HOST CELL SIGNALING BY EHEC INTIMIN PROTEIN

肠出血性大肠大肠埃希菌（EHEC）在美国已成为腹泻病的重要病原体和小儿肾衰竭的主要原因。在最初的宿主细胞附着后，细菌将许多分子注入宿主细胞，这些分子触发信号通路并导致真核细胞骨架的破坏。在注射的蛋白质中有Tir，这是一种定位于宿主细胞膜并作为细菌外膜蛋白内膜蛋白受体的蛋白质。由eae基因编码的内膜蛋白是形成高度组织化的细胞骨架结构所必需的，这种高度组织化的细胞骨架结构包含直接位于结合的细菌下方的丝状肌动蛋白，其将细菌提升到宿主细胞膜平面之上的“基座”上。eae的缺失突变体不能诱导该基座的形成，因此不能在肠道定植。因此，我们推测Tir-intimin相互作用是肠出血性大肠杆菌引起的疾病发展的重要早期事件。我们已经确定了相互作用的intimin和Tir区域，并表明intimin的Tir结合区域足以诱导宿主细胞在用E.杆菌需要详细了解Tir-内膜结合以及这种相互作用下游的分子信号，以深入了解EHEC如何在肠道定植并促进损伤。因此，以下问题将得到解决：1。真核生物膜中Tir的拓扑图是什么？2. Tir与intimin的结合是否足以触发肌动蛋白在感染前细胞上的凝聚？将测试人工结合TIR的乳胶珠在预感染的真核细胞上诱导肌动蛋白凝聚的能力。3.那么，李明博和李明博是如何认识彼此的呢？遗传和生物化学方法，包括晶体学研究，将被追求，以了解这种相互作用的分子基础。4. TIR-intimin相互作用对促进肠道定植是必要的吗？将在EHEC感染的动物模型中检测eae和tir中破坏或恢复tir-内膜结合的点突变对定植的影响。5.哪些哺乳动物细胞因子与Tir的胞质区域相互作用？将鉴定直接从Tir接收肌动蛋白丝形成的生化信号的哺乳动物细胞因子。拟议的实验可能提供新的目标，治疗干预在肠出血性大肠杆菌感染，以及提供洞察的一般细胞机制，肌动蛋白组装控制。

Enterohemorrhagic E. coli (EHEC) has emerged as an important agent of diarrheal disease and the leading cause of pediatric renal failure in the U.S. Intimate attachment to host cells is an essential step during intestinal colonization by EHEC. After initial host cell attachment, the bacterium injects into the host cell a number of molecules that trigger signaling pathways and result in the disruption of the eukaryotic cytoskeleton. Among the injected proteins is Tir, a protein that becomes localized in the host cell membrane and acts as a receptor for the bacterial outer membrane protein intimin. Intimin, encoded by the eae gene, is required for the formation of a highly organized for the formation of a highly organized cytoskeletal structure containing filamentous actin directly beneath the bound bacterium that lifts the bacterium above the plane of the host cell membrane on a "pedestal". Deletion mutants of eae, which cannot induce the formation of this pedestal, are deficient for intestinal colonization. Thus, we postulate that Tir-intimin interaction is an essential early event in the development of disease caused by EHEC. We have identified regions of intimin and Tir that interact with each other , and have shown that the Tir-binding region of intimin is sufficient to induce actin condensation after pre-infection of host cells with E. coli. A detailed understanding of Tir-intimin binding, as well as of the molecular signals immediately downstream of this interaction, are required to gain insight into how EHEC colonizes the intestine and promotes damage. Thus, the following questions will be addressed: 1. What is the topological map of Tir in the eukaryotic membrane? 2. Is Tir binding by intimin sufficient to trigger actin condensation on preinfected cells? Latex beads that artificially bind TIR will be tested for the ability to induce actin condensation on preinfected eukaryotic cells. 3. How does intimin and Tir recognize each other? Genetic and biochemical approaches, including crystallographic studies, will be pursued to understand the molecular basis for this interaction. 4. Is Tir-intimin interaction essential to promote intestinal colonization? Point mutations in eae and tir that disrupt or restore Tir-intimin binding will be tested for their effect on colonization in an animal model for EHEC infection. 5. What mammalian cell factors interact with the cytoplasmic region(s) of Tir? Mammalian cell factors that directly receive from Tir the biochemical signal for actin filament formation will be identified. The proposed experiments may provide novel targets for therapeutic intervention during EHEC infection, as well as provide insight into the general cellular mechanisms by which actin assembly controlled.