Dispersin from Enteroaggregative E. coli

来自肠聚集性大肠杆菌的分散素

• 批准号: 6813738
• 负责人: James P. Nataro
• 金额: $ 31.32万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6813738
• 关键词: Escherichia coli; bacteria infection mechanism; bacterial genetics; bacterial proteins; biological models; chemical models; emerging infectious disease; gastrointestinal infection; genetic manipulation; host organism interaction; membrane channels; membrane structure; membrane transport proteins; nuclear magnetic resonance spectroscopy; pilus; protein localization; protein structure function; protein transport; secretion; site directed mutagenesis; structural biology; tissue /cell culture; virulence

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.

性状（由申请方提供）：肠聚集性大肠杆菌。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。我们已经表明，EAEC粘附到人肠粘膜是由聚集粘附菌毛（AAF）介导的，但由AAF菌毛介导的强自身凝集表型是由非共价连接到细菌表面的亲水性蛋白质外壳调节的。外壳蛋白是一种10.2 kDa的物质，我们将其命名为分散蛋白，它是EAEC毒力质粒上aap基因的产物。我们最近解决了分散素的溶液结构，并已表明它是一个紧密折叠的棒状结构，具有两个突出的β-片层。我们已经发现，易位的分散到细菌细胞表面需要一个假定的ABC转运蛋白复合物，其中包括一个同源的外膜通道TolC。我们提出了一个基于TolC晶体结构的AatA通道的分子模型。鉴于分散蛋白的新奇和重要性，EAEC发病机制，以及新出现的重要性TolC家族成员和ABC转运蛋白，我们建议进一步表征分散蛋白的结构-功能方面以及其分泌的功能特性。这些研究将有助于了解这些重要领域中的每一个。将按照三个不同的目标组织工作。目的1：分散素的结构与功能分析。在这里，我们将讨论分泌和功能的要求。NMR结构将用于建立假设，NMR光谱将用于评价诱变的影响。目的2：AatA的结构与功能分析。在这个目标中，我们将验证基于TolC的AatA转运蛋白的扩散模型。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，并赋予与TolC本身相比的功能多样性。目的3：内膜ABC复合物的表征。在目的，我们将表征所需的内膜复合物的分散素分泌，非典型的ABC转运蛋白AatPBCD为代表。每种蛋白质将被定位，并使用诱变和生化策略解决其作用。