Genetic Analysis of Bordetella Pathogenesis

描述（由申请人提供）：感染发展的早期且通常是关键的步骤是病原体粘附于其宿主的上皮表面。由革兰氏阴性杆菌属的成员表达的丝状血凝素（FHA）是无菌毛粘附素大家族的原型，所述无菌毛粘附素既与表面结合又释放到周围环境中。这种高免疫原性蛋白是所有无细胞百日咳疫苗的主要成分。使用B的体外研究。从B中纯化的百日咳和FHA。百日咳的研究人员已经鉴定了FHA内的各种结合结构域，并表明该蛋白质在表面结合时可作为粘附素（通过介导与纤毛呼吸道上皮的附着），在释放时可作为免疫调节剂（通过与上皮细胞和巨噬细胞表面上的整联蛋白受体相互作用）。然而，由于缺乏研究人类适应性病原体B的自然宿主动物模型，对FHA的体内作用了解甚少。百日咳。我们已经开发了遗传工具、各种体外试验和高度敏感的动物模型，用于使用B研究FHA及其各种结合结构域在天然细菌-宿主相互作用背景下的作用。支气管败血症和兔、大鼠和小鼠。我们的初步数据是B。表达来自B的FHA的支气管败血症菌株。对百日咳的免疫学研究表明，这些敏感的测定将使我们能够剖析特异性FHA结合结构域在粘附和免疫调节中的作用。我们建议1）构建表达嵌合FHA分子的Bb菌株，并使用它们来确定各种FHA结合结构域如何在体内促进粘附、定殖以及先天性和体液免疫应答的发展，2）使用体外测定来研究FHA介导的宿主应答的分子基础，3）研究FHA加工和定位的机制，（4）探讨FHA加工和定位在呼吸道感染发生中的重要性。

DESCRIPTION (provided by applicant): An early and often crucial step in the development of infection is adherence of the pathogen to epithelial surfaces of its host. Filamentous hemagglutinin (FHA), expressed by members of the gram negative genus Bordetella, is prototypical of a large family of afimbrial adhesins that are both surface-associated and released into the surrounding milieu. This highly immunogenic protein is a primary component of all acellular pertussis vaccines. In vitro studies using B. pertussis and FHA purified from B. pertussis have identified various binding domains within FHA and suggest that this protein may function as an adhesin when surface-associated (by mediating attachment to ciliated respiratory epithelia) and an immunomodulator when released (by interacting with integrin receptors on the surface of epithelial cells and macrophages). In vivo roles for FHA, however, are poorly understood due to lack of natural-host animal models for studies with the human-adapted pathogen B. pertussis. We have developed genetic tools, a variety of in vitro assays, and highly sensitive animal models for studying the role(s) of FHA and its various binding domains in the context of a natural bacterial-host interaction using B. bronchiseptica and rabbits, rats and mice. Our preliminary data with a B. bronchiseptica strain expressing FHA from B. pertussis indicate that these sensitive assays will allow us to dissect the roles of specific FHA binding domains in adherence and immunomodulation. We propose to 1) construct Bb strains expressing chimeric FHA molecules and to use them to determine how the various FHA binding domains contribute to adherence, colonization and the development of innate and humoral immune responses in vivo, 2) investigate the molecular bases for FHA-mediated host responses using in vitro assays, 3) investigate the mechanism of FHA processing and localization, and 4) investigate the importance of FHA processing and localization in the development of respiratory infection.