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Recombinant Yersinia YopT Leads to Uncoupling of RhoA-Effector Interaction

机译：重组耶尔森氏菌YopT导致RhoA-效应子相互作用的解偶联

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摘要

Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis deliver different Yop (Yersinia outer proteins) effector proteins into mammalian cells by a type III secretion mechanism. Recently, it was shown thatYersinia producing YopT leads to disruption of the actin cytoskeleton of HeLa cells (M. Iriarte and G. R. Cornelis, Mol. Microbiol. 29:915–929, 1998). To analyze the molecular mechanism of YopT, we cloned and expressed YopT as a glutathioneS-transferase fusion protein. Recombinant YopT caused rounding up of embryonic bovine lung cells and redistribution of the actin cytoskeleton rapidly after microinjection. The Escherichia coli cytotoxic necrotizing factor (CNF1), which constitutively activates Rho proteins, was not able to inhibit or revert YopT-induced cell rounding. YopT caused release of RhoA from embryonic bovine lung membranes and released recombinant isoprenylated RhoA from artificial PE or PE/PIP2 vesicles. Incubation of lysate or cytosol with YopT caused inhibition of the RhoA-rhotekin interaction but led to increased RhoA-RhoGDI interaction. It is suggested that inhibition of the interaction between RhoA and effectors is the underlying mechanism of the YopT action on the cytoskeleton.
机译：小肠结肠炎耶尔森氏菌，假结核耶尔森氏菌和鼠疫耶尔森菌通过III型分泌机制将不同的Yop（外耶尔森氏菌外部蛋白）效应蛋白传递到哺乳动物细胞中。最近发现，产生耶尔森氏菌的YopT导致HeLa细胞肌动蛋白细胞骨架的破坏（M. Iriarte和G. R. Cornelis，Mol。Microbiol。29：915-929，1998）。为了分析YopT的分子机制，我们克隆并表达了YopT为谷胱甘肽 S -转移酶融合蛋白。重组YopT在显微注射后迅速引起胚胎牛肺细胞的聚集和肌动蛋白细胞骨架的重新分布。组成性激活Rho蛋白的大肠杆菌细胞毒性坏死因子（CNF1）不能抑制或逆转YopT诱导的细胞变圆。 YopT导致RhoA从胚胎牛肺膜释放，并从人工PE或PE / PIP2囊泡释放重组异戊二烯化RhoA。将裂解物或细胞质与YopT一起孵育会抑制RhoA-rhotekin相互作用，但会导致RhoA-RhoGDI相互作用增加。提示抑制RhoA和效应子之间的相互作用是YopT作用于细胞骨架的潜在机制。

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