Global analysis of disulfide bond proteins in Pseudomonas aeruginosa exposed to hydrogen peroxide and gamma rays

Purpose:To isolate and characterise the oxidative stress-responsible disulfide bond proteins (DSBP) from Pseudomonas aeruginosa, under hydrogen peroxide (H2O2) and gamma ray-induced stress conditions. Materials and methods:Oxidative stress in P. aeruginosa was induced by H2O2 or ⁶⁰Co γ-rays. DSBP of P. aeruginosa were isolated by using a thiol affinity purification method and liquid chromatography-mass spectrometry (LC-MS/MS). Induction of DSBP and their transcriptional levels were analysed by Western blot and quantitative-polymerase chain reaction (q-PCR), respectively. Results:We isolated and identified 40 DSBP in P. aeruginosa. A comparison of DSBP patterns under different stress conditions, including exposure to 0.5 mM H2O2 and 30 Gy of gamma rays, was carried out. We selected 13 DSBP based on their increased intensity under oxidative stress and investigated their expression levels using q-PCR. Of these 13 proteins, P. aeruginosa (PA) 0126 and PA0277 transcriptional levels were strongly induced (4- to 9-fold) by gamma ray. A corresponding induction in the translational levels of PA0126 was confirmed by Western blot analysis. PA0126 was identified is a hypothetical protein with 206 amino acids and contained three cysteine residues. A shift in molecular weight of PA0126 under oxidised/reduced state indicated its homo-oligomeric structure under non-reducing condition. Conclusion:PA0126, a hypothetical protein with disulfide bonds was identified as a key responder to oxidative stress along with other known DSBP of P. aeruginosa. We determined that all DSBP underwent a redox cycling. Furthermore, our results give deeper insight into the relationship between oxidative stress and DSBP in P. aeruginosa.