Duché Denis, Frenkian Aurélie, Prima Valérie, Lloubès Roland
Laboratoire d'Ingéniérie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
J Bacteriol. 2006 Dec;188(24):8593-600. doi: 10.1128/JB.00941-06. Epub 2006 Sep 29.
Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.
产生核酸内切酶大肠杆菌素的细菌受到一种小免疫蛋白的保护，免受细胞毒性活性的影响，该免疫蛋白以高亲和力和特异性结合，使核酸内切酶失活。这种复合物被释放到细胞外培养基中，并且当复合物与易感细胞结合时，免疫蛋白被抛弃。然而，目前尚不清楚免疫蛋白在感染过程中如何以及在哪个阶段释放。在这里，我们构建了一种由增强型绿色荧光蛋白（EGFP）与大肠杆菌素E2免疫蛋白（Im2）融合而成的杂交免疫蛋白，以增强其检测效果。EGFP-Im2蛋白以1:1的化学计量比结合游离的大肠杆菌素E2，并特异性抑制其DNase活性。将这种杂交复合物添加到易感细胞中表明，杂交免疫蛋白的释放是一个时间依赖性过程。在将复合物添加到细胞后20分钟实现这一过程。我们表明，复合物的解离需要由BtuB蛋白和一种孔蛋白（OmpF或OmpC）形成的功能性转运体，以及由Tol蛋白形成的功能性导入机制。细胞分级分离和蛋白酶敏感性实验表明，在大肠杆菌素导入过程中，免疫蛋白不会穿过细胞包膜。这些观察结果表明，免疫蛋白的解离发生在外膜表面，并且需要大肠杆菌素E2 N末端结构域的完全转运。
