Structure & function of bacterial adhesion pili

描述(申请人提供)：病原菌在宿主中的存活率与它们与宿主组织保持附着的能力相关。通常，细菌表面的丝状粘附毛促进了这种粘连，就像ETEC(产肠毒素大肠杆菌)。ETEC导致严重腹泻，对世界范围内的健康构成重大风险，特别是对婴幼儿。此外，ETEC引起的腹泻很容易通过受污染的食物或水传播，这使国际旅行者在进入感染流行的地区时面临高风险。为了预防或限制疫情的爆发，我们必须了解细菌持续附着的机制，这可能导致定植和疾病。 有关粘连菌毛的结构信息将为合理设计防止细菌结合的新疗法或清除已与人类宿主结合的病原菌提供基础。该项目的长期目标是阐明菌毛的结构如何支持它们作为致病细菌的毒力因子的作用。在拟议的项目期间，将通过对ETEC菌毛的结构和功能的研究、对肾盂肾炎大肠杆菌(可导致肾脏的尿路感染)表面表达的P-菌毛的深入计算机模拟、菌毛损伤/恢复实验和I型粘附素的定位来研究粘连菌毛的结构和功能之间的关系。将采用一种综合的方法来1)阐明致病性ETEC菌毛的结构特征，使它们能够抵抗蠕动和其他肠道清洁系统。研究将包括电子显微镜和图像处理的负染和冷冻水合ETEC菌毛。2)研究P-菌毛螺旋丝可以解开为其原始长度的五倍的纤细结构的机制。能量最小化和空间约束将与遗传算法一起被用来建模，从单个单体亚基，到完整和受损的菌毛细丝原型。3)研究一种通过破坏菌毛来减少细菌结合的方法，目的是减少细菌负荷，从而允许身体的自然防御系统根除剩余的细菌。研究将使用光学镊子来测量破坏泌尿系致病细菌上表达的P-菌毛所需的力，并调查是否发生恢复。4)用免疫电子显微镜定位I型菌毛上的粘附素。

DESCRIPTION (provided by applicant): Survival of pathogenic bacteria in their host is correlated with their capacity to maintain attachment to host tissue. Often this adherence is facilitated by the presence of filamentous adhesion pili on the bacterial surface, as in ETEC (enterotoxigenic Escherichia coli). ETEC cause severe diarrhea, presenting a significant worldwide health risk, particularly for infants and small children. In addition, the ease with which ETEC-caused diarrhea spreads via tainted food or water, places international travelers at high risk when entering regions where infection is endemic. In order to prevent or limit outbreaks, it is vital that we understand the mechanism of sustained bacterial attachment that can lead to colonization and illness. Structural information about adhesion pili will provide a basis for rational design of new therapies for prevention of bacterial binding or for removal of pathogenic bacteria already bound to the human host. The long-term goal of this project is to elucidate how the structure of pili supports their role as a virulence factor for pathogenic bacteria. In the proposed project period, studies on the structure and function of ETEC pili, in-depth computer modeling of P-pili expressed on the surface of pyelonephritic Escherichia coli (which cause urinary tract infections involving the kidneys), pilus damage/recovery experiments, and localization of type 1 adhesins will be used to examine the relationship between the structure and the function of adhesion pili. A combined approach will be employed to 1) elucidate the structural features of virulent ETEC pili that enable them to withstand peristaltic motility and other intestinal cleansing systems. Studies will include electron microscopy and image processing of negatively stained and frozen-hydrated ETEC pili. 2) examine the mechanism by which the P-pilus helical filament can unwind to a thin fibrillar structure five times its original length. Energy minimization and spatial constraints will be used with genetic algorithms to model, from individual monomeric subunits, this prototypic pilus filament into both intact and damaged pili. 3) investigate a means for reducing bacterial binding through damage to pili, with the aim of reducing the bacterial load and thus permitting the body's natural defenses to eradicate the remainder. Studies will use optical tweezers to measure the forces necessary to damage P-pili expressed on uropathogenic bacteria, and to investigate whether recovery occurs. 4) localize the adhesins on type 1 pili using immunoelectron microscopy.