Genetic Analysis of Bordetella Pathogenesis

描述(由申请人提供)：在感染发展的早期，通常也是关键的一步是病原体附着在其宿主的上皮表面。丝状血凝素(FHA)是由革兰氏阴性杆菌属的成员表达的，是一大类表面相关并释放到周围环境中的粘附素家族的原型。这种高度免疫原性的蛋白是所有无细胞百日咳疫苗的主要成分。使用百日咳杆菌和从百日咳杆菌中提纯的FHA进行的体外研究发现，FHA中有多种结合域，并表明该蛋白可能在表面相关时作为粘附素(通过介导与纤毛呼吸道上皮细胞的附着)和在释放时作为免疫调节剂(通过与上皮细胞和巨噬细胞表面的整合素受体相互作用)。然而，由于缺乏研究人类适应的病原体百日咳杆菌的自然宿主动物模型，对FHA在体内的作用知之甚少。我们已经开发了遗传工具、各种体外分析方法和高灵敏度的动物模型，用于研究FHA及其各种结合结构域在自然细菌-宿主相互作用中的作用(S)，使用支气管败血症杆菌和兔子、大鼠和小鼠。我们对一株表达百日咳杆菌FHA的支气管败血症杆菌的初步数据表明，这些敏感的检测方法将使我们能够剖析特定的FHA结合域在黏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株，并利用它们来确定不同的FHA结合域如何在体内对黏附、定植以及天然和体液免疫反应的发展起作用；2)用体外实验研究FHA介导的宿主反应的分子基础；3)研究FHA加工和定位的机制；4)研究FHA加工和定位在呼吸道感染发展中的重要性。

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.