Biology of the HMW1 and HMW2 Adhesins of H. Influenzae

描述（由申请人提供）：不可分型的流感嗜血杆菌是局部呼吸道疾病的常见病因，特别是中耳炎、鼻窦炎、肺炎和支气管炎。每次急性中耳炎发作后，中耳积液数周至数月，伴有严重的听力缺陷，进而影响语言习得、言语发育和学习成绩。不可分型流感嗜血杆菌引起的疾病发病机制的第一步涉及上呼吸道上皮的定植。我们已经证明了两种相关的高分子量蛋白HMW1和HMW2促进了对人上皮的附着，这是定植过程中的一个重要步骤。值得注意的是，HMW1-和hmw2 -1样蛋白存在于75-80%的非分型流感嗜血杆菌菌株中。在最近的工作中，我们已经确定HMW1和HMW2蛋白是自转运蛋白家族的变体成员，并且被糖基化。根据对HMW1的研究，糖基化需要一种名为HMW1C的蛋白质和一种参与LOS生物合成的磷酸葡萄糖糖化酶。此外，我们发现HMW1和HMW2的表达是阶段性的，使生物体能够适应不同的环境，逃避宿主的免疫反应。在本提案中，我们计划阐明HMW1和HMW2糖基化的分子细节。特别是，我们将定义修饰HMW1和HMW2的碳水化合物的化学结构，HMW1和HMW2糖基化的生物合成途径，以及糖基化与免疫原性的关系。在进一步的实验中，我们将阐明调控HMW1和HMW2表达的机制，特别关注位于HMW1和HMW2结构基因启动子内的7碱基对串联重复序列的功能，这些重复序列在数量上发生自发变化。我们还将研究一个保守的19碱基对序列的作用，该序列位于HMW1和HMW2结构基因启动子重复序列的上游，以及hmwB和hmwC基因的上游。最后，利用微阵列技术，我们将鉴定被HMW1/ hmw2介导的粘附激活的流感嗜血杆菌基因，然后评估这些基因在鼠中耳炎模型中的作用。从实际的角度来看，这些研究的结果可能与开发新型抗菌剂和有效治疗和预防非分型流感嗜血杆菌病的许可疫苗直接相关。更普遍的是，它们可以为宿主-微生物关系、蛋白质分泌、蛋白质糖基化和基因调控提供基本的见解。

DESCRIPTION (provided by applicant): Nontypable Haemophilus influenzae is a common cause of localized respiratory tract disease, especially otitis media, sinusitis, pneumonia, and bronchitis. Following each episode of acute otitis media, fluid remains in the middle ear for weeks to months and is associated with significant hearing deficit, which in turn can impair language acquisition, speech development, and school performance. The initial step in the pathogenesis of disease due to nontypable H. influenzae involves colonization of the upper respiratory epithelium. We have demonstrated that two related high-molecular-weight proteins called HMW1 and HMW2 promote attachment to human epithelium, an essential step in the process of colonization. Of note, HMW1- and HMW2-1ike proteins are present in 75-80% of all nontypable H. influenzae strains. In recent work, we have established that the HMW1 and HMW2 proteins are variant members of the autotransporter family and are glycosylated. Based on studies with HMW1, glycosylation requires a protein called HMW1C and a phosphoglucomutase involved in LOS biosynthesis. In addition, we have discovered that expression of HMW1 and HMW2 is phase variable, enabling the organism to adapt to diverse environments and evade the host immune response. In the present proposal we plan to elucidate the molecular details of HMW1 and HMW2 glycosylation. In particular, we will define the chemical structure of the carbohydrate modifying HMW1 and HMW2, the biosynthetic pathway involved in glycosylation of HMW 1 and HMW2, and the relationship between glycosylation and immunogenicity. In additional experiments, we will elucidate the mechanism by which expression of HMW1 and HMW2 is regulated, focusing in particular on the function of 7-base pair tandem repeats that lie within the promoters of the HMW1 and HMW2 structural genes and undergo spontaneous variation in number. We will also examine the role of a conserved 19-base pair sequence that is upstream of the repeats in the promoters of the HMW1 and HMW2 structural genes and upstream of the hmwB and hmwC genes. Finally, using microarray technology, we will identify H. influenzae genes that are activated by HMW1/HMW2-mediated adherence, then assess the effect of these genes in the chinchilla otitis media model. From a practical perspective, the results of these studies may be directly relevant to the development of novel antimicrobials and a licensed vaccine effective in the treatment and prevention of nontypable H. influenzae disease. More generally, they may provide fundamental insights into host-microbial relationships, protein secretion, protein glycosylation, and gene regulation.