MOLECULAR AND FUNCTIONAL STUDIES OF H INFLUENZAE PILI

菌毛流感病毒的分子和功能研究

• 批准号: 6170312
• 负责人: JANET R GILSDORF
• 金额: $ 35.57万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170312
• 关键词: Haemophilus influenzae; adhesin; bacterial genetics; cell adhesion; epithelium; epitope mapping; erythrocytes; genetic strain; host organism interaction; immunoelectron microscopy; laboratory mouse; molecular pathology; monoclonal antibody; pilus; polymerase chain reaction; protein sequence; protein structure function; receptor binding; virulence; western blottings

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.

描述（改编自申请人的摘要）：流感嗜血杆菌 （嗨）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。 第一步是在 Hi 感染的发展是鼻咽组织的定植， 这取决于Hi粘附鼻咽细胞的能力。 到 为此，Hi 进化出了与含有神经节苷脂的受体结合的菌毛 对上皮细胞和人红细胞。 Hi pili 结构复杂 由至少三种蛋白质组成，包括 HifA（主要菌毛 亚基）、HifD（其功能定义不明确）和 HifE，其中包含 实际的上皮细胞粘附素。 该项目的长期目标是确定干扰策略 坚持 Hi 治疗，从而预防 Hi 感染。 这个目标将会实现 通过了解 HifE 粘附素的基本机制 Hi 介导对人体组织的粘附。 具体目标 I 旨在鉴定以下物质的受体结合域： HifE通过三种途径： 1. 分析氨基酸序列 来自 Hi 呼吸道和侵袭性菌株的 HifE 保留区域； 2. 到 识别由干扰菌毛的单克隆抗体定义的表位 介导 Hi 与红细胞和上皮细胞的结合； 3. 构建 通过丙氨酸替换候选受体结合域的突变和 测试突变体在粘附测定中的功能。 Specific Aim II 旨在比较 Hi 的受体结合域 占据独特地位的菌株（生物型 IV 和生物群埃及菌株） 人类体内的生态位，可能具有独特的受体结合域。 Specific Aim III 旨在识别受体的组织特异性 结合结构域，通过在竞争性抑制测定中测试结合 多种人类上皮细胞的受体结合域突变体 并确定菌毛柄在结合特异性中的作用。 Specific Aim IV 旨在鉴定该物质的免疫原性潜力 受体结合结构域。 兔抗血清将上调领域 并通过全细胞斑点印迹评估抗体与 HifE 的结合 测定、蛋白质印迹测定和免疫电子显微镜。 受体 将测试结合域抗体的干扰能力 pilied Hi 对人类红细胞和呼吸细胞的粘附。