VirB, a transcriptional activator of virulence in Shigella flexneri, uses CTP as a cofactor

VirB is a transcriptional activator of virulence in the gram-negative bacterium Shigella flexneri encoded by the large invasion plasmid, pINV. It counteracts the transcriptional silencing by the nucleoid structuring protein, H-NS. Mutations in virB lead to loss of virulence. Studies suggested that VirB binds to specific DNA sequences, remodels the H-NS nucleoprotein complexes, and changes DNA supercoiling. VirB belongs to the superfamily of ParB proteins which are involved in plasmid and chromosome partitioning often as part of a ParABS system. Like ParB, VirB forms discrete foci in Shigella flexneri cells harbouring pINV. Our results reveal that purified preparations of VirB specifically bind the ribonucleotide CTP and slowly but detectably hydrolyse it with mild stimulation by the virS targeting sequences found on pINV. We show that formation of VirB foci in cells requires a virS site and CTP binding residues in VirB. Curiously, DNA stimulation of clamp closure appears efficient even without a virS sequence in vitro. Specificity for entrapment of virS DNA is however evident at elevated salt concentrations. These findings suggest that VirB acts as a CTP-dependent DNA clamp and indicate that the cellular microenvironment contributes to the accumulation of VirB specifically at virS sites.

VirB是编码于大型侵袭质粒pINV的革兰氏阴性菌福氏志贺菌（Shigella flexneri）的毒力转录激活因子，可拮抗核区结构蛋白H-NS（H-NS）介导的转录沉默。virB基因突变会导致菌株毒力丧失。已有研究表明，VirB能够结合特异性DNA序列，重塑H-NS核蛋白复合物，并改变DNA超螺旋状态。VirB属于ParB蛋白超家族，该家族蛋白常作为ParABS系统的组分参与质粒与染色体的分离过程。与ParB类似，携带pINV的福氏志贺菌细胞内的VirB可形成离散聚点。本研究结果显示，纯化的VirB蛋白制剂可特异性结合核糖核苷酸CTP（CTP），并能缓慢但可检测地水解该核苷酸，且pINV上的virS靶向序列可对其水解活性产生轻度刺激。研究证实，细胞内VirB聚点的形成需要virS位点以及VirB的CTP结合残基。有趣的是，即便在体外无virS序列的情况下，DNA对夹闭合过程的刺激仍较为高效；但在高盐浓度条件下，VirB对virS DNA的捕获特异性会显著显现。上述研究结果表明，VirB可作为一种CTP依赖的DNA夹发挥功能，同时提示细胞微环境可促进VirB特异性聚集于virS位点。