Actin Pedestal Formation by EHEC O157:H7

性状（由申请方提供）：肠出血性大肠杆菌。O 157：H7型大肠杆菌（EHEC）是大肠杆菌病的重要病原体，它能在细菌附着部位的肠上皮细胞上形成丝状肌动蛋白。产生肌动蛋白酶的能力促进晚期肠定植并允许在上皮表面上形成大的聚集体。EHEC通过III型分泌系统向哺乳动物细胞中注射两种效应物Tir和EspFU，以产生分泌物。Tir插入宿主细胞膜，并作为细菌外膜蛋白内膜蛋白的受体。Tir的C-末端胞质结构域被IRTKS（胰岛素受体酪氨酸激酶底物）识别，IRTKS是一种哺乳动物衔接子/效应子，其促进质膜处F-肌动蛋白和膨胀膜结构的形成。IRTKS还结合PI（4，5）P2并使膜变形，并结合GTCRac Rac，其也已知刺激肌动蛋白组装。重要的是，C-末端IRTKS SH 3结构域结合EspFU，潜在地将其连接到Tir。EspFU含有多个47-残基富含脯氨酸的重复序列，并通过模拟和置换自抑制性N-WASP肽来激活肌动蛋白成核促进因子（NPF）N-WASP。Tir和EspFU的有效易位需要N-WASP，但如果克服了这种阻断，EspFU可能通过与替代的哺乳动物肌动蛋白NPF相互作用而触发肌动蛋白组装的N-WASP独立途径。这些研究结果表明，宿主肌动蛋白组装最初促进Tir和EspFU易位的模型，这两者都结合IRTKS组装在质膜上的复合物，通过与细菌内膜的相互作用聚集，这有力地刺激了两条肌动蛋白组装途径。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连接到EspFU之外，还可能在基座形成中起作用，因为初步实验表明EspFU的IRTKS结合序列增强基座形成，即使当EspFU直接融合到Tir时。本论文的主要目的是研究N-WASP依赖和非依赖的肌动蛋白基座形成机制，并探讨在哺乳动物感染过程中肌动蛋白基座形成的潜在作用：（1）确定Tir-、EspFU-、PI（4，5）P2-和/或Rac-结合活性对IRTKS促进肌动蛋白基座形成是否重要;（2）鉴定对于基台形成的N-WASP依赖性和/或非依赖性途径有缺陷的EspFU突变体;（3）确定N-WASP依赖性和N-WASP非依赖性途径在极化肠上皮细胞上的基座形成中的相对重要性，和（4）研究在体外，基座的形成是否促进稳定的细菌附着、紧密连接的破坏和/或易位，以及感染期间肠上皮上小菌落的克隆扩增。

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.