VI型分泌系统(T6SS)是细菌大分子复合物,将效应蛋白分泌到靶细胞或细胞外环境中,导致相邻细胞的死亡并提供生存优势。尽管研究表明铜绿假单胞菌中的T6SS受群体感应(QS)系统和第二信使c-di-GMP的调控,潜在的分子机制在很大程度上仍然未知。在这项研究中,我们发现,c-di-GMP结合衔接蛋白PA0012对铜绿假单胞菌PAO1中T6SSHSI-I基因的表达具有抑制作用。要探测PA0012(重命名为TssZ,六型分泌系统相关PilZ蛋白)调节HSI-I基因的表达,我们进行了酵母双杂交筛选,并鉴定了HinK,LasR型转录调节因子,作为TssZ的绑定部分。通过共免疫沉淀测定进一步证实了HinK和TssZ之间的直接蛋白质-蛋白质相互作用。我们的实验结果表明,在高c-di-GMP浓度下,HinK-TssZ相互作用减弱,与其中c-di-GMP增强PilZ蛋白与其配偶体之间的相互作用的当前范例相反。电泳迁移率变化分析显示,非c-di-GMP结合突变体TssZR5A/R9A直接与HinK相互作用,并阻止其与群体感应调节因子pqsR的启动子结合。TssZ和HinK之间的功能联系进一步得到了TssZ和HinK影响成群运动的观察的支持,以PqsR依赖性方式生产绿脓杆菌的绿脓苷和T6SS介导的细菌杀伤活性。一起,这些结果揭示了一种新的调节机制,其中TssZ作为与HinK相互作用以控制基因表达的抑制剂发挥作用。
Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.