IPAC-MEDIATED INVASION OF EPITHELIAL CELLS BY SHIGELLA

简介(摘自申请者摘要)：志贺氏菌属革兰氏杆菌 引起细菌性痢疾的肠道阴性病原体，严重的 世界范围内的健康威胁。志贺氏菌病的一个重要特征是细菌性。 进入非吞噬宿主细胞。侵袭质粒抗原(IPA) 蛋白质)已被确定为这一过程的入侵素；然而， 它们的确切生化功能尚不清楚。远景目标 这项研究的目的是确定其结构和功能特征 这些来自福氏志贺氏菌的入侵因子。为了实现这些目标，IPA 基因的克隆、表达、产物的纯化和生化 所研究的蛋白质中。IPAC被发现具有体外性质 与细菌内化有关。因此，该计划的具体目标 建议的工作是：1)定义IPAC之后的生化事件 与宿主细胞的结合；2)确定IPAC引起 强毒和无毒志贺氏菌的摄取；3)使用纯化的福氏杆菌 IPAB、C和D来研究促进蛋白质复合体的形成 体内侵袭；4)确定IPAC的功能结构域。这些 AIMS将使用细胞生物学和 生物化学与专门的荧光技术相结合。IPAC 功能域将使用单抗和 识别已定义表位的多克隆免疫血清 具有抑制或刺激……活动的特点 志贺氏菌侵袭性。这些研究将增进对S. 福氏杆菌入侵并用于识别重要的新生化靶标 用于预防和治疗细菌性痢疾。

DESCRIPTION (Adapted from the applicant's abstract): Shigella are gram negative enteric pathogens that cause bacillary dysentery, a serious world-wide health threat. An important feature of shigellosis is bacterial entry into nonphagocytic host cells. The invasion plasmid antigens (Ipa proteins) have been identified as the invasins of this process; however, their precise biochemical functions are not known. The long-range objective of this research is to determine the structural and functional features of these invasins from Shigella flexneri. To achieve these goals, the ipa genes were cloned, expressed, their products purified, and the biochemistry of the proteins investigated. IpaC was found to possess in vitro properties related to bacterial internalization. Therefore, the specific aims of the proposed work are: 1) to define the biochemical events that follow IpaC binding to host cells; 2) to determine mechanisms by which IpaC causes uptake of virulent and avirulent strains of S. flexneri; 3) to use purified IpaB, C, and D to investigate formation of protein complexes that facilitate invasion in vivo; and 4) to identify the functional domains of IpaC. These aims will be completed using diverse methodologies in cell biology and biochemistry in conjunction with specialized fluorescence techniques. IpaC functional domains will be identified using monoclonal antibodies and polyclonal immune sera which recognize defined epitopes and are characterized with respect to inhibitory or stimulatory activities on Shigella invasiveness. These studies will enhance understanding of S. flexneri invasion and serve to identify important new biochemical targets for prevention and treatment of bacillary dysentery.