Autotransporters of enterobacteriaceae

革兰氏阴性菌向细胞外环境分泌大量蛋白质， 这些蛋白质中的许多都参与了发病机制。关于蛋白质的作用机制 分泌，自动转运蛋白(AT)途径被认为是最常见的，但有很多 仍有待阐明。本申请是一项申请，要求继续资助 生产性R01奖，其主要贡献包括发现一个大型 分泌型革兰氏阴性毒力因子组(丝氨酸蛋白酶自身转运体 肠杆菌科[Spates])，至少作用模式的特征 其中的两个蛋白质，以及一个新的C-末端AT结构域的特征 催化外膜转位。本申请代表了一种跨学科 发病机制、细菌学、蛋白质生化和结构之间的关系 生物实验室。申请的目的如下。 目的1.唾液毒素的结构与功能。这一目标将解决以下功能 Spates和这种多样性的结构基础。三个相互关联的次级目标将是 追求：a)对Spate结构-功能关系的剖析。B)可以使用幽灵或 FAK裂解是Pet？的作用机制。此子目标将使用RNAi 评估幽灵蛋白和FAK在上皮细胞中丢失的作用的技术。C)澄清 作为殖民因素的斯皮特的作用。 目的2.外膜自体转运蛋白的转运。这一目标将是 追求四个相互关联的次级目标：a)Spateβ-螺旋的作用。我们会工作的 帕特里夏·克拉克博士，他是ATβ-螺旋研究的先驱。b) 散布AC区域的特征。C)中间蛋白的特性 AT易位中的相互作用，包括周质伴侣的作用。d) AT易位中蛋白质内相互作用的特征。 这项工作将扩展关于一组非常常见的毒力因子的知识，并得出 革兰氏菌最常见外膜转运方式的重要特征 阴性菌。

Gram negative bacteria secrete a large number of proteins to the extracellular milieu, and many of these proteins are involved in pathogenesis. Of the mechanisms of protein secretion, the autotransporter (AT) pathway is thought to be the most common, yet much remains to be elucidated. This application is a request for continuation of funding of a productive R01 award, the major contributions of which included the discovery of a large groups of secreted gram negative virulence factors (the serine protease autotransporters of Enterobacteriaceae [SPATEs]), the characterization of the modes of action of at least two of these proteins, and the characterization of a novel C-terminal AT domain that catalyzes outer membrane translocation. The present application represents an interdisciplinary effort among pathogenesis, bacteriology, protein biochemistry, and structural biology laboratories. The aims of the application are as follows. Aim 1. Structure-function of SPATE toxins. This aim will address the functions of SPATEs and the structural basis for this diversity. Three inter-related subaims will be pursued: a) Dissection of SPATE structure-function relationships. b) Can spectrin or FAK cleavage be the mechanism of action of Pet?. This subaim will use RNAi technology to assess the role of spectrin and FAK loss in epithelial cells. c) Elucidation of the role of the SPATEs as colonization factors. Aim 2. Autotransporter translocation through the outer membrane. This aim will be pursued as four inter-related subaims: a) Role of the SPATE beta-helix. We will work with Dr. Patricia Clark, who has pioneered the study of the AT beta-helix. b) Characterization of the SPATE AC region. c) Characterization of inter-protein interactions in AT translocation, including the roles of periplasmic chaperones. d) Characterization of intra-protein interactions in AT translocation. This work will extend knowledge on a very common set of virulence factors and elicidate important features of the most common means of outer membrane translocation in gram negative bacteria.