Dispersin from Enteroaggregative E. coli

描述（由申请人提供）：肠聚集性大肠杆菌是一种新兴的肠道病原体，其毒力机制仅部分阐明。我们已经证明EAEC对人肠粘膜的粘附是由聚集粘附菌毛（AAFs）介导的，但是由AAF菌毛介导的强自凝集表型是由非共价附着在细菌表面的亲水性蛋白外壳调节的。外衣蛋白是一个10.2 kDa的物种，我们将其命名为分散蛋白，它是aap基因在EAEC毒力质粒上的产物。我们最近解决了分散素的溶液结构，并表明它是一个紧密折叠的棒状结构，有两个突出的β片。我们已经发现分散蛋白在细菌细胞表面的易位需要一个假定的ABC转运蛋白复合物，其中包括外膜通道TolC的同源同源物。我们提出了一种基于TolC晶体结构的AatA通道分子模型。鉴于分散蛋白在EAEC发病机制中的新颖性和重要性，以及TolC家族成员和ABC转运蛋白的重要性，我们建议进一步表征分散蛋白的结构-功能方面及其分泌的功能特征。这些研究将有助于理解这些重要领域中的每一个。工作将按照三个不同的目标来组织。目的1：分散素的结构-功能分析。在这里，我们将讨论分泌和功能的需求。核磁共振结构将用于建立假设，核磁共振波谱将用于评估诱变的效果。目的2:AatA的结构功能分析。在这个目的中，我们将验证基于tolc的AatA分散剂模型。我们还将对AatA的特定区域进行定点诱变，这些区域被认为是重要的，并且与TolC本身相比具有功能多样性。目的3：内膜ABC复合物的表征。在目的上，我们将表征分散素分泌所需的内膜复合物，即以AatPBCD为代表的非典型ABC转运体。每个蛋白质将定位和他们的作用解决使用诱变和生化策略。

DESCRIPTION (provided by applicant): Enteroaggregative E. coli is an emerging enteric pathogen, whose virulence mechanisms are only partially elucidated. We have shown that EAEC adherence to the human intestinal mucosa is mediated by Aggregative Adherence Fimbriae (AAFs), but that the strong autoagglutination phenotype mediated by AAF fimbriae is modulated by a hydrophilic protein coat that is non-covalently attached to the surface of the bacterium. The coat protein is a 10.2 kDa species which we have named dispersin, the product of the aap gene on the EAEC virulence plasmid. We have recently solved the solution structure of dispersin and have shown it to be a tightly folded rod-like structure with two prominent beta-sheets. We have discovered that translocation of dispersin to the bacterial cell surface requires a putative ABC transporter complex, which includes a cognate homolog of the outer membrane channel TolC. We have proposed a molecular model for the AatA channel based on the TolC crystal structure. Given the novelty and importance of dispersin to EAEC pathogenesis, and the emerging importance of TolC family members and ABC transporters, we propose to characterize further both the structure-function aspects of the dispersin protein as well as functional characteristics of its secretion. These studies will provide contributions to understanding of each of these important areas. Work will be organized in three distinct aims. Aim 1: Structure-function analysis of dispersin. Here, we will address requirements for secretion and for function. The NMR structure will be used to build hypotheses and NMR spectroscopy will be used to evaluate the effects of mutagenesis. Aim 2: Structure-function analysis of AatA. In this aim, we will verify the TolC-based model of the AatA translocator of dispersin. We will also perform site-directed mutagenesis of specific regions of AatA suggested to be important and to confer diversity of function compared with TolC itself. Aim 3: Characterization of the inner membrane ABC complex. In aim, we will characterize the inner membrane complex required for dispersin secretion, the atypical ABC transporter represented by AatPBCD. Each protein will be localized and their roles addressed using mutagenesis and biochemical strategies.