IPAC-MEDIATED INVASION OF EPITHELIAL CELLS BY SHIGELLA

描述（改编自申请人摘要）：志贺氏菌属革兰氏阳性菌 导致细菌性痢疾的肠道病原体阴性， 全球健康威胁。 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）已被鉴定为该过程的侵袭素;然而， 其精确的生物化学功能尚不清楚。 远景目标 这项研究的目的是确定结构和功能特点， 这些来自福氏志贺菌的侵袭。 为了实现这些目标， 基因被克隆，表达，其产物纯化，和生物化学 研究的蛋白质。 发现IpaC具有体外性质 与细菌内化有关。 因此， 建议的工作是：1）定义IpaC之后的生化事件 与宿主细胞结合; 2）确定IpaC引起 S. 3）使用纯化的 IpaB、C和D，以研究蛋白质复合物的形成， 4）鉴定IpaC的功能结构域。 这些 目标将使用细胞生物学的各种方法完成， 生物化学结合专门的荧光技术。 IPAC 将使用单克隆抗体鉴定功能结构域， 多克隆免疫血清识别确定的表位， 以抑制或刺激活性为特征的， 志贺氏菌的侵袭性。 这些研究有助于加深对S. 福氏杆菌入侵并用于鉴定重要的新生化靶标 用于防治细菌性痢疾。

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.