Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS One. 2011;6(8):e23876. doi: 10.1371/journal.pone.0023876. Epub 2011 Aug 31.
A type VI secretion system (T6SS) was recently shown to be required for full virulence of Vibrio cholerae O37 serogroup strain V52. In this study, we systematically mutagenized each individual gene in T6SS locus and characterized their functions based on expression and secretion of the hemolysin co-regulated protein (Hcp), virulence towards amoebae of Dictyostelium discoideum and killing of Escherichia coli bacterial cells. We group the 17 proteins characterized in the T6SS locus into four categories: twelve (VipA, VipB, VCA0109-VCA0115, ClpV, VCA0119, and VasK) are essential for Hcp secretion and bacterial virulence, and thus likely function as structural components of the apparatus; two (VasH and VCA0122) are regulators that are required for T6SS gene expression and virulence; another two, VCA0121 and valine-glycine repeat protein G 3 (VgrG-3), are not essential for Hcp expression, secretion or bacterial virulence, and their functions are unknown; the last group is represented by VCA0118, which is not required for Hcp expression or secretion but still plays a role in both amoebae and bacterial killing and may therefore be an effector protein. We also showed that the clpV gene product is required for Dictyostelium virulence but is less important for killing E. coli. In addition, one vgrG gene (vgrG-2) outside of the T6SS gene cluster was required for bacterial killing but another (vgrG-1) was not. However, a bacterial killing defect was observed when vgrG-1 and vgrG-3 were both deleted. Several genes encoded in the same putative operon as vgrG-1 and vgrG-2 also contribute to virulence toward Dictyostelium but have a smaller effect on bacterial killing. Our results provide new insights into the functional requirements of V. cholerae's T6SS in the context of secretion as well as killing of bacterial and eukaryotic phagocytic cells.
一种 VI 型分泌系统（T6SS）最近被证明是霍乱弧菌 O37 血清群菌株 V52 完全毒力所必需的。在这项研究中，我们系统地突变了 T6SS 基因座中的每个基因，并根据溶血素共调节蛋白（Hcp）的表达和分泌、对变形虫的毒力以及对大肠杆菌细胞的杀伤来表征它们的功能。我们将 T6SS 基因座中鉴定的 17 种蛋白分为四类：12 种（VipA、VipB、VCA0109-VCA0115、ClpV、VCA0119 和 VasK）对于 Hcp 分泌和细菌毒力是必需的，因此可能作为装置的结构成分发挥作用；两种（VasH 和 VCA0122）是 T6SS 基因表达和毒力所必需的调节剂；另外两种，VCA0121 和缬氨酸-甘氨酸重复蛋白 G3（VgrG-3），对于 Hcp 的表达、分泌或细菌毒力不是必需的，其功能未知；最后一组由 VCA0118 代表，它不参与 Hcp 的表达或分泌，但在变形虫和细菌杀伤中仍发挥作用，因此可能是一种效应蛋白。我们还表明，clpV 基因产物是变形虫毒力所必需的，但对大肠杆菌的杀伤作用则不那么重要。此外，T6SS 基因簇外的一个 vgrG 基因（vgrG-2）是细菌杀伤所必需的，而另一个（vgrG-1）则不是。然而，当 vgrG-1 和 vgrG-3 都被删除时，观察到细菌杀伤缺陷。与 vgrG-1 和 vgrG-2 编码在同一假定操纵子中的几个基因也有助于变形虫的毒力，但对细菌杀伤的影响较小。我们的研究结果为霍乱弧菌 T6SS 在分泌以及杀伤细菌和真核吞噬细胞方面的功能要求提供了新的见解。
