The virulence regulator VirB from Shigella flexneri uses a CTP-dependent switch mechanism to activate gene expression

Previous studies on the transcriptional antisilencer VirB from S. flexneri have shown that it activates virulence gene expression by counteracting the nucleoid-organizing protein H-NS in a virS-dependent manner. The loading and spreading on the virS site lead to a local decrease in negative supercoiling, thereby destabilizing the H-NS complexes to activate gene expression. However, the underlying process remains unclear. In contrast to conventional transcriptional activators, VirB does not directly interact with the RNA polymerase (RNAP). VirB belongs to a novel class of molecular switches, which act as DNA sliding clamps that use cytidine triphosphate (CTP) binding and hydrolysis to control their opening and closing state. The newly discovered CTPase activity of ParB that allows its binding and sliding along the DNA raises the question of whether VirB uses a similar mode of action. Here, we show that VirB binds CTP, embraces DNA in a clamp-like fashion upon its CTP-dependent loading at virS sites and slides laterally on DNA after clamp closure. CTP-binding mutations inhibit VirB loading in vitro, and abolish the formation of VirB nucleoprotein complexes as well as virulence gene expression in vivo. Therefore, VirB functions as a CTP-dependent molecular switch that regulates transcription through a loading-and-sliding mechanism during bacterial pathogenesis.