Dual Expression Profile of Type VI Secretion System Immunity Genes Protects Pandemic Vibrio cholerae

The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.霍乱弧菌VI型分泌系统（type VI secretion system, T6SS）可组装为分子注射器，将毒性蛋白效应因子注入真核与原核细胞内。本团队此前曾报道，霍乱弧菌O37血清群菌株V52可维持组成型激活的T6SS，以杀伤其他革兰氏阴性细菌，同时可抵御同源细菌的攻击。大流行株O1 El Tor型霍乱弧菌菌株C6706在实验室条件下呈T6SS沉默状态，无法合成T6SS结构组分与效应因子，也无法杀伤大肠杆菌（Escherichia coli）靶标菌。但当被具有T6SS活性的V52菌株攻击时，C6706却可展现出完全的抗性。上述研究结果表明，抵御T6SS介导的毒力并不依赖于活性T6SS本身。本研究针对T6SS免疫蛋白编码基因tsiV1、tsiV2与tsiV3的双重表达模式展开阐述，该模式可赋予大流行株霍乱弧菌T6SS免疫能力，同时使T6SS沉默菌株能够抵御具有T6SS活性的邻近细菌的攻击。这种双重表达模式可使这三个免疫蛋白编码基因的转录过程，独立于同一操纵子内编码的其他T6SS蛋白进行。其中一种免疫蛋白TsiV2可抵御T6SS效应因子VasX，而VasX的编码位点紧邻tsiV2的上游区域。VasX是一种分泌型脂结合蛋白，本团队此前曾针对其介导T6SS对社会性变形虫盘基网柄菌（Dictyostelium discoideum）的毒力展开过功能表征。本研究数据表明，vasX的开放阅读框（open reading frame, ORF）内存在一个内部启动子，可驱动下游基因tsiV2的表达。此外，研究证实VasX可与其辅助蛋白VasW协同作用，破坏原核靶细胞的细胞膜内膜。T6SS免疫蛋白编码基因tsiV1、tsiV2与tsiV3的双重调控模式，使霍乱弧菌能够在维持T6SS活性免疫能力的同时，严格调控T6SS的基因表达。