MOLECULAR AND FUNCTIONAL STUDIES OF H INFLUENZAE PILI

简介(摘自申请者摘要)：流感嗜血杆菌 (Hi)是呼吸道感染的重要原因，包括 鼻窦炎、中耳炎、支气管炎和肺炎。第一步就是 Hi感染的发展是鼻咽组织的定植， 这取决于Hi黏附于鼻咽细胞的能力。至 为此，Hi进化出了与神经节苷脂受体结合的菌毛 在上皮细胞和人类红细胞上。Hi菌毛是复杂的结构 至少由三种蛋白质组成，包括HIFA(主要菌毛 子单元)、HifD(其功能定义不明确)和HifE，其包含 真正的上皮细胞粘附素。 该项目的长期目标是确定干预战略 与Hi坚持，从而防止Hi感染。这一目标将会实现 通过了解HifE粘附素的潜在机制 HI调节对人体组织的黏附。 特异性目的I用于鉴定沙门氏菌的受体结合域(S) HIFE通过三种方法：1.分析其氨基酸序列 来自Hi呼吸道和保守区侵袭性菌株的HifE； 确定由干扰菌毛的单抗定义的表位 介导的Hi与红细胞和上皮细胞的结合；3.构建 丙氨酸置换致候选受体结合区(S)突变 检测突变体在贴壁试验中的功能。 特异靶II用于比较Hi的受体结合结构域(S) 菌株(生物型IV型和埃及生物群菌株) 在人类内的利基，并可能具有独特的受体结合域。 特异性AIM III用于鉴定受体的组织特异性 结合结构域(S)，通过检测在竞争抑制试验中的结合 受体结合区突变体对多种人上皮细胞的作用 并通过确定菌毛柄在结合特异性中的作用。 特定的目的IV旨在确定免疫原性的潜力 受体结合域(S)。兔抗血清将被提升到结构域 用全细胞斑点印迹法检测抗体与HifE的结合 分析、Western印迹分析和免疫电子显微镜。感受器 结合结构域抗体将被测试其干扰能力 纤毛Hi与人红细胞和呼吸系统细胞的粘附性

DESCRIPTION (Adapted from the applicant's abstract): Haemophilus influenzae (Hi) are significant cause of respiratory tract infections, including sinusitis, otitis media, bronchitis, and pneumonia. The first step in the development of Hi infections is colonization of nasopharyngeal tissues, which depends on the ability of Hi to adhere to nasopharyngeal cells. To this end, Hi have evolved pili that bind to ganglioside-containing receptors on epithelial cells and human red cells. Hi pili are complicated structures composed of at least three proteins, including HifA (the major pilus subunit), HifD (whose function is poorly defined), and HifE which contains the actual epithelial cell adhesin. The long-term goal of this project is to identify strategies to interfere with Hi adherence and, thus, prevent Hi infections. This goal will be met through understanding the underlying mechanisms by which the HifE adhesin of Hi mediates adherence to human tissue. Specific Aim I is designed to identify the receptor binding domain(s) of HifE through three approaches: 1. To analyze the amino acid sequences of HifE from Hi respiratory and invasive strains for conserved regions; 2. To identify epitopes defined by monoclonal antibodies that interfere with pilus mediated Hi binding to erythrocytes and epithelial cells; 3. To construct mutations in candidate receptor binding domain(s) by alanine-replacement and to test the function of the mutants in adherence assays. Specific Aim II is designed to compare the receptor binding domain(s) of Hi strains (biotype IV and biogroup aegyptius strains) that occupy unique niches within humans and may possess distinctive receptor binding domains. Specific Aim III is designed to identify tissue specificity of the receptor binding domain(s), by testing in competitive inhibition assays the binding of receptor binding domain mutants to a variety of human epithelial cells and by determining the role of the pilus stalk in binding specificity. Specific Aim IV is designed to identify the immunogenic potential of the receptor binding domain(s). Rabbit antisera will be raised to the domains and binding of the antibodies to HifE assessed by whole cell dot blot assays, Western blot assays, and immunoelectron microscopy. The receptor binding domain antibodies will be tested for their ability to interfere with the adherence of piliated Hi to human erythrocytes and respiratory cells.