Implications of the Evolutionary Past on the Activities of VirB: A Key Transcriptional Regulator of Virulence Genes in Shigella flexneri

Shigella species are bacterial agents that are the leading cause of diarrheal diseases, the second leading cause of child mortality worldwide. One key protein in Shigella is VirB, a transcriptional regulator of virulence genes that is essential for pathogenicity. VirB functions to antagonize H-NS, a histone-like nucleoid structuring protein that silences many genes found on the large virulence plasmid (pINV). The interplay between H-NS, the silencer, and VirB, the anti-silencer, is fundamental for Shigella virulence. Furthermore, this mechanism of gene regulation appears widespread in many bacteria including other enteric pathogens. The goal of my dissertation was to explore the activities of VirB to gain a better understanding of the mechanism used for anti-silencing. An early indication that VirB might not act like a traditional transcriptional regulator came from comparing its amino acid sequence to other proteins. VirB does resemble other transcription factors, but shares homology with a group of proteins involved in DNA partitioning (exemplified by ParB). As such, key questions were raised about whether VirB exhibits some of the activities of its homolog, ParB, and whether these identified activities are associated with or required for its mechanism of anti-silencing. My studies reveal that GFP-VirB is like ParB proteins in that it forms discrete foci in the bacterial cytoplasm in live S. flexneri. Focus formation is dependent upon VirB-DNA interactions, whereby VirB binding to a plasmid containing a 25 bp recognition sequence is necessary and sufficient. Additionally, GFP-VirB focus formation is maintained in closely related bacterial species (Escherichia coli and Salmonella typhimurium), which suggests that VirB could be used as a molecular marker to track the precise location of a DNA target site in live bacterial cells of other distantly related species. Since published phylogenetic trees of the ParB superfamily have not included VirB, I present a VirB-centric phylogenetic analysis of the ParB protein family. This analysis reveals that VirB was likely acquired from a bacteriophage, rather than having evolved from pINV-encoded ParB. While exploring the similarities and differences of VirB and pINV ParB, I discovered that ParB (characterized to silence downstream genes) can function as an anti-silencer, like VirB, when provided its cognate binding site in the context of a VirB-dependent promoter. These findings provide evidence that other proteins in the ParB superfamily may also act to modulate gene regulation, a previously underappreciated role. Altogether, my studies show that VirB, a key virulence gene regulator in Shigella, forms foci (functional hubs) in the cell cytoplasm that resemble the higher order structures observed for ParB. While the relevance of these hubs for countering H-NS remains unclear, it is tantalizing to consider that VirB, like ParB, may be remodeling the DNA that it engages and that this is responsible for relieving transcriptional silencing imparted by H-NS. Additionally, my PhD research highlights that the evolutionary past of a protein may have implications on in its current activities. Although VirB and ParB serve distinct cellular roles, the activities shared by these family members once bound to DNA seem to have allowed VirB to adopt a new role as an anti-silencing protein. In conclusion, my dissertation research provides novel insight into the large, diverse ParB superfamily