A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition

The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic target cells to cause host damage or as a weapon for interbacterial competition. Pseudomonas aeruginosa encodes three distinct T6SS clusters (H1-, H2-, and H3-T6SS). The H1-T6SS-dependent substrates have been identified and well characterized; however, only limited information is available for the H2- and H3-T6SSs since relatively fewer substrates for them have yet been established. Here, we obtained P. aeruginosa H2-T6SS-dependent secretomes and further characterized the H2-T6SS-dependent copper (Cu2+)-binding effector azurin (Azu). Our data showed that both azu and H2-T6SS were repressed by CueR and were induced by low concentrations of Cu2+. We also identified the Azu-interacting partner OprC, a Cu2+-specific TonB-dependent outer membrane transporter. Similar to H2-T6SS genes and azu, expression of oprC was directly regulated by CueR and was induced by low Cu2+. In addition, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. Our findings provide insights for understanding the diverse functions of T6SSs and the role of metal ions for P. aeruginosa in bacteria-bacteria competition.

VI型分泌系统（Type VI secretion system, T6SS）广泛分布于革兰氏阴性菌中，其功能为将底物转运至真核与原核靶细胞以引发宿主损伤，或作为细菌间竞争的武器。铜绿假单胞菌（Pseudomonas aeruginosa）编码三套独立的T6SS基因簇（H1-T6SS、H2-T6SS与H3-T6SS）。目前H1-T6SS依赖的底物已被鉴定并得到充分表征；但针对H2-T6SS与H3-T6SS的研究信息较为有限，因为其对应的底物尚少被确认。本研究获取了铜绿假单胞菌H2-T6SS依赖的分泌组，并进一步对H2-T6SS依赖的铜离子（Cu²+）结合效应蛋白天青蛋白（azurin, Azu）进行了表征。实验数据显示，azu基因与H2-T6SS均受到铜离子应答调节因子（CueR）的抑制，并可被低浓度Cu²+诱导表达。本研究同时鉴定出Azu的互作伴侣外膜蛋白C（OprC），一种Cu²+特异性TonB依赖型外膜转运蛋白。与H2-T6SS基因及azu类似，oprC的表达直接受CueR调控，并可被低浓度Cu²+诱导。此外，Azu-OprC介导的Cu²+转运系统对于铜绿假单胞菌的细菌间竞争能力与毒力至关重要。本研究结果为理解T6SS的多样功能，以及金属离子在铜绿假单胞菌细菌间竞争中的作用提供了新的见解。