Page A L, Fromont-Racine M, Sansonetti P, Legrain P, Parsot C
Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Paris Cedex 15, France.
Mol Microbiol. 2001 Nov;42(4):1133-45. doi: 10.1046/j.1365-2958.2001.02715.x.
The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.
革兰氏阴性病原菌的III型分泌（TTS）系统由组装成TTS机制的蛋白质、由该机制分泌的蛋白质以及这些蛋白质储存和有时分泌所需的特定分子伴侣组成。TTS途径涉及许多连续的蛋白质相互作用，以将效应蛋白递送至宿主细胞。我们使用酵母双杂交系统来研究福氏志贺氏菌效应蛋白和分子伴侣的相互作用伙伴。使用效应蛋白和分子伴侣作为诱饵，筛选含有与TTS蛋白片段随机融合的猎物文库。通过该方法检测到效应蛋白IpaB和IpaC与其分子伴侣IpgC之间的相互作用，并鉴定了相互作用结构域。使用His标签的IpgC蛋白共纯化截短的IpaB和IpaC蛋白，我们表明分子伴侣结合结构域是独特的，位于这些蛋白的N端。该结构域对于重组蛋白的分泌不是必需的，但参与IpaC的稳定性和IpaB的不稳定性。用诱饵IpaA、IpaB和IpaC鉴定了同型相互作用。还选择了效应蛋白与TTS机制成分之间的相互作用，这可能有助于深入了解TTS过程的调控。
