Bacterial Membrane Proteins in Gram-Negative Sepsis

描述（由申请人提供）： 在感染期间，膜组分以复合物形式从细菌脱落，所述复合物包括LPS和两种细菌膜脂蛋白：胞壁素脂蛋白（MLP）和肽聚糖相关脂蛋白（PAL）。该更新建议旨在研究这些细菌膜组分中的每一种彼此之间以及与宿主脂蛋白之间的物理相互作用，以及每一种在通过Toll样受体（TLR）介导的细胞和全身炎症反应的激活中的相对贡献。我们的中心假设是，MLP和PAL有助于脓毒症的发病机制，并且它们在体内的活性可以通过彼此、与LPS以及与宿主蛋白质和脂蛋白的相互作用而改变。虽然大多数以前的工作已经研究了化学纯化或合成的TLR激动剂，这项工作将尽可能研究MLP和PAL的天然形式。第一个具体的目的是研究MLP和PAL从细菌细胞壁释放到细菌膜复合物和血清中的低密度形式的动力学。体外研究将使用人血清。将在两种小鼠感染模型中进行体内研究：腹膜E.杆菌感染 盲肠结扎和穿孔。随着时间的推移，将研究MLP和PAL与含有LPS和apoA1的脂蛋白颗粒亚群的物理联系以及MLP和PAL在器官和组织细胞中的分布。第二个具体目的是比较血清释放形式的MLP和PAL与各自的化学纯化形式在体外和体内的作用。将使用巨噬细胞和内皮细胞评价效应，因为这些细胞整合了脓毒症中的炎症反应。将使用MLP和PAL缺陷的细菌菌株以及TLR2和TLR4缺陷的小鼠来区分每种细菌脂蛋白和LPS对细胞活化的贡献。我们的第三个具体目标是研究抗MLP IgG对上述过程和两种模型中的存活率的影响。该实验旨在评估MLP和PAL的生理相关形式在感染期间脓毒症的病理生理学中发挥的重要性。

DESCRIPTION (provided by applicant): During infection, membrane components are shed from bacteria in complexes that include LPS and two bacterial membrane lipoproteins: murein lipoprotein (MLP), and peptidoglycans associated lipoprotein (PAL). This renewal proposal seeks to study the physical interactions that each of these bacterial membrane components have with each other and with host lipoproteins, and the relative contribution of each in activation of cellular and systemic inflammatory responses that are mediated through Toll-like receptors (TLRs). Our central hypothesis is that MLP and PAL contribute to the pathogenesis of sepsis, and that their activity in vivo may be altered by interactions with each other, with LPS, and with host proteins and lipoproteins. Whereas most previous work has studied chemically purified or synthetic TLR agonists, this work will study natural forms of MLP and PAL wherever possible. The first specific aim is to study the kinetics of release of MLP and PAL from the bacterial cell wall into bacterial membrane complexes and into low density forms in serum. In vitro studies will utilize human serum. In vivo studies will be performed in two models of infection in mice: peritoneal E. coli infection and cecal ligation and puncture. The physical association of MLP and PAL with subsets of lipoprotein particles containing LPS and apoA 1 and the distribution of MLP and PAL in organs and cells in tissues will be studied over time. The second specific aim is to compare the effects of serum released forms of MLP and PAL with chemically purified forms of each in vitro and in vivo. Effects will be evaluated using both macrophages and endothelial cells because these cells integrate the inflammaory response in sepsis. Bacterial strains deficient in MLP and PAL, and mice deficient in TLR2 and TLR4, will be used to distinguish the contributions of cellular activation by each bacterial lipoprotein and by LPS. Our third specific aim is to study the effect of anti-MLP IgG on the processes above and on survival in the two models. The experiments are designed to evaluate the importance that physiologically relevant forms of MLP and PAL play in the pathophysiology of sepsis during infection.