No abstract

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.

Recently, the prevalence of Aeromonas hydrophila antibiotic-resistant strains has been reported in aquaculture, but its intrinsic antibiotic resistance mechanisms are largely unknown. In the present study, a label-free proteomics technology was used to compare the differential protein abundances in response to norfloxacin (NOR) stress in A. hydrophila. The results showed that there were 186 proteins decreasing and 220 proteins increasing abundances in response to NOR stress. Bioinformatics analysis showed that the differentially expressed proteins were enriched in several biological processes, such as sulfur metabolism and homologous recombination. Furthermore, the antibiotic sensitivity assays showed that the deletion of AHA_0904, cirA, and cysI significantly decreased the resistance against NOR, whereas ΔAHA_1239, ΔcysA, ΔcysD, and ΔcysN significantly increased the resistance against NOR. Our results provide insights into NOR resistance mechanisms and indicate that AHA_0904, cirA, AHA_1239, and sulfur metabolism may play important roles in NOR resistance in A. hydrophila.

Bacterial antibiotic resistance is a serious global problem; the underlying regulatory mechanisms are largely elusive. The earlier reports states that the vital role of transcriptional regulators (TRs) in bacterial antibiotic resistance. Therefore, we have investigated the role of TRs on enoxacin (ENX) resistance in Aeromonas hydrophila in this study. A label-free quantitative proteomics method was utilized to compare the protein profiles of the ahslyA knockout and wild-type A. hydrophila strains under ENX stress. Bioinformatics analysis showed that the deletion of ahslyA triggers the up-regulated expression of some vital antibiotic resistance proteins in A. hydrophila upon ENX stress and thereby reduce the pressure by preventing the activation of SOS repair system. Moreover, ahslyA directly or indirectly induced at least 11 TRs, which indicates a complicated regulatory network under ENX stress. We also deleted six selected genes in A. hydrophila that altered in proteomics data in order to evaluate their roles in ENX stress. Our results showed that genes such as AHA_0655, narQ, AHA_3721, AHA_2114, and AHA_1239 are regulated by ahslyA and may be involved in ENX resistance. Overall, our data demonstrated the important role of ahslyA in ENX resistance and provided novel insights into the effects of transcriptional regulation on antibiotic resistance in bacteria.

The colicin I receptor (CirA) is a well‐studied outer membrane protein that has been reported to play important roles in antibiotic resistance, virulence, and iron homeostasis, although its exact physiological roles require further investigation. In this study, differentially expressed proteins between the ΔahcirA and wild‐type (WT) strains of Aeromonas hydrophila were compared using quantitative proteomics. Bioinformatics analysis revealed that the expression of peptide, histidine, and arginine ATP‐binding cassette (ABC) transporter system‐related proteins was significantly higher in the ΔahcirA strain. Subsequent growth assays revealed that ΔahcirA grew slower than the WT strain in nutrient‐limited medium when supplemented with dipeptide, histidine, and arginine as the carbon source. Far‐western blot analysis further confirmed that AhCirA can directly bind to histidine/arginine and dipeptide small‐molecule substrates in addition to their periplasmic‐binding proteins, AhDppA and AhHisJ, respectively. These results indicate that AhCirA may play an important role in the uptake of amino acids and peptides as a channel‐forming porin while also directly interacting with ABC transporters to transport nutrient substances into the plasma membrane. Overall, this study demonstrates that AhCirA is a multifunctional protein in A. hydrophila and extends our understanding of known nutrient transport mechanisms among bacteria.