An Integrated Quantitative and Targeted Proteomics Reveals Fitness Mechanisms of Aeromonas hydrophila under Oxytetracycline Stress

To date, above ten thousand tons of antibiotics are used in aquaculture each year that lead to the deterioration of natural resources. However, knowledge is limited on the molecular biological behavior of common aquatic pathogens against antibiotics stress. In this study, proteomics profiles of Aeromonas hydrophila, which were exposed to different levels of oxytetracycline (OXY) stress, were displayed and compared using iTRAQ labeling and SWATH-MS based LC–MS/MS methods. A total 1383 proteins were identified by SWATH-MS method, and 2779 proteins were identified from iTRAQ labeling samples. There are 152 up-regulated and 52 down-regulated proteins overlapped in 5 μg/mL OXY stress and both 83 up- and down-regulated proteins overlapped in 10 μg/mL OXY stress in both methods, respectively. Results show that many protein synthesis and translation related proteins increased, while energy generation related proteins decreased in OXY stress. The varieties of selected proteins involved in both pathways were further validated by sMRMHR, q-PCR, and enzyme activity assay. Furthermore, the concentrations of NAD+ and NADH were measured to verify the characteristic of energy generation process in OXY stress and OXY resistance strain. We demonstrate that the down-regulation of energy generation related metabolic pathways and up-regulation of translation may play an important role in antibiotics fitness or resistance of aquatic pathogens.