Structure & function of bacterial adhesion pili

性状（由申请方提供）：病原菌在其宿主中的存活与其保持与宿主组织附着的能力相关。通常，细菌表面的丝状粘附皮利的存在促进了这种粘附，如ETEC（产肠毒素大肠杆菌）。ETEC引起严重腹泻，在全球范围内构成重大健康风险，特别是对婴儿和幼儿。此外，ETEC引起的腹泻通过受污染的食物或水传播的容易性，使国际旅行者在进入感染流行地区时处于高风险之中。为了预防或限制疫情，我们必须了解可导致定植和疾病的持续细菌附着机制。 关于粘附皮利的结构信息将为合理设计新的治疗方法提供基础，以防止细菌结合或去除已经与人类宿主结合的病原菌。这个项目的长期目标是阐明皮利的结构如何支持它们作为致病菌的毒力因子的作用。在建议的项目期间，将通过对ETEC皮利结构和功能的研究、对肾盂肾炎大肠杆菌（导致累及肾脏的尿路感染）表面表达的P-皮利的深入计算机建模、皮利损伤/恢复实验以及1型粘附素的定位来检查粘附皮利结构和功能之间的关系。将采用联合方法1）阐明毒性ETEC皮利的结构特征，使其能够承受蠕动和其他肠道清洁系统。研究将包括负染色和冷冻水合ETEC皮利的电子显微镜和图像处理。2)研究P-菌毛螺旋丝可以展开成其原始长度五倍的细纤维状结构的机制。能量最小化和空间约束将与遗传算法一起使用，从单个单体亚基将该原型菌毛丝建模为完整和受损的皮利。3)研究一种通过破坏皮利来减少细菌结合的方法，目的是减少细菌负荷，从而使身体的自然防御能力消除剩余的细菌。研究将使用光学镊子来测量破坏在尿路致病细菌上表达的P-菌毛所需的力，并调查是否会发生恢复。4)使用免疫电镜定位1型皮利上的粘附素。

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.