LIPOOLIGOSACCHARIDES OF HAEMOPHILUS INFLUENZA

流感嗜血杆菌的脂寡糖是重要的 毒力因子 脂寡糖的脂质A部分 (LOS)是造成这种毒性的部分原因 有机体 LOS的寡糖部分在 致病性H.流感感染不太清楚。 我们 进度报告中概述的初步研究表明， 这些来自Hib和NTHi的寡糖含有这样的结构， 模拟至少三种不同的人表面糖脂抗原。 在 此外，我们还克隆了两个不同的生物合成基因簇， 与这些寡糖的表达相关。 A 7.2 kb 这些簇之一的一部分已被测序，并且缺失或 定点H.产生流感病毒突变体。 这种转变 将DNA修饰到Hib菌株A2中产生了同基因Hib菌株 具有稳定的LOS突变，其在人体组织中的侵入性较小 培养细胞系比同基因野生型Hib菌株。 我们 研究已经导致了结构的物理化学分析 来自Hib和NTHi菌株的寡糖。 这一分析表明， 独特的深层核心结构的H.流感LOS寡糖。 同时取代支链三庚糖在这个 结构可以导致合成一系列复杂寡糖 侧链。 此外，物理化学研究提供了 产生的寡糖修饰的结构确认 通过一个寡糖生物合成簇 鉴定 我们的假设在这个重新提交的国家，H。 流感病毒LOS寡糖通过免疫途径在发病机制中发挥作用 通过分子模拟逃避，并促进近距离 粘附于人细胞并可能促进人细胞侵入。 为了解决这个假设，我们计划继续定义 参与Hib和NTHi生物合成的过程 低聚糖。 此外，我们建议从一个H。 流感生物群埃及人（HIA）菌株已被证明是 人鼻咽器官培养模型的侵袭性。 通过 产生表达有限的Hib、NTHi和HIA突变体， 低聚糖的库，我们将定义低聚糖 负责粘附和侵入人类细胞的残基。 最后，我们将完成服务水平的结构分析 Hib菌株A2和NTHi菌株2019的寡糖和研究所 分析了H.埃及型流感 这些目标 将通过以下具体目标来实现：1） H.流感LOS寡糖生物合成 基因簇 2)理化特性 Hib、NTHi和HIA LOS的寡糖部分。 3)定义 参与粘附的LOS寡糖结构， 在组织培养和器官培养系统中的入侵。 开展 这些特定目标的结构方面，我们将使用NMR 和质谱技术。 具体来说，我们将采用 电喷雾离子化-基质辅助激光解吸质谱 光谱法来评估LOS的结构异质性。 串联MSIMS 然后将用于四扇区仪器或通过低 能量ESI MSIMS，以确定特定的寡糖结构， 这些物种。

The lipooligosaccharides of Haemophilus influenzae are important virulence factors. The lipid A portion of the lipooligosaccharide (LOS) is responsible in part for the toxicity associated with this organism. The role of the oligosaccharide portion of the LOS in pathogenesis of H. influenzae infection is less clear. our preliminary studies outlined in the progress report have shown that these oligosaccharides from Hib and NTHi contain structures which mimic at least three different human surface glycolipid antigens. In addition, we have cloned two distinct clusters of biosynthesis genes associated with expression of these oligosaccharides. A 7.2 kb portion of one of these clusters has been sequenced and deletion or site directed H. influenzae mutants generated. Transformation of this modified DNA into Hib strain A2 has resulted in isogenic Hib strains with stable LOS mutations which are less invasive in human tissue culture cell lines than the isogenic wild type Hib strain. Our studies have resulted in physicochemical analysis of the structure of oligosaccharides from Hib and NTHi strains. This analysis has shown a unique deep core structure for the H. influenzae LOS oligosaccharide. Simultaneous substitution of the branched tri heptose in this structure can lead to synthesis of a complex array of oligosaccharide side chains. In addition, physicochemical studies have provided structural confirmation of the oligosaccharide modifications produced by one of the oligosaccharide biosynthesis clusters we have identified. Our hypothesis in this resubmission states that H. influenzae LOS oligosaccharides play a role in pathogenesis by immune evasion through molecular mimicry and act to promote close range adherence to human cells and possibly facilitate human cell invasion. To resolve this hypothesis, we plan to continue to define the nature of the process involved in the biosynthesis of the Hib and NTHi oligosaccharides. In addition, we propose the study of LOS from a H. influenzae biogroup aegyptius (HIA) strain which has been shown to be invasive for the human nasopharyngeal organ culture model. By generation of Hib, NTHi and HIA mutants expressing a limited repertoire of oligosaccharides, we will define the oligosaccharide residues responsible for adherence and invasion of human cells. Finally, we will complete the structural analysis of the LOS oligosaccharide of Hib strain A2 and NTHi strain 2019 and institute analysis of the H. influenzae biogroup aegyptius LOS. These goals will be accomplished by the following Specific Aims: 1) Characterization of H. influenzae LOS oligosaccharide biosynthesis gene clusters. 2) Physiochemical characterization of the oligosaccharide portion of Hib, NTHi and HIA LOS. 3) Definition of the LOS oligosaccharide structure(s) involved in adherence and invasion in tissue culture and organ culture systems. To carry out the structural aspects of these specific aims, we will be using NMR and mass spectrometry techniques. Specifically, we will employ electrospray ionization and matrix-assisted laser desorption mass spectrometry to assess structural heterogeneity of LOS. Tandem MSIMS will then be used either on a four sector instrument or through low energy ESI MSIMS to determine specific oligosaccharide structures in these LOS species.