Bacteria have evolved distinct molecular mechanisms as a defense against oxidative stress. The foremost regulator of oxidative stress response has been found to be OxyR. However, the molecular details of regulation upstream of OxyR remain largely unknown and need further investigation. Here, we characterize a oxidant stress and antibiotic tolerance regulator, OsaR (PA0056), produced by Pseudomonas aeruginosa. Mutation of osaR increased bacterial tolerance to aminoglycoside and beta-lactam antibiotics, as well as to hydrogen peroxide. Expression of the oxyR regulon genes oxyR, katAB, and ahpBCF was increased in the osaR mutant. However, the OsaR protein does not regulate the oxyR regulon genes through direct binding to their promoters. PA0055, osaR, PA0057 and dsbM are in the same gene cluster, and we provide evidence that expression of these genes involved in oxidant tolerance is controlled by binding of OsaR to intergenic region between osaR and PA0057, which contain two divergent promoters. The gene cluster is also regulated by PA0055 via an indirect effect. We further discovered that OsaR formed intramolecular disulfide bonds when exposed to oxidative stress, resulting in a change of its DNA binding affinity. Taken together, our results indicate that OsaR is inactivated by oxidative stress and plays a role in the tolerance of P. aeruginosa to aminoglycoside and beta-lactam antibiotics.
IMPORTANCE
As opportunistic pathogen, Pseudomonas aeruginosa can cause serious infections which are hard to eradicate because of antibiotic resistance in immunodeficient patients. We found that OsaR is involved in oxidative stress and antibiotics resistance by regulation of downstream genes via redox state change. Research on factors affecting the transcriptional level of oxyR is very limited, but important since it has implications on antibiotic resistance. In this study, it was found that OsaR can indirectly inhibit transcription of oxyR. In addition the gene cluster composed of PA0055, osaR, PA0057 and dsbM was identified, and the associated regulatory mechanisms and functions were elucidated. Our work not only provides a mechanistic understanding of antibiotic tolerance regulation in P. aeruginosa, but also has significant implications for redox regulation in human pathogens in general.
Redox protein OsaR (PA0056) regulates dsbM and the oxidative stress response in Pseudomonas aeruginosa
