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The poor stability of antibacterial peptide to protease limits its clinical application. Among these limitations, trypsin mainly exists in digestive tract, which is an insurmountable obstacle to orally delivered peptides. OM19R is a random curly polyproline cationic antimicrobial peptide, which has high antibacterial activity against some gram-negative bacteria, but its stability against pancreatin is poor. According to the structure-activity relationship of OM19R, all cationic amino acid residues (l-arginine and l-lysine) at the trypsin cleavage sites were replaced with corresponding d-amino acid residues to obtain the designed peptide OM19D, which not only maintained its antibacterial activity but also enhanced the stability of trypsin. Proceeding high concentrations of trypsin and long-time (such as 10 mg/mL, 8 h) treatment, it still had high antibacterial activity (MIC = 16–32 µg/mL). In addition, OM19D also showed high stability to serum, plasma and other environmental factors. It is similar to its parent peptide in secondary structure and mechanism of action. Therefore, this strategy is beneficial to improve the protease stability of antibacterial peptides.

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IntroductionThe continued emergence and spread of multidrug-resistant (MDR) bacterial pathogens require a new strategy to improve the efficacy of existing antibiotics. Proline-rich antimicrobial peptides (PrAMPs) could also be used as antibacterial synergists due to their unique mechanism of action.MethodsUtilizing a series of experiments on membrane permeability, In vitro protein synthesis, In vitro transcription and mRNA translation, to further elucidate the synergistic mechanism of OM19r combined with gentamicin.ResultsA proline-rich antimicrobial peptide OM19r was identified in this study and its efficacy against Escherichia coli B2 (E. coli B2) was evaluated on multiple aspects. OM19r increased antibacterial activity of gentamicin against multidrug-resistance E. coli B2 by 64 folds, when used in combination with aminoglycoside antibiotics. Mechanistically, OM19r induced change of inner membrane permeability and inhibited translational elongation of protein synthesis by entering to E. coli B2 via intimal transporter SbmA. OM19r also facilitated the accumulation of intracellular reactive oxygen species (ROS). In animal models, OM19r significantly improved the efficacy of gentamicin against E. coli B2.DiscussionOur study reveals that OM19r combined with GEN had a strong synergistic inhibitory effect against multi-drug resistant E. coli B2. OM19r and GEN inhibited translation elongation and initiation, respectively, and ultimately affected the normal protein synthesis of bacteria. These findings provide a potential therapeutic option against multidrug-resistant E. coli.