Saulino E T, Thanassi D G, Pinkner J S, Hultgren S J
Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO 63110, USA.
EMBO J. 1998 Apr 15;17(8):2177-85. doi: 10.1093/emboj/17.8.2177.
The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone-adhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone-adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.
多种革兰氏阴性菌中不同黏附结构的生物合成依赖于伴侣蛋白/外膜蛋白途径。关于外膜蛋白如何将蛋白质亚基转运穿过外膜，以及这些黏附结构如何组装，我们所知甚少。我们发现了几种机制，外膜蛋白通过这些机制来调节1型菌毛的有序组装，特别是通过伴侣蛋白-黏附素复合物与外膜蛋白的关键相互作用。利用表面等离子体共振实时研究伴侣蛋白-亚基复合物与外膜蛋白的结合和解离事件，结果表明伴侣蛋白-黏附素复合物与外膜蛋白的结合最紧密且最快。这表明伴侣蛋白-黏附素复合物向外膜蛋白的动力学分配是黏附素在菌毛尖端定位的决定性因素。此外，我们鉴定并纯化了一种在体内形成的伴侣蛋白-黏附素-外膜蛋白组装中间体。胰蛋白酶消化试验表明，该复合物中的外膜蛋白处于改变的构象，这种构象在菌毛组装过程中得以维持。这些数据支持了一个模型，即伴侣蛋白-黏附素复合物与外膜蛋白的结合使外膜蛋白稳定在一种能够组装的构象，并允许菌毛组装的起始。
