The Yersinia Yop virulon:: LcrV is required for extrusion of the translocators YopB and YopD
The Yersinia Yop virulon:: LcrV is required for extrusion of the translocators YopB and YopD
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DOI：
10.1128/jb.180.5.1207-1214.1998
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发表时间：
1998-03-01
影响因子：
3.2
通讯作者：
Cornelis, GR
中科院分区：
文献类型：
--
作者：
Sarker, MR;Neyt, C;Cornelis, GR
LcrV 是 Yop 病毒的重要片段，由大型 lcrGVsycDyopBD 操纵子编码。尽管经过多次努力，LcrV 在 Yop 病毒中的作用仍然难以捉摸。为了澄清这一点，我们在小肠结肠炎耶尔森氏菌 E40 的 pYV 质粒中设计了 lcrV 的完全缺失，并表征了突变体的表型。互补实验表明，yopB 和 yopD 的突变不是极性的。然而，突变消除了YopB和YopD的分泌，而其他Yops的分泌不受影响甚至增加。 Northern印迹分析显示yopD的转录不受影响。 YopD可以在细菌内部检测到，这证明其分泌不足并不是由于缺乏翻译或蛋白水解而引起的，这表明LcrV专门参与了YopB和YopD的释放过程。然后我们研究了 LcrV 和 YopB 或 YopD 之间可能的相互作用。我们构建了谷胱甘肽 S-转移酶-LcrV 杂合蛋白，并观察到 YopB 或 YopD 可以与其共纯化。同样的方法表明，LcrV 也与 LcrG 相互作用，但不与伴侣 SycD 相互作用。然后，使用 lcrV 的缺失体，我们在 LcrV 的 C 末端鉴定了明确的 LcrG 结合结构域。
LcrV, an essential piece of the Yop virulon, is encoded by the large lcrGVsycDyopBD operon. In spite of repeated efforts, the role of LcrV in the Yop virulon remains elusive. In an attempt to clarify this, we engineered a complete deletion of lcrV in the pYV plasmid of Yersinia enterocolitica E40 and characterized the phenotype of the mutant. Complementation experiments showed that the mutation was not polar with regard to yopB and yopD. Nevertheless the mutation abolished secretion of YopB and YopD, while secretion of the other Yops was unaffected or even increased. Northern blot analysis showed that transcription of yopD was not affected. YopD could be detected inside the bacteria, shelving that the lack of its secretion was not due to a lack of translation or to proteolysis, This indicated that LcrV is specifically involved in the process of release of YopB and YopD. We then investigated the possible interactions between LcrV and YopB or YopD. We constructed a glutathione S-transferase-LcrV hybrid protein, and we observed that either YopB or YopD could be copurified with it. The same approach showed that LcrV also interacts with LcrG but not with the chaperone SycD, Using deletants of lcrV, we then identified a definite LcrG-binding domain in the C terminus of LcrV.