MOBILIZATION AND DELIVERY OF LIPOOLIGOSACCHARIDES TO HOST TARGETS

宿主对革兰氏阴性菌（GNB）的脂多糖（LPS）或低脂寡糖（LOS）的反应被认为在防御许多侵袭性GNB感染中起重要作用。如果控制不当，这些正常的保护性反应也会导致危及生命的系统性炎症性疾病。这在暴发性脑膜炎球菌败血症中得到了显著的说明，这是一种极端急性疾病，似乎与异常高水平的LOS积累有关。该项目的长期目标是更好地了解内毒素动员的分子决定因素及其与确定的宿主靶标的相互作用。一个主要的焦点将是释放的细菌膜泡的生物发生和生物学特性，被认为是体内弥散性内毒素的重要代表，也是体外培养脑膜炎奈瑟菌的一个特别突出的特征。我们的具体目标是：1)确定脑膜炎奈瑟菌与血管内宿主防御组分相互作用对脑膜炎球菌LOS合成和动员的影响；II)表征纯化LOS、膜泡和完整细菌向多形核白细胞（PMN）和内皮细胞传递的分子决定因素；III)比较纯化的LOS聚集体、膜泡和完整细菌对宿主介导的清除和降解的敏感性。我们将采用几种实验方法来定量分析脑膜炎球菌LOS的代谢和相互作用，包括定义细菌突变体，以便对细菌（糖）脂进行选择性放射性标记，TLC/图像分析，HPLC， GC-MS和MALTI-TOF，纯化内毒素结合蛋白和中和抗体，以及亲和纯化以寻找细菌膜出血-宿主相互作用的新介质。我们将在这个项目中大量地利用合作研究者，他们提供专业知识和工具，以一种不同寻常的系统和控制的方式研究脑膜炎球菌（内毒素）-宿主相互作用的动力学。这些研究可能为两个基本问题提供新的见解：1)在细菌与宿主相互作用期间，内毒素的动员过程中，是什么控制了内毒素的动员？2)脑膜炎球菌的LOS和含LOS碎片的无细胞膜的独特物理特性如何影响内毒素与宿主机制的相互作用，从而介导促炎或抗炎反应？

Host responses to lipopolysaccharides (LPS) or lipooligosaccharides (LOS) of Gram-negative bacteria (GNB) are believed to play a major role in defense against many invasive GNB infections. These normally protective responses can also cause life-threatening systematic inflammatory disorders when inadequately controlled. This is dramatically illustrated in fulminant meningococcal septicemia, an extreme acute disease that appears linked to the accumulation of extraordinary high levels of LOS. The broad long-term objectives of this project are to better understand the molecular determinants of endotoxin mobilization and of its interaction with defined host targets. A major focus will be on the biogenesis and biological properties of released bacterial membrane blebs, believed to represent an important of disseminated endotoxin in vivo and in a particularly prominent feature of growing N. meningitidis in vitro. Our specific aims are to: I) Determine the effect of interaction of N. meningitidis with components of intravascular host defenses on the synthesis and mobilization of meningococcal LOS; II) Characterize the molecular determinants of delivery of purified LOS, membrane blebs and intact bacteria to polymorphonuclear leukocytes (PMN) and endothelial cells; and III) Compare the susceptibility of LOS presented in the forms of aggregates of purified LOS, membrane blebs and intact bacteria to host-mediated clearance and degradations. We will employ several experimental approaches to quantitatively analyze the metabolism and interactions of meningococcal LOS including define bacterial mutants to permit selective radiolabeling of bacterial (glyco)lipids, TLC/image analysis, HPLC, GC-MS and MALTI-TOF, purified endotoxin-binding proteins and neutralizing antibodies, and affinity purification to search for novel mediators of bacterial membrane bled-host interactions. We will draw heavily on collaborating investigators in this program who provide expertise and tools to study in an unusually systematic and controlled fashion the dynamics of meningococcal (endotoxin)-host interactions. These studies are likely to provide new insights concerning two fundamental questions: 1) What controls the mobilization of endo during the mobilization of endotoxin during bacterial interaction with the host? 2) How do the unique physical characteristics meningococcal LOS and of cell-free LOS-containing membrane from fragments affect the interaction of endotoxin with host machinery that mediate either pro- or anti- inflammatory responses?