Cu2+ regulated sulfonamides resistance gene (sul) via reactive oxygen species induced ArcA in a pathogenic Vibrio splendidus

Abstract

To detect the sulfonamide resistance of Vibrio splendidus strain Vs, and characterize the factors that regulate the expression of sulfonamide resistance gene sulVs. Minimal inhibitory concentration was measured using growth inhibition method, gene expression was measured using real-time reverse transcriptase PCR, reactive oxygen species (ROS) production under Cu2+ or Cu2+/NAC conditions was measured using an excitation wavelength of 485 nm, and an emission wavelength of 525 nm, and the binding of sulVs promoter and the transcriptional factor ArcA was measured using electrophoretic mobility shift assay (EMSA). sulVs gene was cloned and its expression was upregulated to 6.98- and 3.57-fold in the presence of sulfadiazine and sulfamethoxazole, respectively. Moreover, Cu2+ could also upregulate the expression of sulVs to 14.27-fold, the production of ROS to 4.37-fold, and the expressions of antioxidant-related genes and a transcriptional regulator arcA gene. After addition of N-Acetyl-L-cysteine, a ROS inhibitor, the production of ROS decreased to 53.1%, and the expression of arcA gene was also downregulated to 26%. EMSA showed that the purified recombinant ArcA could directly bind to the promoter region of sulVs with specificity. V. splendidus strain Vs showed sulfonamide resistance due to sulVs, and Cu2+ could increase the level of ROS followed by ArcA activation as a transcriptional factor to increase the expression of sulVs.