LptA-mediated transport of LPS

描述（由申请方提供）：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）外小叶的主要成分。LPS，也称为内毒素，是这一大类细菌生存所必需的，并作为抵御宿主感染期间遇到的敌对环境的第一道防线。考虑到LPS在革兰氏阴性菌存活中的重要作用，如果LPS转运的任何步骤不发生，细菌细胞就会死亡--以及它产生的独特的细胞表面，对LPS合成和转运中涉及的蛋白质和机制的详细理解将是开发针对这些有希望的新药靶点的新型抗生素的基础。 通过最近的遗传学研究已经鉴定了许多参与LPS转运的蛋白质，表明一组七种内膜（IM）、周质和OM蛋白（命名为LptA、LptB、LptC、LptD、LptE、LptF和LptG）直接参与将LPS从IM移动到OM。然而，这组蛋白如何将LPS转运到OM的机制尚不清楚。关于这个过程最引人注目的问题之一是LPS的疏水结构域如何穿过周质。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如何在OM处将LPS传递给LptDE。拟议研究的成功完成将包括开发一种新的LptA功能评估工具，建立一个全面的体内生长测定结果库，以鉴定对其结构或功能至关重要的LptA氨基酸，鉴定与LPS结合有关的特定LptA位点和构象变化，以及表征LptA及其结合伴侣LptC之间的相互作用，LptDE和LPS。新的遗传筛选，激光光散射分析，创新的电子顺磁共振（EPR）光谱研究，和等温滴定量热法测量的结果将提供详细的见解LPS运输的机制，革兰氏阴性菌的周质。这一独特的知识将大大提高我们对细菌中LPS转运的理解，并为未来对其他未知结构和功能的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.