Goosney D L, DeVinney R, Pfuetzner R A, Frey E A, Strynadka N C, Finlay B B
Biotechnology Laboratory, The Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Curr Biol. 2000 Jun 15;10(12):735-8. doi: 10.1016/s0960-9822(00)00543-1.
Enteropathogenic Escherichia coli (EPEC) triggers a dramatic rearrangement of the host epithelial cell actin cytoskeleton to form an attaching and effacing lesion, or pedestal. The pathogen remains attached extracellularly to the host cell through the pedestal for the duration of the infection. At the tip of the pedestal is a bacterial protein, Tir, which is secreted from the bacterium into the host cell plasma membrane, where it functions as the receptor for an EPEC outer membrane protein, intimin [1]. Delivery of Tir to the host cell results in its tyrosine phosphorylation, followed by Tir-intimin binding. Tir is believed to anchor EPEC firmly to the host cell, although its direct linkage to the cytoskeleton is unknown. Here, we show that Tir directly binds the cytoskeletal protein alpha-actinin. alpha-Actinin is recruited to the pedestal in a Tir-dependent manner and colocalizes with Tir in infected host cells. Binding is mediated through the amino terminus of Tir. Recruitment of alpha-actinin occurs independently of Tir tyrosine phosphorylation. Recruitment of actin, VASP, and N-WASP, however, is abolished in the absence of this tyrosine phosphorylation. These results suggest that Tir plays at least three roles in the host cell during infection: binding intimin on EPEC; mediating a stable anchor with alpha-actinin through its amino terminus in a phosphotyrosine-independent manner; and recruiting additional cytoskeletal proteins at the carboxyl terminus in a phosphotyrosine-dependent manner. These findings demonstrate the first known direct linkage between extracellular EPEC, through the transmembrane protein Tir, to the host cell actin cytoskeleton via alpha-actinin.
肠致病性大肠杆菌（EPEC）会引发宿主上皮细胞肌动蛋白细胞骨架的剧烈重排，形成黏附和抹除损伤，即基座。在感染期间，病原体通过基座在细胞外与宿主细胞保持附着。基座顶端是一种细菌蛋白Tir，它从细菌分泌到宿主细胞质膜中，在那里作为EPEC外膜蛋白紧密黏附素的受体发挥作用[1]。Tir传递到宿主细胞会导致其酪氨酸磷酸化，随后发生Tir-紧密黏附素结合。尽管Tir与细胞骨架的直接联系尚不清楚，但人们认为它能将EPEC牢固地锚定在宿主细胞上。在这里，我们表明Tir直接结合细胞骨架蛋白α-辅肌动蛋白。α-辅肌动蛋白以Tir依赖的方式被招募到基座，并在受感染的宿主细胞中与Tir共定位。结合是通过Tir的氨基末端介导的。α-辅肌动蛋白的招募独立于Tir酪氨酸磷酸化。然而，在没有这种酪氨酸磷酸化的情况下，肌动蛋白、血管舒张刺激蛋白（VASP）和神经Wiskott-Aldrich综合征蛋白（N-WASP）的招募被消除。这些结果表明，Tir在感染期间在宿主细胞中至少发挥三种作用：结合EPEC上的紧密黏附素；通过其氨基末端以磷酸酪氨酸非依赖的方式与α-辅肌动蛋白介导稳定的锚定；并以磷酸酪氨酸依赖的方式在羧基末端招募额外的细胞骨架蛋白。这些发现证明了细胞外EPEC通过跨膜蛋白Tir经由α-辅肌动蛋白与宿主细胞肌动蛋白细胞骨架之间的首个已知直接联系。
