Doctor of Philosophy (PhD)
Organic peroxides (OPs) are highly toxic oxidants. They directly react with cellular macromolecules rendering them inactive. Soil bacteria live in an oxidative environment rich in OPs from plant exudates and other xenobiotic compounds. Bacteria also face a burst of organic peroxides (lipid peroxides) in the macrophages during host invasion. Prokaryotes sense oxidative changes in environment and modulate their gene expression as an adaptive response for survival. MarR (multiple antibiotic resistance regulators) proteins enable bacteria to respond to such stress. Resistance to organic oxidants has been hypothesized to help bacteria survive in presence of disinfectants. This increases nosocomial infection rate.
Bacteria belonging to the Burkholderia cepacia complex (Bcc)., are mainly associated with OP resistance. Being opportunistic, they infect immunocompromised individuals, especially those suffering from cystic fibrosis. Genome wide analysis revealed an ohr-ohrR locus in these bacteria. OhrR (organic hydroperoxide reductase regulator) has been shown to regulate expression of ohr (encoding organic hydroperoxide reductase) in some bacterial species. Ohr degrades organic oxidants in a thiol-dependent manner. OhrR senses changes in the environment by virtue of its redox-active cysteines.
In this study, Burkholderia thailandensis was the model system chosen due to the oxidative environment it thrives in. Modeled structure of B. thailandensis OhrR (Bt OhrR) revealed two cysteines (C16 and C121) predicted to be involved in oxidant sensing and transcriptional regulation. Based on SDS-PAGE, Bt OhrR responds to organic and inorganic oxidants, contrary to conventional OhrRs. Oxidized species were formed as a result of disulfide bonding between cysteines. Site-directed mutagenesis showed initial oxidation of C16 key for disulfide bond formation with the neighboring C121. DNA binding assays revealed OhrR binds the ohr and ohrR promoters, with binding to ohr promoter attenuated in presence of oxidants (cumene hydroperoxide and hydrogen peroxide). Experimental analysis and structural modeling supported the premise that OhrR derepresses ohr as a result of intermonomer disulfide bonding.
Footprinting showed C16 residues formed an oxidized product sufficient for attenuating DNA binding by OhrR, indicating C16 to be “crucial” for peroxide sensing and gene regulation in B. thailandensis. Upregulation in ohr and ohrR expression was observed in the presence of organic oxidants, confirming OhrR’s role as a redox sensor-regulator. With disinfectants being one of the last resorts for maintaining hygiene in hospitals, understanding bacterial mechanisms of resistance to such compounds is critical.
Pande, Anuja Rajiv, "Breaking Conventions: OhrR, A Transcriptional Regulator from Burkholderia thailandensis" (2018). LSU Doctoral Dissertations. 4537.
Available for download on Tuesday, December 31, 2019