Insights into transcriptional silencing and anti-silencing in Shigella flexneri: a detailed molecular analysis of the icsP virulence locus.

Transcriptional silencing and anti-silencing mechanisms modulate bacterial physiology and virulence in many human pathogens. In Shigella species, many virulence plasmid genes are silenced by the histone-like nucleoid structuring protein H-NS and anti-silenced by the virulence gene regulator VirB. Despite the key role that these regulatory proteins play in Shigella virulence, their mechanisms of transcriptional control remain poorly understood. Here, we characterize the regulatory elements and their relative spacing requirements needed for the transcriptional silencing and anti-silencing of icsP, a locus that requires remotely located regulatory elements for both types of transcriptional control. Our findings highlight the flexibility of the regulatory elements' positions with respect to each other, and yet, a molecular roadblock docked between the VirB binding site and the upstream H-NS binding region abolishes transcriptional anti-silencing by VirB, providing insight into transcriptional anti-silencing. Our study also raises the need to re-evaluate the currently proposed VirB binding site. Models of transcriptional silencing and anti-silencing at this genetic locus are presented, and the implications for understanding these regulatory mechanisms in bacteria are discussed.

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 a functional virB gene, 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 the best-characterized 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 at a VirB-dependent promoter, its role in generating a Congo red positive phenotype in Shigella , and the ability of the VirB protein to form foci in the bacterial cytoplasm when fused to GFP. Thus, this work is the first to show that VirB is a bona fide CTP-binding protein and links Shigella virulence phenotypes to the nucleoside triphosphate, CTP. Importance: Shigella species cause bacillary dysentery (shigellosis), the second leading cause of diarrheal deaths worldwide. With growing antibiotic resistance, 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, primarily plasmid-borne clade of the ParB superfamily, which has diverged from versions that have a distinct cellular role - DNA partitioning. We are the first to 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. This study i) reveals that VirB binds CTP, ii) provides a link between VirB-CTP interactions and Shigella virulence phenotypes, and iii) broadens our understanding of the ParB superfamily, a group of bacterial proteins that play critical roles in many different bacteria.

Investigating the DNA-Binding Site for VirB, a Key Transcriptional Regulator of Shigella Virulence Genes, Using an In Vivo Binding Tool.

The transcriptional anti-silencing and DNA-binding protein, VirB, is essential for the virulence of Shigella species and, yet, sequences required for VirB-DNA binding are poorly understood. While a 7-8 bp VirB-binding site has been proposed, it was derived from studies at a single VirB-dependent promoter, icsB. Our previous in vivo studies at a different VirB-dependent promoter, icsP, found that the proposed VirB-binding site was insufficient for regulation. Instead, the required site was found to be organized as a near-perfect inverted repeat separated by a single nucleotide spacer. Thus, the proposed 7-8 bp VirB-binding site needed to be re-evaluated. Here, we engineer and validate a molecular tool to capture protein-DNA binding interactions in vivo. Our data show that a sequence organized as a near-perfect inverted repeat is required for VirB-DNA binding interactions in vivo at both the icsB and icsP promoters. Furthermore, the previously proposed VirB-binding site and multiple sites found as a result of its description (i.e., sites located at the virB, virF, spa15, and virA promoters) are not sufficient for VirB to bind in vivo using this tool. The implications of these findings are discussed.