Okada Ryu, Matsuda Shigeaki, Iida Tetsuya
Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
PLoS One. 2017 Nov 17;12(11):e0187846. doi: 10.1371/journal.pone.0187846. eCollection 2017.
Vibrio parahaemolyticus is a Gram-negative pathogen that causes food-borne gastroenteritis. A major virulence determinant of the organism is a type III secretion system (T3SS2) encoded on a pathogenicity island, Vp-PAI. Vp-PAI gene expression is regulated by two transcriptional regulators, VtrA and VtrB, whose N-terminal regions share homology with an OmpR-family DNA-binding domain. VtrA activates the gene expression of VtrB, which in turn activates Vp-PAI gene expression; however, the mechanism of this transcriptional activation by VtrA is not well understood. In this study, we determined that VtrA is a membrane protein with a transmembrane (TM) domain, which was required for its transcriptional regulatory activity. Although the N-terminal region of VtrA alone is insufficient for its transcriptional regulatory activity, forced oligomerization using the leucine-zipper dimerization domain of yeast GCN4 conferred transcriptional regulatory activity and a greater affinity for the promoter region of vtrB. A ToxR-based assay demonstrated that VtrA oligomerizes in vivo. We also showed that bile, a host-derived activator of VtrA, induces the oligomerization of VtrA, which requires the C-terminal domain. The promoter region of vtrB contained repetitive T-rich DNA elements, which are important for vtrB transcriptional activation and are conserved among T3SS2-possessing Vibrio species. These findings propose that VtrA is active as oligomers, which may facilitate its N-terminus binding the target DNA, thus enhancing its transcriptional regulatory activity.
副溶血性弧菌是一种革兰氏阴性病原体，可引起食源性肠胃炎。该生物体的一个主要毒力决定因素是位于致病岛Vp-PAI上编码的III型分泌系统（T3SS2）。Vp-PAI基因表达受两种转录调节因子VtrA和VtrB调控，其N端区域与OmpR家族DNA结合域具有同源性。VtrA激活VtrB的基因表达，而VtrB又反过来激活Vp-PAI基因表达；然而，VtrA这种转录激活的机制尚不清楚。在本研究中，我们确定VtrA是一种具有跨膜（TM）结构域的膜蛋白，这是其转录调节活性所必需的。虽然单独的VtrA N端区域不足以产生其转录调节活性，但使用酵母GCN4的亮氨酸拉链二聚化结构域进行强制寡聚化赋予了转录调节活性以及对vtrB启动子区域更高的亲和力。基于ToxR的检测表明VtrA在体内会发生寡聚化。我们还表明，胆汁作为VtrA的一种宿主来源激活剂，可诱导VtrA的寡聚化，这需要C端结构域。vtrB的启动子区域包含富含T的重复DNA元件，这些元件对vtrB转录激活很重要，并且在具有T3SS2的弧菌属物种中保守。这些发现表明VtrA以寡聚体形式发挥作用，这可能有助于其N端结合靶DNA，从而增强其转录调节活性。
