Thursday, November 21, 2024
11/21/2024
Project Summary Nucleoid structuring proteins (NSPs), like the histone-like nucleoid structuring protein H-NS, bind to DNA and transcriptionally silence genes in bacteria. DNA-binding proteins, known as anti-silencers, counter NSP- mediated silencing leading to gene expression. These opposing processes govern DNA management and control transcription in all bacterial cells, impacting many aspects of bacterial physiology, including virulence. In the bacterial pathogen Shigella flexneri, genes on the 230 kb virulence plasmid are silenced by H-NS and, at 37°C, are anti-silenced by VirB, a required regulator of Shigella virulence. The overarching goal of this project is to fully understand transcriptional silencing and anti-silencing mechanisms of virulence genes in the bacterial pathogen Shigella. Over the last project period, a three-step model of anti-silencing has been developed, in which VirB i/ binds to its site, ii/ spreads along DNA, and iii/ causes a localized change in DNA supercoiling that evicts or remodels proximal H-NS:DNA complexes, allowing transcription to proceed. Here, our model will be explored further, based on our new finding that VirB specifically binds an unusual ligand, the nucleoside triphosphate CTP, like distant relatives in the ParB superfamily. We hypothesize that “The CTP ligand of VirB is essential for its role as an anti-silencing protein that controls Shigella virulence.” This hypothesis will be tested using three independent aims. In Aim 1, the role of CTP in VirB:DNA interactions, that underpin VirB-dependent anti-silencing, will be characterized using biochemical and genetic approaches with innovative elements. Based on preliminary data, this work will likely identify which of the three mechanistic steps of anti-silencing requires the CTP ligand. In Aim 2, CTP hydrolysis by VirB will be measured and the effect this has on VirB:DNA interactions and anti-silencing will be characterized. While the ParB/Spo0J literature, suggests that hydrolysis of CTP by VirB may cause a conformational change that impacts VirB:DNA interactions, structural differences highlighted by a new VirB model (collaborator A. Iyer, NIH) make it uncertain if hydrolysis of CTP by VirB has been retained. Finally, in Aim 3, VirB mutants with intermediate CTP binding activity will be examined to determine the effects on Shigella virulence. Here, a novel genetic screen allowing the identification of VirB derivatives with intermediate CTP binding activity will be used so that these VirB derivatives can then be characterized in assays that measure Shigella virulence. This aim is likely to i/ strengthen the link between CTP binding by VirB and Shigella virulence and may ii/ provide the first evidence that CTP pools regulate virulence gene expression. In sum, the proposed work will bring us closer to achieving our stated long-term goal (above) by specifically examining the role of CTP in the anti-silencing of Shigella virulence genes by VirB. Furthermore, since the anti-silencer, VirB, is a fast-evolving member of the ParB superfamily that does not participate in DNA segregation, this work will broaden our understanding of this superfamily; an important group of proteins that play critical roles in many different bacteria, including those that cause human disease.
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