In Gram-negative bacteria, the outer membrane proteins (OMPs) perform a crucial role in antibiotic resistance, but it is largely unknown how they behave in response to antibiotic stress. In this study, we treated Aeromonas hydrophila with two different doses of oxytetracycline (OXY) to induce antibiotic stress. Proteins were isolated from sarcosine-insoluble fractions and quantitatively examined by using tandem mass tag labeling-based mass spectrometry to identify differentially expressed proteins. As a result, we identified 125 differential proteins in the 5 μg/ml OXY treatment group, including 20 OMPs, and 150 proteins from the 10 μg/ml OXY group, including 22 OMPs. Gene ontology analysis showed that translation-related proteins, including 30S and 50S ribosome proteins, were significantly enriched in increasing abundance under OXY stress; whereas the downregulated proteins were associated with the transport process, such as maltodextrin, maltose, and oligosaccharide transport. We then validated a subset of the identified differential proteins by using Western blot and quantitative polymerase chain reaction analyses. Finally, the quantitative real-time PCR (qPCR) results showed that at the transcription level, the expression of five OMP genes, including AHA_1280 (protein name A0KHS0), AHA_1281 (A0KHS1), AHA_1447 (A0KI84, BamE), AHA_1861 (A0KJE1), and AHA_2766 (A0KLX3), and one lipoprotein gene AHA_1740 (A0KJ25) was consistent with proteomic results under 5 and 10 μg/ml OXY treatment, respectively. In addition, the Western blotting also demonstrated that two altered OMP proteins A0KHS1 and A0KHH2 were upregulated for both OXY treatment groups. This study indicates that bacteria regulate the expression levels of OMPs in response to antibiotic stress and further contribute to our understanding of the functions of OMPs in antibiotic resistance. Moreover, our results suggest that the upregulation of translation and downregulation of the transport process may affect bacterial fitness during OXY stress. These findings may provide new clues to the antibiotic resistance mechanism in A. hydrophila.
Keywords: Aeromonas hydrophila; antibiotic stress; outer membrane protein; quantitative proteomics.