Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that affects immune-compromised patients. Some strains display high resistance to a great range of antibiotics, therefore, identification of novel targets for drug development is desirable. OhrR-Ohr system is central in the bacterial response to organic hydroperoxides. Ohr (Organic Hydroperoxide Resistance Protein) is a dithiol, Cys-based peroxidase that efficiently reduces organic hydroperoxides, but not H2O2. Expression of Ohr gene is regulated by OhrR (Organic Hydroperoxide Resistance Regulator Protein), a transcriptional repressor from the MarR family. The ability of OhrR to bind DNA in Ohr promoter is modulated by the oxidation of a critical Cys residue. The present work aims to characterize the kinetics of OhrR oxidation by distinct hydroperoxides and the reduction by distinct thiols. Emphasis was given to fatty acids hydroperoxides that are intermediates in host-pathogen interactions and are reduced at extraordinary rates by Ohr. We have produced recombinant OhrR from Pseudomonas aeruginosa and the three Cys residues were individually replaced by Ser (OhrRCys9Ser, OhrRCys19Ser and OhrRCys121Ser). OhrR fluorescence changed between the oxidized and reduced states, allowing kinetic characterization by means of a fluorimeter equipped with a Stopped Flow device. The second order for the reaction of reduced OhrR with tert-butyl hydroperoxide was in the 103 M-1 s-1 range, while similar rate constant for the reaction with the fatty acid hydroperoxides were in the 106M-1 s-1 range. Cys19 appeared to be the reactive cysteine, responsible for sensing organic hydroperoxides, since no change in the fluorescence was observed for the interaction between Cys19Ser mutant with the hydroperoxides. Similar results were obtained for OhrR de C. violaceum. Next, we intend to characterize the reduction of oxidized OhrR by distinct thiols. The kinetic characterization for OhrR proteins can improve our knowledge on the mechanisms underlying bacteria's response to fatty acid peroxides.
Published Version
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