Interaction between the SifA Virulence Factor and Its Host Target SKIP Is Essential for Salmonella Pathogenesis
Interaction between the SifA Virulence Factor and Its Host Target SKIP Is Essential for Salmonella Pathogenesis
复制标题
DOI：
10.1074/jbc.m109.034975
复制
发表时间：
2009-11-27
影响因子：
4.8
通讯作者：
Meresse, Stephane
中科院分区：
文献类型：
--
作者：
Diacovich, Lautaro;Dumont, Audrey;Meresse, Stephane
SifA是一种沙门氏菌效应子，通过致病岛2编码的3型分泌系统易位到感染细胞中。SifA是一个重要的毒力因子。先前的研究表明，在易位后，SifA结合真核宿主蛋白SKIP的普列克底物蛋白同源基序。反过来，SifA-SKIP复合物调节细菌液泡上分子运动驱动蛋白-1的动员。SifA具有包含功能基序的多个结构域。在这里，我们进行了分子解剖和SifA的突变研究，以评估不同结构域的SifA功能的相对贡献。生物化学和晶体学分析证实，SifA的N-末端结构域足以与SKIP的普列克底物蛋白同源结构域相互作用，形成具有微摩尔解离常数的1：1复合物。WXXXE基序中的色氨酸残基的突变（其已被提出来模拟GTdR的活性形式）深刻地影响SifA的稳定性和易位，而谷氨酸残基的突变没有功能影响。不结合SKIP的SifA L130 D突变体在感染的细胞和小鼠感染模型中都显示出Delta sifA样表型。我们的结论是，WXXXE基序是必不可少的维持SifA的三级结构，其功能需要与真核蛋白SKIP的相互作用。
SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a Delta sifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.