LIPOOLIGOSACCHARIDES OF HAEMOPHILUS INFLUENZA

流感嗜血杆菌的脂寡糖很重要 毒力因素。 脂寡糖的脂质A部分 （LOS）对与此相关的毒性负有部分责任 有机体。 LOS寡糖部分的作用 流感嗜血杆菌感染的发病机制尚不清楚。 我们的 进度报告中概述的初步研究表明 这些来自 Hib 和 NTHi 的寡糖含有以下结构 模拟至少三种不同的人类表面糖脂抗原。 在 此外，我们克隆了两个不同的生物合成基因簇 与这些寡糖的表达有关。 7.2 kb 这些簇之一的一部分已被测序并删除或 产生定点流感嗜血杆菌突变体。 对此的改造 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，对人体组织的侵袭性较小 培养细胞系与同基因野生型 Hib 菌株相比。 我们的 研究人员对结构进行了物理化学分析 来自 Hib 和 NTHi 菌株的寡糖。 这项分析表明 流感嗜血菌 LOS 寡糖独特的深层核心结构。 同时取代支链三庚糖 结构可以导致合成一系列复杂的寡糖 侧链。 此外，物理化学研究还表明 所产生的寡糖修饰的结构确认 通过我们拥有的寡糖生物合成簇之一 确定。 我们在这次重新提交中的假设表明 H. 流感病毒LOS寡糖通过免疫在发病机制中发挥作用 通过分子拟态躲避并促进近距离行动 粘附于人体细胞并可能促进人体细胞侵袭。 为了解决这个假设，我们计划继续定义性质 Hib 和 NTHi 生物合成过程的概述 寡糖。 此外，我们建议研究 H. 埃及流感生物群 (HIA) 菌株已被证明 侵入性人类鼻咽器官培养模型。 由 产生表达有限的 Hib、NTHi 和 HIA 突变体 低聚糖的全部内容，我们将定义低聚糖 负责人体细胞粘附和侵袭的残基。 最后，我们将完成LOS的结构分析 Hib A2 株和 NTHi 2019 株寡糖及研究所 流感嗜血杆菌生物群 aegyptius LOS 的分析。 这些目标 将通过以下具体目标来实现：1) 流感嗜血菌 LOS 寡糖生物合成的表征 基因簇。 2) 理化特性 Hib、NTHi 和 HIA LOS 的寡糖部分。 3）定义 LOS寡糖结构参与粘附和 侵入组织培养和器官培养系统。 进行 这些具体目标的结构方面，我们将使用 NMR 和质谱技术。 具体来说，我们将聘请 电喷雾电离和基质辅助激光解吸质量 光谱测定法评估 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 测定特定寡糖结构 这些 LOS 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.