Quantitative Proteomics Reveals That the Protein Components of Outer Membrane Vesicles (OMVs) in Aeromonas hydrophila Play Protective Roles in Antibiotic Resistance

In recent years, the intracellular mechanisms that contribute to antibiotic resistance have received increasing attention, and outer membrane vesicles (OMVs) have been reported to be related to antibiotic resistance in several Gram-negative bacterial species. However, the intrinsic molecular mechanisms and the form of such antibiotic resistance are still largely unknown. In this study, OMVs from an oxytetracycline (OXY) sensitive aquatic pathogen, Aeromonas hydrophila (OXY-S), were found with significantly increased OXY resistance. Interestingly, the OXY-resistant strain (OXY-R) had a more protective role in OXY resistance. Therefore, a DIA-based quantitative proteomics analysis was performed to compare the differential expression of OMV proteins between OXY-R (OMVsR) and OXY-S (OMVsS). The results showed that seven proteins increased and five proteins decreased in OMVsR vs OMVsS. A subsequent antibiotics susceptibility assay showed that the deletion of icd, rpsF, and iscS significantly increased OXY sensitivity. Moreover, the exogenous addition of the crude OMV fractions of overexpressed recombinant proteins in E. coli with rRpsF, rIcd, rIscS, rOmpA, rPepA, rFrdA, and rRplQ demonstrated that these proteins promoted the OXY resistance of A. hydrophila. Overall, our results indicate the important protective role of OMVs in antibiotic resistance in A. hydrophila and provide novel insights on bacterial antibiotic resistance mechanisms.