Actin pedestal formation by EHEC O157:H7

描述(由申请人提供)：肠出血性和致肠性大肠杆菌(分别为EHEC和EPEC)是腹泻疾病的重要病原体，可在细菌附着部位下方的肠道上皮细胞上诱导肌动蛋白底座。他们通过向宿主细胞注入蛋白质来实现这一点，这些蛋白质最终激活了宿主细胞肌动蛋白组装的调节因子N-WASP。对于这两种病原体来说，一个关键的效应因子是TIR，它是一种插入宿主细胞膜的细菌蛋白，充当细菌外膜蛋白内膜的受体。尽管有这些相似之处，但EHEC和EPEC的基座形成涉及根本不同的机制。对于EPEC，TIR是唯一被传递到宿主细胞的诱导基座形成所需的细菌蛋白。这种蛋白在宿主细胞膜上的聚集促进了与Nck的结合，Nck是一种宿主适配器蛋白，进而激活N-WASP。相反，EHEC的TIR既不与Nck结合，也不需要Nck来形成基座，并且不是EHEC基座形成所需的唯一转位细菌蛋白。相反，EHEC需要第二种转位的细菌蛋白，称为EspFu，这是我们实验室最近发现的。我们目前的数据支持一个模型，在该模型中，EspFu直接与N-WASP相互作用，促进TIR/N-WASP复合体的形成。然而，与Nck不同，EspFu似乎并不直接与TIR相互作用。这一观察结果表明，TIR/N-WASP复合体的最终形成可能需要一个未知的宿主因素。我们建议通过以下方法来测试和完善这一模型：(1)描述肠出血性大肠杆菌TIR细胞质结构域中对肌动蛋白组装至关重要的元件；(2)定义TIR、EspFu和N-WASP之间的基本相互作用，并确定TIR-EspFu相互作用所需的潜在宿主蛋白，如果我们确认其存在的话；(3)概述TIR/EspFu介导的肌动蛋白在无细胞提取物中的组装；(4)评估肌动蛋白底座形成在肠道定植和诱导EHEC感染过程中组织损伤中的作用。

DESCRIPTION (provided by applicant): Enterohemorrhagic and enteropathogenic E. coli (EHEC and EPEC, respectively) are important agents of diarrheal disease that induce actin pedestals on intestinal epithelial cells beneath sites of bacterial attachment. They do so by injecting the host cell with proteins that ultimately activate a host cell regulator of actin assembly known as N-WASP. One critical effector for both pathogens is Tir, a bacterial protein that is inserted into the host cell membrane and acts as a receptor for the bacterial outer membrane protein intimin. Despite these similarities, pedestal formation by EHEC and EPEC involve fundamentally different mechanisms. For EPEC, Tir is the only bacterial protein delivered to host cells that is required to induce formation of pedestals. Clustering of this protein in the host cell membrane promotes binding to Nck, a host adaptor protein that in turn activates N-WASP. In contrast, the Tir of EHEC neither binds to nor requires Nck for pedestal formation, and is not the only translocated bacterial protein required for pedestal formation by EHEC. Instead, EHEC requires a second translocated bacterial protein, termed EspFU, recently identified by our laboratory. Our current data support a model in which EspFU interacts directly with N-WASP to promote the formation of Tir/N-WASP complexes. However, unlike Nck, EspFU does not appear to interact directly with Tir. This observation implies that an unidentified host factor is likely to be required for the ultimate formation of Tir/N-WASP complexes. We propose to test and refine this model by: (1) delineating the elements of the cytoplasmic domain of EHEC Tir essential for actin assembly; (2) defining the essential interactions between Tir, EspFU and N-WASP, and identifying the putative host protein that is required for Tir-EspFU interaction, should we confirm its existence; (3) recapitulating Tir/EspFU-mediated actin assembly in cell-free extracts; (4) assessing the role of actin pedestal formation in intestinal colonization and the induction of tissue damage during EHEC infection.