Boosting Synergistic Effects of Short Antimicrobial Peptides With Conventional Antibiotics Against Resistant Bacteria

Abstract

The global spread of antibiotic-resistant infections has meant that there is an urgent need to develop new antimicrobial alternatives. In this study, we developed a strategy to boost and/or synergize the activity of conventional antibiotics by combination with antimicrobial peptides tagged with the bulky non-natural amino acid β-naphthylalanine (Nal) to their N- or C-terminus. A checkerboard method was used to evaluate synergistic effects of the parent peptide and the Nal-tagged peptides. Moreover, boron-dipyrro-methene labeled vancomycin was used to characterize the synergistic mechanism of action between the peptides and vancomycin on the bacterial strains. These Nal-tagged antimicrobial peptides also reduced the antibiotic-induced release of lipopolysaccharide from Gram-negative bacteria by more than 99.95%. Our results demonstrate that Nal-tagged peptides could help in developing antimicrobial peptides that not only have enhanced antibacterial activities but also increase the synergistic effects with conventional antibiotics against antibiotic-resistant bacteria.

Keywords: end-tagging; antibiotic resistance; antimicrobial peptides (AMP); bulky non-natural amino acid; synergism.

FIGURE 1

The membrane permeabilization of Enterococcus faecium BCRC 15B0132, Acinetobacter baumannii BCRC 14B0091, A. baumannii BCRC 14B0097, A. baumannii BCRC 14B0100, Escherichia coli BCRC 13B0198, and E. coli BCRC 13B0207, by S1, S1-Nal, and S1-Nal-Nal for 60 min exposure. Calcein-AM loaded cells (10⁷ CFU/ml) were resuspended by PBS, and the aliquots of 100 μl were added into a sterile black-wall 96-well plate, then treated with 0.5 × MIC of peptides in each well and measured the calcein leakage immediately. Each time point for the peptides was repeated three times independently.

FIGURE 2

S-Nal and S1-Nal-Nal increase the uptake of boron-dipyrro-methene (BODIPY)-labeled vancomycin in E. faecium. (A) Fluorescence images of 10⁷ CFU/ml E. faecium BCRC 15B0132 treated with 0.5 × MIC of S1, S1-Nal and S1-Nal-Nal at 37°C for 30 min, then treated BODIPY-labeled vancomycin for 30 min (scale bar represents 5 μm). (B) Mean fluorescence intensity of S1, S1-Nal, and S1-Nal-Nal treated in E. faecium BCRC 15B0132. Samples treated with BODIPY-labeled vancomycin only served as a control. Results are presented as means ± SEM, ***P < 0.001 compared with control. n.s., no significant differences.

FIGURE 3

S1-Nal and S1-Nal-Nal increase the uptake of BODIPY-labeled vancomycin in A. baumannii. (A) Fluorescence images of 10⁷ CFU/ml A. baumannii BCRC 14B0097 treated with 0.5 × MIC of S1, S1-Nal, and S1-Nal-Nal at 37°C for 30 min, then treated BODIPY-labeled vancomycin for 30 min (scale bar represents 5 μm). (B) Mean fluorescence intensity of S1, S1-Nal, and S1-Nal-Nal treated in A. baumannii BCRC 14B0097. Samples treated with BODIPY-labeled vancomycin only served as a control. Results are presented as means ± SEM, ***P < 0.001 compared with control. n.s., no significant differences.

FIGURE 4

S1-Nal and S1-Nal-Nal increase the uptake of BODIPY-labeled vancomycin in E. coli. (A) Fluorescence images of 10⁷ CFU/ml E. coli BCRC 13B0207 treated with 0.5 × MIC of S1, S1-Nal, and S1-Nal-Nal at 37°C for 30 min, then treated BODIPY-labeled vancomycin for 30 mins (scale bar represents 5 μm). (B) Mean fluorescence intensity of S1, S1-Nal, and S1-Nal-Nal treated in E. coli BCRC 13B0207. Samples treated with BODIPY-labeled vancomycin only served as a control. Results are presented as means ± SEM, ***P < 0.001 compared with control.

FIGURE 5

Attenuation of LPS release from E. coli. Escherichia coli BCRC 13B0198 were incubated at the mid-log phase (10⁴ CFU/ml) and treated with peptide alone (at 1 × MIC) or combination with vancomycin (both at 0.5 × MIC) at 37°C for 6 h. The samples were filtered through 0.2 μm pore filter and the endotoxin level was detected by LAL assay. ***P < 0.001 compared with vancomycin only. ns, no significant differences compared with vancomycin only.

FIGURE 6

Schematic illustration of the synergistic mechanism for BODIPY-labeled vancomycin and peptides. S1-Nal-Nal (red) disturbs the bacterial outer membrane and enhances the entry of BODIPY-labeled vancomycin (green circle) into bacteria.