Characterization of the Vibrio vulnificus 1-Cys Peroxiredoxin Prx3 and Regulation of Its Expression by the Fe-S Cluster Regulator IscR in Response to Oxidative Stress and Iron Starvation

Abstract

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that reduce toxic peroxides. A new Vibrio vulnificus Prx, named Prx3, was identified and characterized in this study. Biochemical and mutational analyses revealed that Prx3 reduces H 2 O 2 , utilizing glutaredoxin 3 (Grx3) and glutathione (GSH) as reductants, and requires only N-terminal peroxidatic cysteine for its catalysis. These results, combined with the monomeric size of Prx3 observed under non-reducing conditions, suggested that Prx3 is a Grx3/GSH-dependent 1-Cys Prx and oxidized without forming intermolecular disulfide bonds. The prx3 mutation impaired growth in the medium containing peroxides and reduced virulence in mice, indicating that Prx3 is essential for survival under oxidative stress and pathogenesis of V. vulnificus . The Fe-S cluster regulator IscR activates prx3 by direct binding to a specific binding sequence centered at −44 from the transcription start site. The binding sequence was homologous to the Type 2 IscR-binding sequence, most likely recognized by the Fe-S clusterless apo-IscR in Escherichia coli . The iscR 3CA mutant, chromosomally encoding the apo-locked IscR, exhibited 3-fold higher levels of activation of prx3 than the wild type and accumulated more IscR 3CA protein in cells. The IscR-dependent activation of prx3 by aerobic growth and iron starvation was also associated with the increase in cellular levels of IscR protein. Taken together, the results suggested that IscR senses iron starvation as well as reactive oxygen species and shifts to the apo-form, which leads to the increase of cellular IscR and in turn prx3 expression, contributing to the survival and virulence of V. vulnificus during pathogenesis.ROS and iron availability influence [2Fe-2S] cluster occupancy in IscR. Results Prx3 is a Grx3/glutathione-dependent 1-Cys peroxiredoxin essential for survival under oxidative stress and pathogenesis of Vibrio vulnificus , and IscR directly activates prx3 by sensing ROS and iron starvation. Conclusion IscR-dependent prx3 expression contributes to the pathogenesis of V. vulnificus . Significance This study elucidated the IscR-mediated regulation of an antioxidant enzyme.