Centre for Molecular Microbiology and Infection, Imperial College London, UK.
Cell Microbiol. 2010 May 1;12(5):654-64. doi: 10.1111/j.1462-5822.2009.01423.x. Epub 2009 Dec 21.
We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide-free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H-Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.
我们研究了 III 型分泌系统 WxxxE 效应物肠出血性大肠杆菌 EspM2（触发应激纤维形成）和沙门氏菌血清型鼠伤寒 SifA（参与细胞内存活）如何调节 Rho GTPases。我们发现 EspM2 或 SifA 与无核苷酸 RhoA 之间存在直接相互作用。核磁共振波谱显示 EspM2 的折叠与 SifA 和鸟嘌呤核苷酸交换因子（GEF）效应物 SopE 相似。EspM2 诱导 RhoA 核苷酸交换，但不诱导 Rac1 或 H-Ras 核苷酸交换，而 SifA 则不诱导它们中的任何一种核苷酸交换。突变 WxxxE 基序中的 W70 或环绕催化环的 L118 和 I127 残基会影响 EspM2 的稳定性。位于 EspM2 催化环内的 Q124 被丙氨酸取代，极大地削弱了 EspM2 的体外 RhoA GEF 活性以及在外源表达时诱导应激纤维形成的能力。这些结果表明，SifA 与 RhoA 的结合不会触发核苷酸交换，而 EspM2 是一种独特的 Rho GTPase GEF。
