Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
Microbiol Spectr. 2022 Aug 31;10(4):e0157622. doi: 10.1128/spectrum.01576-22. Epub 2022 Jul 25.
The environmental pathogenic bacterium Chromobacterium violaceum kills Gram-positive bacteria by delivering violacein packed into outer membrane vesicles, but nothing is known about its contact-dependent competition mechanisms. In this work, we demonstrate that C. violaceum utilizes a type VI secretion system (T6SS) containing multiple VgrG proteins primarily for interbacterial competition. The single T6SS of C. violaceum contains six genes, which are located in the main T6SS cluster and four islands. Using T6SS core component-null mutant strains, Western blotting, fluorescence microscopy, and competition assays, we showed that the C. violaceum T6SS is active and required for competition against Gram-negative bacteria such as Pseudomonas aeruginosa but dispensable for C. violaceum infection in mice. Characterization of single and multiple mutants revealed that, despite having high sequence similarity, the six VgrGs show little functional redundancy, with VgrG3 showing a major role in T6SS function. Our coimmunoprecipitation data support a model of VgrG3 interacting directly with the other VgrGs. Moreover, we determined that the promoter activities of T6SS genes increased at high cell density, but the produced Hcp protein was not secreted under such condition. This T6SS growth phase-dependent regulation was dependent on CviR but not on CviI, the components of a C. violaceum quorum sensing (QS) system. Indeed, a Δ but not a Δ mutant was completely defective in Hcp secretion, T6SS activity, and interbacterial competition. Overall, our data reveal that C. violaceum relies on a QS-regulated T6SS to outcompete other bacteria and expand our knowledge about the redundancy of multiple VgrGs. The type VI secretion system (T6SS) is a contractile nanomachine used by many Gram-negative bacteria to inject toxic effectors into adjacent cells. The delivered effectors are bound to the components of a puncturing apparatus containing the protein VgrG. The T6SS has been implicated in pathogenesis and, more commonly, in competition among bacteria. Chromobacterium violaceum is an environmental bacterium that causes deadly infections in humans. In this work, we characterized the single T6SS of C. violaceum ATCC 12472, including its six VgrG proteins, regarding its function and regulation. This previously undescribed C. violaceum T6SS is active, regulated by QS, and required for interbacterial competition instead of acute infection in mice. Among the VgrGs, VgrG3, encoded outside the main T6SS cluster, showed a major contribution to T6SS function. These results shed light on a key contact-dependent killing mechanism used by C. violaceum to antagonize other bacteria.
环境病原菌类色杆菌通过将包裹在外膜泡中的紫色素递送给革兰氏阳性菌来杀死它们，但对于其接触依赖性竞争机制知之甚少。在这项工作中，我们证明类色杆菌利用包含多种 VgrG 蛋白的 VI 型分泌系统（T6SS）主要用于细菌间竞争。类色杆菌的单个 T6SS 包含六个 基因，它们位于主要 T6SS 簇和四个 岛中。使用 T6SS 核心成分缺失突变株、Western blot、荧光显微镜和竞争测定，我们表明类色杆菌 T6SS 是活跃的，并且需要与革兰氏阴性菌（如铜绿假单胞菌）竞争，但在小鼠中对类色杆菌感染是可有可无的。单个和多个 突变体的特征表明，尽管具有很高的序列相似性，但六个 VgrG 显示出很少的功能冗余，VgrG3 在 T6SS 功能中起主要作用。我们的共免疫沉淀数据支持 VgrG3 与其他 VgrG 直接相互作用的模型。此外，我们确定 T6SS 基因的启动子活性在高细胞密度时增加，但在这种条件下不分泌产生的 Hcp 蛋白。这种 T6SS 生长阶段依赖性调节依赖于 CviR，但不依赖于 CviI，CviI 是类色杆菌群体感应 (QS) 系统的组成部分。事实上，与野生型相比，Δ 但不是 Δ 突变体在 Hcp 分泌、T6SS 活性和细菌间竞争方面完全缺陷。总体而言，我们的数据表明，类色杆菌依赖于 QS 调节的 T6SS 来与其他细菌竞争并扩展我们对多个 VgrG 冗余性的认识。VI 型分泌系统（T6SS）是一种由许多革兰氏阴性菌用来将毒性效应器注射到邻近细胞中的收缩纳米机器。递送到的效应物与含有蛋白 VgrG 的穿孔装置的成分结合。T6SS 已被牵连到发病机制中，更常见的是在细菌之间的竞争中。类色杆菌是一种环境细菌，会导致人类致命感染。在这项工作中，我们对 ATCC 12472 型类色杆菌的单个 T6SS 进行了功能和调控的描述，包括其六个 VgrG 蛋白。这个以前未被描述的类色杆菌 T6SS 是活跃的，受 QS 调节，并且需要细菌间竞争而不是在小鼠中急性感染。在 VgrG 中，编码在主要 T6SS 簇之外的 VgrG3 对 T6SS 功能有主要贡献。这些结果揭示了类色杆菌用来拮抗其他细菌的关键接触依赖性杀伤机制。
