Ma Jiale, Pan Zihao, Huang Jinhu, Sun Min, Lu Chengping, Yao Huochun
a Department of Veterinary Microbiology and Immunology, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing , China.
b Key Lab of Animal Bacteriology, Ministry of Agriculture , Nanjing , China.
Virulence. 2017 Oct 3;8(7):1189-1202. doi: 10.1080/21505594.2017.1279374. Epub 2017 Jan 6.
The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3-4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4) genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors.
VI型分泌系统（T6SS）是许多细菌物种广泛使用的一种分子武器，用于靶向真核宿主细胞或竞争细菌。通过动态注射机制，T6SS可以将多种效应蛋白直接递送到受体细胞中。在此，我们报道了一个由携带不同毒素结构域的扩展Hcp编码的新型T6SS效应蛋白家族。生物信息学分析表明，这些具有C末端延伸毒素的Hcp（命名为Hcp-ET）广泛存在于肠杆菌科中。为了验证我们的发现，我们测试了Hcp-ET1的抗菌作用，结果表明它通过T6SS依赖性递送，凭借预测的HNH-DNase活性有效抑制了靶细胞的生长。进一步的研究表明，Hcp-ET2通过Tle1磷脂酶（由DUF2235结构域编码）活性介导细菌间的拮抗作用。值得注意的是，对蛋白质同源性和基因组邻域的综合分析表明，Hcp-ET3-4在C末端与2个毒素结构域（Pyocin S3和Colicin-DNase）融合，其编码基因后面跟着同源免疫基因的3次重复。然而，由于Pyocin S3和Colicin-DNase编码片段之间融合区域的终止密码子突变，一些细菌编码了一个分离的hcp-et3和一个孤儿et4（et4）基因。我们的结果表明，这两种毒素都具有抗菌作用。此外，同源免疫蛋白的所有重复都有助于中和Pyocin S3和Colicin的DNase毒性，这在以前尚未见报道。总之，我们提出Hcp-ET蛋白是多态性T6SS效应蛋白，因此呈现了一种新型的T6SS效应蛋白编码模式。
