Zoued Abdelrahim, Cassaro Chloé J, Durand Eric, Douzi Badreddine, España Alexandre P, Cambillau Christian, Journet Laure, Cascales Eric
Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM, UMR 7255), Institut de Microbiologie de la Méditerranée (IMM), Aix-Marseille Univ. - Centre National de la Recherche Scientifique (CNRS), 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Architecture et Fonction des Macromolécules Biologiques (AFMB, UMR 6098), Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France; Architecture et Fonction des Macromolécules Biologiques (AFMB, UMR 6098), Aix-Marseille Univ., Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France.
J Mol Biol. 2016 Nov 6;428(22):4413-4423. doi: 10.1016/j.jmb.2016.08.030. Epub 2016 Sep 4.
The type VI secretion system (T6SS) is a multiprotein complex that delivers toxin effectors in both prokaryotic and eukaryotic cells. It is constituted of a long cytoplasmic structure-the tail-made of stacked Hcp hexamers and wrapped by a contractile sheath. Contraction of the sheath propels the inner tube capped by the VgrG spike protein toward the target cell. This tubular structure is built onto an assembly platform-the baseplate-that is composed of the TssEFGK-VgrG subunits. During the assembly process, the baseplate is recruited to a trans-envelope complex comprising the TssJ outer membrane lipoprotein and the TssL and TssM inner membrane proteins. This membrane complex serves as a docking station for the baseplate/tail and as a channel for the passage of the inner tube during sheath contraction. The baseplate is recruited to the membrane complex through multiple contacts including interactions of TssG and TssK with the cytoplasmic loop of TssM and of TssK with the cytoplasmic domain of TssL, TssL. Here, we show that TssL interacts also with the TssE baseplate subunit. Based on the available TssL structures, we targeted conserved regions and specific features of TssL in enteroaggregative Escherichia coli. By using bacterial two-hybrid analysis and co-immunoprecipitation, we further show that the disordered L3-L4 loop is necessary to interact with TssK and that the L6-L7 loop mediates the interaction with TssE, whereas the TssM cytoplasmic loop binds the conserved groove of TssL. Finally, competition assays demonstrated that these interactions are physiologically important for T6SS function.
VI型分泌系统（T6SS）是一种多蛋白复合体，可将毒素效应蛋白递送至原核细胞和真核细胞中。它由一个长的细胞质结构——尾——组成，尾由堆叠的Hcp六聚体构成，并被一个收缩鞘包裹。鞘的收缩将由VgrG刺突蛋白封端的内管推向靶细胞。这种管状结构构建在一个组装平台——底板——上，底板由TssEFGK-VgrG亚基组成。在组装过程中，底板被招募至一个跨膜复合体，该复合体由TssJ外膜脂蛋白以及TssL和TssM内膜蛋白组成。这种膜复合体作为底板/尾的对接站，并在鞘收缩期间作为内管通过的通道。底板通过多种接触被招募至膜复合体，包括TssG和TssK与TssM细胞质环的相互作用以及TssK与TssL细胞质结构域TssL的相互作用。在此，我们表明TssL也与底板亚基TssE相互作用。基于现有的TssL结构，我们针对肠聚集性大肠杆菌中TssL的保守区域和特定特征进行研究。通过细菌双杂交分析和免疫共沉淀，我们进一步表明无序的L3-L4环对于与TssK相互作用是必需的，而L6-L7环介导与TssE的相互作用，而TssM细胞质环结合TssL的保守凹槽。最后，竞争分析表明这些相互作用对于T6SS功能在生理上是重要的。
