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

Abstract

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.

Keywords:

• Pseudomonas aeruginosa PAO1
• disulfide bond proteins
• oxidative stress
• thiol affinity purification method
• hydrogen peroxide
• gamma ray

Acknowledgements

This project has been carried out under the Nuclear R&D Program by the Ministry of Science and Technology, Republic of Korea.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.