Actin Pedestal Formation by EHEC O157:H7

描述(申请人提供)：肠出血性大肠杆菌(EHEC)O157：H7血清型，腹泻疾病的重要病原体，在细菌附着部位下方的肠道上皮细胞上触发丝状肌动蛋白基座的形成。产生肌动蛋白基座的能力促进了晚期肠道的定植，并允许在上皮表面形成大的聚集体。为了产生底座，EHEC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，并作为细菌外膜蛋白内膜的受体。胰岛素受体酪氨酸激酶底物(IRTKS)识别TIR的C末端细胞质结构域，IRTKS是哺乳动物的一种适配器/效应器，促进质膜上F-肌动蛋白和突起膜结构的形成。IRTKS还与PI(4，5)P2结合，使膜变形，并与GTPase Rac结合，后者也是已知的刺激肌动蛋白组装的酶。重要的是，C-末端的IRTKS SH3结构域与EspFu结合，可能将其与TIR联系起来。EspFu含有多个47个残基的富含脯氨酸的重复序列，通过模仿和取代自身抑制的N-WASP多肽来激活肌动蛋白成核促进因子(NPF)N-WASP。N-WASP是TIR和EspFu有效转运所必需的，但如果这一障碍被克服，EspFu可以触发一条N-WASP非依赖的肌动蛋白组装途径，推测是通过与另一种哺乳动物肌动蛋白NPF相互作用实现的。这些发现提出了一种模型，在该模型中，宿主肌动蛋白组装最初促进TIR和EspFu的易位，这两种结合都能与IRTKS结合在质膜上组装一个复合体，通过与细菌内膜的相互作用而聚集，从而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型移位，并通过未知的方式在体内促进上皮定植。除了将TIR与EspFu联系起来外，IRTKS还可能在基座形成中发挥作用，因为初步实验表明，即使当EspFu直接与TIR融合时，EspFu的IRTKS结合序列也能促进基座的形成。我们将致力于研究N-WASP依赖和非依赖肌动蛋白底座形成的机制，并研究哺乳动物感染过程中底座形成的潜在作用：(1)确定TIR-、EspFu-、PI(4，5)P2-和/或RAC结合活性对IRTKS促进肌动蛋白底座形成是否重要；(2)鉴定N-WASP依赖和/或非依赖底座形成途径缺陷的EspFu突变体；(3)确定N-WASP依赖和N-WASP非依赖途径在极化肠上皮细胞基座形成中的相对重要性，以及(4)在感染过程中，基座形成是否促进细菌的稳定附着、紧密连接的破坏和/或体外易位，以及肠道上皮微克隆的克隆性扩张。

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.