YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells:: evidence of a structural domain necessary for translocation
YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells:: evidence of a structural domain necessary for translocation
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DOI：
10.1046/j.1365-2958.1998.00973.x
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发表时间：
1998-08-01
影响因子：
3.6
通讯作者：
Wolf-Watz, H
中科院分区：
文献类型：
--
作者：
Francis, MS;Wolf-Watz, H
假结核耶尔森菌YopB和YopD蛋白是Yop效应蛋白转运到靶细胞细胞质中所必需的。尽管YopD的功能尚不清楚，但YopB被认为介导靶细胞膜的孔形成，使Yop效应蛋白易位。为了研究YopD在易位中的作用，我们构建了一株假结核杆菌突变株，该突变株的框架内缺失了整个YopD基因。正如最近对鼠疫杆菌YopD蛋白的研究显示，我们发现体外低钙反应控制毒力基因表达受到YopD的负调控。这个yopD零突变体（YPIII/plB621）对HeLa细胞单层也没有细胞毒性，支持yopD在易位过程中的作用。虽然耶尔森氏菌转位酶装置的其他成分（YopB、YopK和YopN）没有易位到宿主细胞质中，但分离感染的HeLa细胞使我们能够通过野生型菌株（YPIII/plB102）鉴定YopD的胞质定位，而不是通过YopD或YopB缺陷菌株鉴定，YopD也可以通过免疫荧光在感染了多个yop突变菌株（YPIII/plB29MEKA）的HeLa细胞单层细胞质中鉴定。这些结果表明，YopD在负调控Yap产生和Yop效应物易位（包括YopD蛋白本身）方面具有双重功能。为了研究YopD c端附近的两亲结构域是否参与易位过程，构建了一个缺乏该区域的突变株（YPIII/ plB155 Delta D278-292），从表型上看，这个小帧内的Delta YopD（278-292）缺失突变体与YopD无突变体难以区分。截断的YopD蛋白和Yop效应物没有转移到感染该突变体的HeLa细胞单层的细胞质中。框架内较小的Delta yopD（278-292）缺失和Delta yopD零突变所显示的可比较的调控和易位表型表明，Yop合成的调控和Yop易位是密切耦合的。我们对耶尔森菌感染过程提出了一个有趣的场景，强调了YopD极化易位的必要性，以特异性地建立Yop效应物的易位。这些观察结果与先前的建议相反，即易位酶装置的成员没有易位到宿主细胞的细胞质中。
Yersinia pseudotuberculosis YopB and YopD proteins are essential for translocation of Yop effector proteins into the target cell cytosol. YopB is suggested to mediate pore formation in the target cell plasma membrane, allowing translocation of Yop effector proteins, although the function of YopD is unclear. To investigate the role in translocation for YopD, a mutant strain in Y. pseudotuberculosis was constructed containing an in frame deletion of essentially the entire yopD gene. As shown recently for the Y. pestis YopD protein, we found that the in vitro low calcium response controlling virulence gene expression was negatively regulated by YopD. This yopD null mutant (YPIII/plB621) was also non-cytotoxic towards HeLa cell monolayers, supporting the role for YopD in the translocation process. Although other constituents of the Yersinia translocase apparatus (YopB, YopK and YopN) are not translocated into the host cell cytosol, fractionation of infected HeLa cells allowed us to identify the cytosolic localization of YopD by the wild-type strain (YPIII/plB102), but not by strains defective in either YopD or YopB, YopD was also identified by immunofluorescence in the cytoplasm of HeLa cell monolayers infected with a multiple yop mutant strain (YPIII/plB29MEKA). These results demonstrate a dual function for YopD in negative regulation of Yap production and Yop effector translocation, including the YopD protein itself. To investigate whether an amphipathic domain near the C-terminus of YopD is involved in the translocation process, a mutant strain (YPIII/ plB155 Delta D278-292) was constructed that is devoid of this region, Phenotypically, this small in frame Delta yopD(278-292) deletion mutant was indistinguishable from the yopD null mutant. The truncated YopD protein and Yop effecters were not translocated into the cytosol of HeLa cell monolayers infected with this mutant. The comparable regulatory and translocation phenotypes displayed by the small in frame Delta yopD(278-292) deletion and Delta yopD null mutants suggest that regulation of Yop synthesis and Yop translocation are intimately coupled. We present an intriguing scenario to the Yersinia infection process that highlights the need for polarized translocation of YopD to specifically establish translocation of Yop effecters. These observations are contrary to previous suggestions that members of the translocase apparatus were not translocated into the host cell cytosol.