HOST CELL SIGNALING BY EHEC INTIMIN PROTEIN

肠出血性大肠埃希菌(EHEC)已成为腹泻病的重要病原体，也是美国儿童肾功能衰竭的主要原因。在最初的宿主细胞附着后，细菌向宿主细胞注入一些分子，这些分子触发信号通路，导致真核细胞骨架的破坏。注射的蛋白质中有TIR，这是一种定位于宿主细胞膜的蛋白质，充当细菌外膜蛋白内膜的受体。由eae基因编码的内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丝状肌动蛋白，直接位于结合细菌的下方，将细菌提升到“基座”上宿主细胞膜的平面之上。EAE的缺失突变体不能诱导这种基座的形成，因此缺乏肠道定植。因此，我们推测TIR-内膜相互作用在EHEC引起的疾病的发展过程中是一个重要的早期事件。我们已经确定了内膜和TIR相互作用的区域，并证明了在宿主细胞与大肠杆菌预感染后，内膜的TIR结合区域足以诱导肌动蛋白凝聚。为了深入了解EHEC如何定植肠道并促进损伤，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。因此，以下问题将被解决：1.真核膜上TIR的拓扑图是什么？2.TIR通过内膜结合是否足以引发预先感染细胞上的肌动蛋白凝聚？人工结合TIR的乳胶珠将被测试在预感染的真核细胞上诱导肌动蛋白凝聚的能力。3.Intiin和TIR是如何相互识别的？将采用遗传和生化方法，包括结晶学研究，以了解这种相互作用的分子基础。4.TIR-内膜相互作用是促进肠道定植的必要因素吗？破坏或恢复TIR-内膜结合的EAE和TIR的点突变将在EHEC感染的动物模型中测试它们对定植的影响。5.哪些哺乳动物细胞因子与TIR的细胞质区域(S)相互作用？将确定直接从TIR接收肌动蛋白细丝形成的生化信号的哺乳动物细胞因子。拟议的实验可能为EHEC感染期间的治疗干预提供新的靶点，并提供对肌动蛋白组装控制的一般细胞机制的洞察。

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.