Cappello Renato E, Estrada-Gutierrez Guadalupe, Irles Claudine, Giono-Cerezo Silvia, Bloch Robert J, Nataro James P
Institutional Program in Molecular Biomedicine, National School of Homeopathy and Medicine, Instituto Politecnico Nacional, Mexico City, Mexico.
FEMS Immunol Med Microbiol. 2011 Apr;61(3):301-14. doi: 10.1111/j.1574-695X.2010.00776.x. Epub 2011 Feb 7.
Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication.
肠聚集性大肠杆菌（EAEC）是一种新出现的腹泻病原体。许多EAEC菌株产生质粒编码毒素（Pet），该毒素对人肠道组织具有细胞毒性作用。Pet中毒的HEp-2细胞呈现圆形并从基质上脱离，同时伴有F-肌动蛋白应力纤维的丧失和含血影蛋白的膜细胞骨架的凝聚。尽管研究表明Pet可直接切割血影蛋白，但尚不清楚这是否为该毒素的基本作用模式。此外，尚未报道Pet对肌动蛋白和血影蛋白以外的细胞骨架成分的影响。在此，我们通过免疫荧光证明，在Pet中毒后，HEp-2和HT29细胞失去粘着斑复合物（FAC），这一过程包括粘着斑激酶（FAK）、α-辅肌动蛋白、桩蛋白、纽蛋白、F-肌动蛋白和血影蛋白本身的重新分布。这种重新分布与粘着斑处磷酸酪氨酸标记的减少相关。免疫印迹和免疫沉淀实验表明，在FAK和血影蛋白重新分布之前，FAK的酪氨酸去磷酸化。此外，磷酸酶抑制可阻断细胞回缩，提示酪氨酸去磷酸化是FAK切割之前发生的事件。最后，我们表明体外酪氨酸去磷酸化的FAK易受Pet切割。这些数据表明，在Pet中毒过程中发生了血影蛋白重新分布以外的机制。
