VirB, a key transcriptional regulator of Shigella virulence, requires a CTP ligand for its regulatory activities

The VirB protein, encoded by the large virulence plasmid of Shigella spp., is a key transcriptional regulator of virulence genes. Without virB, Shigella cells are avirulent. On the virulence plasmid, VirB functions to offset transcriptional silencing mediated by the nucleoid structuring protein, H-NS, which binds and sequesters AT-rich DNA, making it inaccessible for gene expression. Thus, gaining a mechanistic understanding of how VirB counters H-NS-mediated silencing is of considerable interest. VirB is unusual in that it does not resemble classic transcription factors. Instead, its closest relatives are found in the ParB superfamily, where the best-characterized members function in faithful DNA segregation before cell division. Here, we show that VirB is a fast-evolving member of this superfamily and report for the first time that the VirB protein binds a highly unusual ligand, CTP. VirB binds this nucleoside triphosphate preferentially and with specificity. Based on alignments with members of the ParB family, we identify amino acids of VirB likely to bind CTP. Substitutions in these residues disrupt several well-documented activities of VirB, including its anti-silencing activity, its ability to generate a Congo red positive phenotype, and the ability of the VirB protein to form foci in the bacterial cytoplasm when fused to GFP. Although stably produced, our CTP binding mutants do not bind DNA in vivo, likely explaining these phenotypes. Thus, this work is the first to show that VirB is a bona fide CTP-binding protein, links Shigella virulence phenotypes to the nucleoside triphosphate CTP, and provides novel insight into VirB-CTP-DNA interactions. IMPORTANCE: Shigella species cause bacillary dysentery, the second leading cause of diarrheal deaths worldwide. There is a pressing need to identify novel molecular drug targets. Shigella virulence phenotypes are controlled by the transcriptional regulator, VirB. We show that VirB belongs to a fast-evolving, plasmid-borne clade of the ParB superfamily, which has diverged from versions with a distinct cellular role—DNA partitioning. We report that, like classic members of the ParB family, VirB binds a highly unusual ligand, CTP. Mutants predicted to be defective in CTP binding are compromised in a variety of virulence attributes controlled by VirB, likely because these mutants cannot engage DNA. This study (i) reveals that VirB binds CTP, (ii) provides a link between VirB-CTP interactions and Shigella virulence phenotypes, (iii) provides new insight into VirB-CTP-DNA interactions, and (iv) broadens our understanding of the ParB superfamily, a group of bacterial proteins that play critical roles in many bacteria.