Quantitative Proteomics Reveals Antibiotics Resistance Function of Outer Membrane Proteins in Aeromonas hydrophila

Abstract

Outer membrane proteins (OMPs) play essential roles in antibiotic resistance, particularly in Gram-negative bacteria; however, they still have many unidentified functions regarding their behavior in response to antibiotic stress. In the current work, quantitative tandem mass tag labeling-based mass spectrometry was used to compare the outer membrane related proteins between an oxytetracycline-resistant (OXY-R) and its original control stain (OXY-O) in Aeromonas hydrophila. Consequently, a total of 261 commonly altered proteins in two biological repeats were identified including 29 proteins that increased and 28 that decreased. Gene ontology analysis showed that the expression of transport proteins was significantly reduced, and translation-related proteins were downregulated in the OXY-R strain. After using western blotting to validate selected altered proteins, eight OMP-related genes were knocked out and their roles in antibiotic resistance were further evaluated. The survival assays showed that some mutants such as ΔAHA_4281, ΔAHA_2766, ΔAHA_2282, ΔAHA_1181, and ΔAHA_1280 affected the susceptibility of A. hydrophila to antimicrobials. Moreover, the minimum inhibitory concentration assay showed that these candidate mutants also respond differently to other types of antibiotics. Our results reveal several novel outer membrane related proteins of A. hydrophila that play important roles in antibiotic resistance, and as such, may be helpful for screening studies to identify novel drug targets.

The characteristics of antibiotic resistance and phenotypes in 29 outer‐membrane protein mutant strains in Aeromonas hydrophila

“…In addition, the antibiotic susceptibilities of five mutants (ΔbamA, ΔAHA_2282, ΔAHA_2766, ΔAHA_1280 and ΔAHA_2699) have been tested in our previous research to nine antibiotics using the liquid dilution MICs assay (Yao et al, 2018). In the present study, we measured their MBCs to other 11 antibiotics except for those antibiotics.…”

“…The A. hydrophila ATCC 7966, E. coli MC1061, E. coli S17-1 and pRE112 plasmid used in this study were kept in our laboratory. The ΔbamA, ΔAHA_2282, ΔAHA_2766, ΔAHA_1280 and ΔAHA_2699 knocked out strains were constructed for our previous work and kept in our laboratory as well (Yao et al, 2018). Mutants were grown in LB medium overnight at 200 rpm, 30 C, and then diluted at 1:100 in fresh LB medium.…”

“…The colonies that grew on the sucrose LB agar plate but were sensitive to chloramphenicol were further verified by PCR and sequencing (Yao et al, 2018). The primer sequences used in this study are provided in Supporting Information Table S1, and the target genes information, protein descriptions and their homology comparison with E. coli or other bacterial species are shown in Table 1.…”

“…hydrophila (Lin et al ., ). The minimum inhibitory concentration (MIC) assay against nine antibiotics showed that at least four OMP mutants, namely AHA_1280 , AHA_2699 , AHA_4281 and AHA_2766 , affect the bacterial antibiotic resistance (Yao et al ., ). However, the physiological functions of OMPs in A .…”

“…In our previous study, several altered OMPs under oxytetracycline stress were quantified in A. hydrophila . The minimum inhibitory concentration (MIC) assay against nine antibiotics showed that at least four OMP mutants, namely AHA_1280, AHA_2699, AHA_4281 and AHA_2766, affect the bacterial antibiotic resistance (Yao et al, 2018). However, the physiological functions of OMPs in A. hydrophila are still largely unknown, especially for their roles in antibiotic resistance.…”