From the CNRS, Aix Marseille Université, Institut de Microbiologie de la Méditerranée (IMM), Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM)/UMR7255, 13009 Marseille, France.
CNRS, Aix Marseille Université, IMM, Laboratoire de Chimie Bactérienne (LCB)/UMR7283, 13009 Marseille, France, and.
J Biol Chem. 2018 Dec 14;293(50):19441-19450. doi: 10.1074/jbc.RA117.001127. Epub 2018 Oct 18.
In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. Despite our knowledge of most T2SS components, the mechanisms underlying effector recruitment and secretion by the T2SS remain enigmatic. Using complementary biophysical and biochemical approaches, we identified here two direct interactions between the secreted effector CbpD and two components, XcpY and XcpZ, of the T2SS assembly platform (AP) in the opportunistic pathogen Competition experiments indicated that CbpD binding to XcpY is XcpZ-dependent, suggesting sequential recruitment of the effector by the periplasmic domains of these AP components. Using a bacterial two-hybrid system, we then tested the influence of the effector on the AP protein-protein interaction network. Our findings revealed that the presence of the effector modifies the AP interactome and, in particular, induces XcpZ homodimerization and increases the affinity between XcpY and XcpZ The observed direct relationship between effector binding and T2SS dynamics suggests an additional synchronizing step during the type 2 secretion process, where the activation of the AP of the T2SS nanomachine is triggered by effector binding.
在许多革兰氏阴性菌中，由于其能够将大量完全折叠的蛋白质效应物递送到细胞外环境中，因此 II 型分泌系统（T2SS）在毒力中起着重要作用。尽管我们对大多数 T2SS 成分有了一定的了解，但 T2SS 效应子募集和分泌的机制仍然是个谜。在这里，我们使用互补的生物物理和生化方法，鉴定了机会性病原体中分泌效应子 CbpD 与 T2SS 组装平台（AP）的两个组件 XcpY 和 XcpZ 之间的两个直接相互作用。竞争实验表明，CbpD 与 XcpY 的结合依赖于 XcpZ，这表明效应子通过这些 AP 成分的周质域依次募集。然后，我们使用细菌双杂交系统测试了效应子对 AP 蛋白-蛋白相互作用网络的影响。我们的研究结果表明，效应子的存在改变了 AP 蛋白-蛋白相互作用网络，特别是诱导 XcpZ 同源二聚体化并增加 XcpY 和 XcpZ 之间的亲和力。观察到的效应子结合与 T2SS 动力学之间的直接关系表明，在 II 型分泌过程中存在额外的同步步骤，其中 T2SS 纳米机器的 AP 的激活是由效应子结合触发的。
