伤寒沙门菌和许多其他重要病原体。脂多糖，也被称为内毒素，对这种大型细菌的生存至关重要，并在宿主感染期间作为抵御恶劣环境的第一道防线。考虑到脂多糖在革兰氏阴性菌存活中的重要作用——即，如果脂多糖转运的任何步骤没有发生，细菌细胞就会死亡——以及它所产生的独特细胞表面，对脂多糖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。通过最近的遗传学研究，许多参与脂多糖运输的蛋白质已经被鉴定出来，表明一组7种内膜（IM）、质周和OM蛋白（命名为LptA、LptB、LptC、LptD、LptE、LptF和LptG）直接参与将脂多糖从IM转移到OM。然而，这组蛋白如何将LPS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脂多糖的疏水区域如何穿过外周质。因此，拟开展的研究将重点关注外质蛋白LptA如何从im相关蛋白LptC接收LPS， LptA如何在LPS穿过外质时保护其疏水酰基链，以及LptA如何将LPS传递到OM处的lpde。成功完成拟议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库，以确定对LptA结构或功能至关重要的LptA氨基酸，确定与LPS结合有关的特定LptA位点和构象变化，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量热测量的结果将为LPS在革兰氏阴性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增强我们对细菌中LPS转运的理解，并为未来对其他结构和功能未知的Lpt蛋白的研究奠定基础。

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.