Actin Pedestal Formation by EHEC O157:H7

描述（由申请人提供）：肠出血性大肠杆菌（EHEC）血清型O157:H7是腹泻疾病的重要病原体，可在细菌附着部位下方的肠上皮细胞上触发丝状肌动蛋白基基的形成。产生肌动蛋白基座的能力促进了后期肠道定植，并允许在上皮表面形成大聚集体。为了产生基座，肠出血性大肠杆菌通过III型分泌系统将两种效应物Tir和EspFU注入哺乳动物细胞。Tir被插入宿主细胞膜，作为细菌外膜蛋白内膜素的受体。Tir的c端胞质结构域被IRTKS（胰岛素受体酪氨酸激酶底物）识别，IRTKS是一种哺乳动物的接头/效应物，可促进f -肌动蛋白和质膜上突起膜结构的形成。IRTKS还结合PI(4,5)P2并使膜变形，并结合GTPase Rac，后者也被认为可以刺激肌动蛋白的组装。重要的是，c端IRTKS SH3结构域与EspFU结合，可能将其与Tir连接。EspFU含有多个富含47个残基脯氨酸的重复序列，通过模仿和取代自身抑制的N-WASP肽来激活肌动蛋白成核促进因子（NPF） N-WASP。N-WASP是Tir和EspFU的有效易位所必需的，但如果克服了这个障碍，EspFU可以触发一个与N-WASP无关的肌动蛋白组装途径，可能是通过与另一种哺乳动物肌动蛋白NPF相互作用。这些发现提示了一个宿主肌动蛋白组装最初促进Tir和EspFU易位的模型，两者结合IRTKS在质膜上组装一个复合体，通过与细菌内膜相互作用聚集，有效地刺激肌动蛋白组装的两条途径。Tir/ espfu介导的肌动蛋白组装可能反过来促进更有效的III型易位，并通过未知的方式促进体内上皮定植。IRTKS除了将Tir与EspFU连接外，还可能在基座形成中发挥作用，因为前期实验表明，即使EspFU直接与Tir融合，EspFU的IRTKS结合序列也会增强基座形成。研究人员将研究N-WASP依赖型和非依赖型肌动蛋白基座形成的机制，并研究基座形成在哺乳动物感染过程中的潜在作用：(1)确定Tir-、EspFU-、PI(4,5)P2-和/或rac -结合活性是否对IRTKS促进肌动蛋白基座形成至关重要；(2)鉴定出在n - wasp依赖和/或不依赖的基座形成途径中存在缺陷的EspFU突变体；(3)确定极化肠上皮细胞中N-WASP依赖通路和N-WASP独立通路的相对重要性；(4)研究基座形成是否促进了细菌在体外的稳定附着、紧密连接的破坏和/或易位，以及感染期间肠上皮上微菌落的克隆扩增。

DESCRIPTION (provided by applicant): Enterohemorrhagic E. coli (EHEC) serotype O157:H7, an important agent of diarrheal disease, triggers the formation of filamentous actin pedestals on intestinal epithelial cells beneath sites of bacterial attachment. The ability to generate actin pedestals promotes late stage intestinal colonization and permits the formation of large aggregates on the epithelial surface. To generate pedestals, EHEC injects two effectors, Tir and EspFU, into mammalian cells via a type III secretion system. Tir is inserted into the host cell membrane and acts as a receptor for the bacterial outer membrane protein intimin. The C-terminal cytoplasmic domain of Tir is recognized by IRTKS (Insulin Receptor Tyrosine Kinase Substrate), a mammalian adaptor/effector that promotes the formation of F-actin and protrusive membrane structures at the plasma membrane. IRTKS also binds PI(4,5)P2 and deforms membranes, and binds the GTPase Rac, which is also known to stimulate actin assembly. Importantly, a C-terminal IRTKS SH3 domain binds to EspFU,, potentially linking it to Tir. EspFU contains multiple 47-residue proline-rich repeats and activates the actin nucleation promoting factor (NPF) N- WASP by mimicking and displacing an autoinhibitory N-WASP peptide. N-WASP is required for efficient translocation of Tir and EspFU, but if this block is overcome, EspFU can trigger an N-WASP-independent pathway for actin assembly, presumably by interacting with an alternative mammalian actin NPF. These findings suggest a model in which host actin assembly initially promotes translocation of Tir and EspFU, both of which bind IRTKS to assemble a complex at the plasma membrane, clustered by interaction with bacterial intimin, that potently stimulates two pathways of actin assembly. Tir/EspFU-mediated actin assembly may in turn promote more efficient type III translocation, and, by unknown means, epithelial colonization in vivo. IRTKS may play a role in pedestal formation in addition to linking Tir to EspFU, since pilot experiments suggest that the IRTKS binding sequence of EspFU enhances pedestal formation even when EspFU is directly fused to Tir. The following aims will be pursued to investigate both N-WASP- dependent and -independent mechanisms of actin pedestal formation, and to examine potential roles of pedestal formation during mammalian infection: (1) Determine whether Tir-, EspFU-, PI(4,5)P2-, and/or Rac-binding activity is important for IRTKS to promote actin pedestal formation; (2) Identify mutants of EspFU that are defective for the N-WASP-dependent and/or -independent pathways of pedestal formation; (3) Determine the relative importance of the N-WASP-dependent and N-WASP- independent pathways in pedestal formation on polarized intestinal epithelial cells, and (4) Investigate whether pedestal formation promotes stable bacterial attachment, disruption of tight junctions and/or translocation in vitro, and the clonal expansion of microcolonies on intestinal epithelium during infection. PUBLIC HEALTH RELEVANCE: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in North America, Europe and Japan (79) that causes both intestinal disease and a life-threatening systemic illness (51, 52) The interaction of intestinal bacteria with the gut wall is a critical step in disease (33), and EHEC triggers the host cell to generate striking pedestal-like structures beneath bound bacteria. These pedestal promote enhanced colonization of the gut later in infection, and these studies investigate EHEC induces pedestals and how this process promotes disease.