Genomics Research Center, College of Pharmacy, Harbin Medical University, Harbin, China.
Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
J Bacteriol. 2022 Jul 19;204(7):e0012222. doi: 10.1128/jb.00122-22. Epub 2022 Jun 23.
Three distinct genetic architectures (GAs) of Type VI secretion systems (T6SSs) have been described in gut Bacteroidales species, each with unique genes and characteristics. Unlike the GA3 T6SSs, potent antagonism has not yet been demonstrated for the GA1 or GA2 T6SSs. We previously showed that the GA2 T6SS loci are contained on integrative and conjugative elements and that there are five subtypes. Collectively, GA2 are the most prevalent Bacteroidales T6SSs in the human populations analyzed. In this study, we provide a comprehensive bioinformatic analysis of the three variable regions of GA2 T6SS loci, which encode toxic effector and immunity proteins. In total, we identified 63 distinct effectors encoded within 31 nonredundant GA2 loci, 18 of which do not have described motifs or predicted functions. We provide experimental evidence for toxin activity for four different GA2 effectors, showing that each functions only when present in the periplasm, and experimentally confirm their cognate immunity proteins. Our data demonstrate that each GA2 locus encodes at least three distinct effectors with targets in both the cytoplasm and the periplasm. The data also suggest that the effectors of a given locus are loaded onto the tube by different mechanisms, which may allow all three effectors encoded within a single GA2 locus with distinct antibacterial activity to be loaded onto a single T6 tube, increasing the antagonistic effect. Humans are colonized with many gut Bacteroidales species at high density, allowing for extensive opportunities for contact-dependent antagonism. To begin to understand the antagonistic potential of the GA2 T6SSs of the gut Bacteroidales, we performed bioinformatic and experimental analyses of the three divergent regions containing the toxin effector and immunity genes. We show that each GA2 T6SS locus encodes at least three distinct toxic effectors including toxins linked to Rhs and Hcp with cytoplasmic targets, and unlinked effectors with targets in the periplasm. The diversity and modality of effectors exceeds that of the GA1 or GA3 T6SS loci (M. J. Coyne, K. G. Roelofs, and L. E. Comstock, BMC Genomics 17:58, 2016, https://doi.org/10.1186/s12864-016-2377-z) and suggests that these T6SSs have the potential to be potent antibacterial weapons in the human gut.
三种不同的 VI 型分泌系统（T6SS）遗传结构（GA）已在肠道拟杆菌物种中描述，每种结构都具有独特的基因和特征。与 GA3 T6SS 不同，GA1 或 GA2 T6SS 尚未表现出强烈的拮抗作用。我们之前表明，GA2 T6SS 基因座包含在整合和共轭元件上，并且有五个亚型。总体而言，GA2 是分析的人群中最普遍的拟杆菌 T6SS。在这项研究中，我们对 GA2 T6SS 基因座的三个可变区进行了全面的生物信息学分析，这些区域编码有毒效因子和免疫蛋白。总共，我们在 31 个非冗余的 GA2 基因座中鉴定了 63 种不同的效应因子，其中 18 种没有描述的基序或预测的功能。我们提供了四种不同 GA2 效应因子的毒素活性的实验证据，表明每种效应因子仅在周质中存在时才起作用，并实验证实了它们的同源免疫蛋白。我们的数据表明，每个 GA2 基因座都至少编码三种不同的效应因子，这些效应因子的靶标在细胞质和周质中。数据还表明，给定基因座的效应因子通过不同的机制加载到管上，这可能允许单个 GA2 基因座中编码的具有不同抗菌活性的三个效应因子加载到单个 T6 管上，从而增强拮抗作用。人类在高密度时被许多肠道拟杆菌物种定植，从而为接触依赖性拮抗作用提供了广泛的机会。为了开始了解肠道拟杆菌 GA2 T6SS 的拮抗潜力，我们对包含毒素效应因子和免疫基因的三个不同区域进行了生物信息学和实验分析。我们表明，每个 GA2 T6SS 基因座至少编码三种不同的有毒效应因子，包括与 Rhs 和 Hcp 相关的细胞质靶标的毒素以及与周质靶标的无关联的效应因子。效应因子的多样性和模态超过了 GA1 或 GA3 T6SS 基因座（M. J. Coyne，K. G. Roelofs 和 L. E. Comstock，BMC Genomics 17:58，2016，https://doi.org/10.1186/s12864-016-2377-z），这表明这些 T6SS 有可能成为人类肠道中的强大抗菌武器。
