Actin pedestal formation by EHEC O157:H7

EHEC O157:H7 形成肌动蛋白基座

• 批准号: 7351828
• 负责人: JOHN M LEONG
• 金额: $ 53.05万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7351828
• 关键词: Actins; Adaptor Signaling Protein; Animals; Back; Bacterial Attachment Site; Bacterial Outer Membrane Proteins; Bacterial Proteins; Binding; Cell membrane; Cell-Matrix Junction; Cells; Classification; Complex; Cytoplasmic Tail; Data; Disease; Elements; Epithelial Cells; Escherichia coli EHEC; Gnotobiotic; In Vitro; Infant; Infection; Inflammatory disease of the intestine; Integration Host Factors; Intestines; Laboratories; Link; Mammalian Cell; Mediating; Modeling; Mutagenesis; Oryctolagus cuniculus; Peptides; Proteins; Role; Signal Transduction; System; Testing; Tissues; Transfection; Tyrosine; Xenopus; base; deletion analysis; egg; enteropathogenic Escherichia coli; mutant; pathogen; receptor; research study

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.

性状（由申请方提供）：肠出血性和肠致病性大肠杆菌。大肠杆菌（EHEC和EPEC，分别为）是大肠杆菌病的重要病原体，其在细菌附着位点下方的肠上皮细胞上诱导肌动蛋白酶。他们通过向宿主细胞注射蛋白质来实现这一点，这些蛋白质最终激活了一种被称为N-WASP的宿主细胞肌动蛋白组装调节剂。两种病原体的一个关键效应物是Tir，一种插入宿主细胞膜并作为细菌外膜蛋白内膜蛋白受体的细菌蛋白。 尽管有这些相似之处，但肠出血性大肠杆菌和肠穿孔性大肠杆菌的基座形成涉及根本不同的机制。对于EPEC，Tir是递送至宿主细胞的诱导细菌形成所需的唯一细菌蛋白。这种蛋白质在宿主细胞膜中的聚集促进与Nck的结合，Nck是一种宿主衔接蛋白，反过来激活N-WASP。相反，肠出血性大肠杆菌的Tir既不结合也不需要Nck来形成基座，并且不是肠出血性大肠杆菌形成基座所需的唯一易位细菌蛋白。相反，肠出血性大肠杆菌需要第二个易位的细菌蛋白，称为EspFU，最近由我们的实验室确定。我们目前的数据支持EspFU直接与N-WASP相互作用以促进Tir/N-WASP复合物形成的模型。然而，与Nck不同的是，EspFU似乎并不直接与Tir相互作用。这一观察结果意味着，一个身份不明的主机因素是可能需要的最终形成的TIR/N-WASP复合物。我们建议通过以下步骤来测试和完善这个模型：（1）描述肌动蛋白组装所必需的EHEC Tir胞质结构域的元件;（2）定义Tir、EspFU和N-WASP之间的基本相互作用，并鉴定Tir-EspFU相互作用所需的假定宿主蛋白，如果我们确认它的存在的话;（3）在无细胞提取物中重现Tir/EspFU介导的肌动蛋白组装;（4）探讨肌动蛋白基座的形成在肠出血性大肠杆菌肠道定植和组织损伤中的作用。