Insights into the Cross-Immunity Mechanism within Effector Families of Bacteria Type VI Secretion System from the Structure of StTae4-EcTai4 Complex

The Gram-negative bacteria type VI secretion system (T6SS) has been found to play an important role in interbacterial competition, biofilm formation and many other virulence-related processes. The bacteria harboring T6SS inject the effectors into their recipient’s cytoplasm or periplasm to kill them and meanwhile, to avoid inhibiting itself, the cognate immunity proteins were produced to acts as the effector inhibitor. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity (EI) pairs. We have recently solved the structures of StTae4-Tai4 and EcTae4-Tai4 complexes from the human pathogens Salmonella typhimurium and Enterobacter cloacae, respectively. It is very interesting and important to discover whether there is cross-neutralization between St- and EcTai4 and whether their effector inhibition mechanism is conserved. Here, we determined the crystal structure of StTae4 in complex with EcTai4. The solution conformation study revealed it is a compact heterotetramer that consists of an EcTai4 homodimer binding two StTae4 molecules in solution, different from that in crystal. A remarkable shift can be observed in both the flexible winding loop of StTae4 and protruding loop of EcTai4 and disulfide bonds are formed to stabilize their overall conformations. The in vitro and in vivo interactions studies showed EcTai4 can efficiently rescue the cells from the toxicity of its cognate effectors StTae4, but can not neutralize the toxic activities of the effectors from other families. These findings provide clear structural evidence to support the previous observation of cross-immunity within T6SS families and provide a basis for understanding their important roles in polymicrobial environments.

革兰氏阴性菌第六型分泌系统（type VI secretion system, T6SS）已被证实可在细菌间竞争、生物被膜形成以及诸多其他毒力相关过程中发挥关键作用。携带T6SS的细菌可将效应蛋白注入受体菌的细胞质或周质空间以杀灭靶菌；同时为避免自身受到抑制，细菌会产生同源免疫蛋白作为效应蛋白抑制剂。Tae4（第六型酰胺酶效应蛋白4，type VI amidase effector 4）与Tai4（第六型酰胺酶免疫蛋白4，type VI amidase immunity 4）是新近发现的T6SS效应蛋白-免疫蛋白（effector-immunity, EI）对。我们近期分别解析了来自人类致病菌鼠伤寒沙门氏菌（Salmonella typhimurium）和阴沟肠杆菌（Enterobacter cloacae）的StTae4-Tai4复合物与EcTae4-Tai4复合物的晶体结构。探究StTai4与EcTai4之间是否存在交叉中和作用，以及二者的效应蛋白抑制机制是否保守，是一项兼具趣味性与重要性的研究课题。本研究解析了StTae4与EcTai4复合物的晶体结构。溶液构象研究显示，该复合物在溶液中为紧凑的异四聚体：由一个EcTai4同源二聚体结合两个StTae4分子构成，这与晶体状态下的构象存在差异。研究观察到StTae4的柔性缠绕环与EcTai4的突出环均发生了显著位移，且形成了二硫键以稳定整体构象。体外与体内相互作用实验表明，EcTai4可有效拯救细胞免受同源效应蛋白StTae4的毒性作用，但无法中和其他家族效应蛋白的毒性活性。本研究结果为T6SS家族内交叉免疫的既往观测提供了明确的结构学证据，同时为理解二者在多微生物环境中的重要功能奠定了基础。