Biology subjects

Karney, M. M.

Publications and source records attributed to Karney, M. M..

3 recordsLinked to original sources

bioRxiv2023

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. ImportanceShigella 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.

molecular biology↗

bioRxiv2023

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp., at [≤] 30 {degrees}C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid. Upon a switch to 37 {degrees}C, VirB, a DNA binding protein and key transcriptional regulator of Shigella virulence, is produced. VirB functions to counter H-NS-mediated silencing in a process called transcriptional anti-silencing. Here, we show that VirB mediates a loss of negative DNA supercoils from our plasmid-borne, VirB-regulated PicsP-lacZ reporter, in vivo. The changes are not caused by a VirB-dependent increase in transcription, nor do they require the presence of H-NS. Instead, the VirB-dependent change in DNA supercoiling requires the interaction of VirB with its DNA binding site, a critical first step in VirB-dependent gene regulation. Using two complementary approaches, we show that VirB:DNA interactions in vitro introduce positive supercoils in plasmid DNA. Subsequently, by exploiting transcription-coupled DNA supercoiling, we reveal that a localized loss of negative supercoils is sufficient to alleviate H-NS-mediated transcriptional silencing, independently of VirB. Together, our findings provide novel insight into VirB, a central regulator of Shigella virulence and more broadly, a molecular mechanism that offsets H-NS-dependent silencing of transcription in bacteria.

molecular biology↗

bioRxiv2022

The AraC/XylS protein MxiE and its co-regulator IpgC control a negative feedback loop in the transcriptional cascade that regulates type III secretion in Shigella flexneri

Members of the AraC Family of Transcriptional Regulators (AFTRs) control the expression of many genes important to cellular processes, including virulence. In Shigella species, the type III secretion system (T3SS), a key determinant for host cell invasion, is regulated by the three-tiered VirF/VirB/MxiE transcription cascade. Both VirF and MxiE belong to the AFTRs and are characterized as positive transcriptional regulators. Here, we identify a novel regulatory activity for MxiE and its co-regulator IpgC, which manifests as a negative feedback loop in the VirF/VirB/MxiE transcription cascade. Our findings show that MxiE and IpgC down-regulate the virB promoter and hence VirB protein production, thus, decreasing VirB-dependent promoter activity at ospD1, one of the nearly 50 VirB-dependent genes. At the virB promoter, regions required for negative MxiE- and IpgC-dependent regulation were mapped and found to be coincident with regions required for positive VirF-dependent regulation. In tandem, negative MxiE- and IpgC-dependent regulation of the virB promoter only occurred in the presence of VirF suggesting that MxiE and IpgC can function to counter VirF activation of the virB promoter. Lastly, MxiE and IpgC do not down-regulate another VirF-activated promoter, icsA, demonstrating that this negative feedback loop targets the virB promoter. Our study provides insight into a mechanism that may reprogram Shigella virulence gene expression following type III secretion and provides the impetus to examine if MxiE and IpgC homologs in other important bacterial pathogens such as Burkholderia pseudomallei and Salmonella enterica serovars Typhimurium and Typhi coordinate similar negative feedback loops. IMPORTANCEThe large AraC Family of Transcriptional Regulators (AFTRs) control virulence gene expression in many bacterial pathogens. In Shigella species, the AraC/XylS protein MxiE and its co-regulator IpgC positively regulate the expression of type III secretion system genes within the three-tiered VirF/VirB/MxiE transcription cascade. Our findings suggest a negative feedback loop in the VirF/VirB/MxiE cascade in which MxiE and IpgC counter VirF-dependent activation of the virB promoter, thus, making this the first characterization of negative MxiE- and IpgC-dependent regulation. Our study provides insight into a mechanism that likely reprograms Shigella virulence gene expression following type III secretion, which has implications for other important bacterial pathogens with functional homologs of MxiE and IpgC.

microbiology↗