LIPOOLIGOSACCHARIDES OF HAEMOPHILUS INFLUENZA

流感嗜血杆菌的脂寡糖是很重要的 毒力因素。脂低聚糖中的脂A部分 (LOS)对与此相关的毒性负有部分责任 有机体。视黄醇低聚糖部分的作用 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的寡糖包含 模拟至少三种不同的人类表面糖脂抗原。在……里面 此外，我们还克隆了两组不同的生物合成基因 与这些寡糖的表达有关。A 7.2 KB 这些集群之一的一部分已被排序并删除或 产生了定点突变的流感嗜血杆菌。这一点的转变 将DNA改造成Hib A2株，获得了等基因的Hib株 具有稳定的LOS突变，这种突变在人体组织中侵袭性较小 培养的细胞系优于同基因野生型Hib株。我们的 研究结果是对其结构进行了物理化学分析 Hib和NTHi菌株的低聚糖。这一分析表明， 流感嗜血杆菌低聚糖独特的深层核心结构。 该化合物中支链三庚糖的同时取代 结构可导致合成一系列复杂的低聚糖 侧链。此外，物理化学研究还提供了 低聚糖修饰产物的结构确认 通过我们拥有的一个低聚糖生物合成簇 已确认身份。我们在重新提交的文件中提出的假设是H。 流感病毒低聚糖通过免疫在发病机制中的作用 通过分子模仿和近距离推动行动来逃避 黏附于人类细胞，并可能促进人类细胞的入侵。 为了解决这一假设，我们计划继续定义 Hib和NTHi的生物合成过程 低聚糖。此外，我们还建议从H。 埃及埃及流感生物群(HIA)菌株已被证明是 侵袭性为人鼻咽部器官培养模型。通过 表达限制性基因的Hib、NTHi和Hia突变体的产生 低聚糖曲目，我们将定义低聚糖 对人体细胞的黏附和入侵负责的残留物。 最后，我们将完成LOS的结构分析。 Hib A2株和NTHi 2019株低聚糖及其研究所 埃及埃及流感嗜血杆菌生物群的分析这些目标 将通过以下具体目标实现1) 流感嗜血杆菌低聚糖生物合成特性的研究 基因簇。2)样品的物理化学表征 Hib、NTHi和Hia Los的低聚糖部分。3)定义 参与黏附的LOS寡糖结构(S) 侵入组织培养和器官培养系统。执行 这些特定目标的结构方面，我们将使用核磁共振 和质谱学技术。具体地说，我们将聘用 电喷雾电离和基质辅助激光解吸质量 用光谱分析来评估LOS的结构异质性。串联MS/MS 然后在四扇区仪器上使用或通过LOW 能量电喷雾电喷雾质谱(ESI-MS/MS)用于确定低聚糖的结构 这些洛城物种。

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 sitedirected 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 MS/MS will then be used either on a four sector instrument or through low energy ESI MS/MS to determine specific oligosaccharide structures in these LOS species.