Monday, May 6, 2024
5/6/2024
Summary Nucleoid-associated proteins (NAPs), 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, function to offset NAP-mediated silencing, leading to gene expression. These frequent and widespread regulatory events in bacteria are functionally akin to eukaryotic chromatin remodeling and control many aspects of bacterial physiology, including virulence. Since bacterial NAPs (“the silencers”) are crucial for the organization and sequestration of bacterial DNA, insight into these key regulatory processes will likely lead to the discovery of novel drug targets and new therapeutics. While many unrelated DNA-binding proteins can serve as anti- silencers, mechanistic underpinnings are likely shared. Consequently, the long-term goal of this project is to fully understand transcriptional silencing and anti-silencing mechanisms in the bacterial pathogen Shigella. In Shigella, at 30°C or lower, H-NS silences many genes on the large virulence plasmid (pINV; 230 kb). Upon a switch to human body temperature (37°C), production of the VirB anti-silencer counters H-NS silencing leading to the up-regulation of key virulence genes. Research findings made through previous R15 awards are foundational to the work currently proposed. The overarching hypothesis of this study is “VirB spreading and/or bridging activities on DNA directly relieve H-NS mediated silencing by triggering a loss of negative supercoils in DNA.” This hypothesis will be tested using two specific aims. In Aim 1, the relationship between DNA supercoiling and transcriptional silencing/anti-silencing will be determined using in vivo and in vitro approaches. First, two creative and complementary in vitro approaches that ‘fix’ VirB-mediated changes in plasmid DNA will be used to determine if VirB:DNA interactions directly trigger a change in DNA supercoiling. Second, the effect of DNA supercoiling on H-NS-mediated silencing is investigated by measuring the activity of an H-NS-silenced promoter in either a set of topoisomerase I mutants or in the presence of the DNA gyrase inhibitor, novobiocin. In Aim 2, the nature of VirB:DNA complexes and their involvement in VirB-dependent transcriptional anti-silencing will be elucidated. First, ChIP-PCR combined with an innovative DNase I treatment step will definitively test if VirB spreads along DNA from its cognate site at the well-characterized icsP locus. Second, VirB mutants likely defective in VirB:VirB interactions will be characterized in terms of binding, spreading, forming foci in the bacterial cytoplasm and anti-silencing activities. Third, the involvement of VirB spreading and/or bridging in the anti-silencing of the icsP promoter is investigated. The work proposed is in line with the stated long-term goal (above) and is likely to provide insight into transcriptional silencing/anti- silencing mechanisms in other bacteria. All experiments are suitable for undergraduate and graduate students, in keeping with the R15 funding mechanism. The proposed research will be conducted at the University of Nevada, Las Vegas; a minority serving institution located in an IDeA state.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
