MOBILIZATION AND DELIVERY OF LIPOOLIGOSACCHARIDES TO HOST TARGETS

宿主对革兰氏阴性菌(GNB)的脂多糖(LPS)或脂低聚糖(LOS)的反应被认为在抵御许多侵袭性GNB感染方面发挥着重要作用。当控制不当时，这些正常的保护性反应也会导致危及生命的全身性炎症障碍。这在暴发性脑膜炎球菌败血症中得到了戏剧性的说明，这是一种似乎与极高水平的LOS积聚有关的极端急性疾病。该项目广泛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。重点将放在释放的细菌膜泡的生物发生和生物学特性上，被认为是体内播散性内毒素的重要代表，以及在体外生长脑膜炎奈瑟菌的一个特别突出的特征。我们的具体目标是：i)确定脑膜炎奈瑟菌与血管内宿主防御系统成分的相互作用对脑膜炎球菌LOS合成和动员的影响；ii)表征将纯化的LOS、膜泡和完整细菌运送到多形核白细胞(PMN)和内皮细胞的分子决定因素；以及iii)比较以纯化的LOS聚集体、膜泡和完整细菌的形式存在的LOS对宿主介导的清除和降解的敏感性。我们将使用几种实验方法来定量分析脑膜炎双球菌LOS的代谢和相互作用，包括定义细菌突变体以允许选择性地标记细菌(糖类)脂类、TLC/图像分析、高效液相、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与宿主相互作用的新媒介。我们将在这个项目中大量利用合作研究人员，他们提供专业知识和工具，以异常系统和受控的方式研究脑膜炎双球菌(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的内毒素动员过程中，是什么控制了Endo的动员？2)脑膜炎球菌LOS和片段中含有LOS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?