IADR Abstract Archives
Bactericidal Activity and In Vitro/Vivo Toxicity of Antimicrobial Peptide D-GL13K
Objectives: GL13K is an antimicrobial peptide derived from the parotid secretory protein BPIFA2. D-GL13K, but not L-GL13K, is active against the oral bacteria Streptococcus gordonii and Porphyromonas gingivalis. Here we tested the bactericidal activity of GL13K enantiomers against additional Gram negative (Pseudomonas aeruginosa) and Gram positive (Staphylococcus aureus) species and evaluated the toxicity of GL13K against human cells and the model organism Galleria mellonella (wax worms).
Methods: Minimum inhibitory concentrations (MIC) were determined in a standard 96-well plate assay. Minimum bactericidal concentrations (MBC) were determined by culturing MIC-assay aliquots. In vitro toxicity was determined by quantitating LDH-release from HEK-blue cells after incubation with serial peptide dilutions. In vivo toxicity testing of D-GL13K was conducted using Galleria mellonella (waxworm) as a model organism. Mortality was determined after overnight incubation with the peptides by melanization and lack of response upon probing.
Results: D-GL13K generally shows a lower MIC than the L-enantiomer. In S. gordonii, the difference was abolished by mutation of dltA, which is responsible for D-alanylation of the cell wall. In both S. aureus and P. aeruginosa, the MIC was identical to the MBC, i.e. the peptides are bactericidal. Both peptide enantiomers caused moderate cytotoxicity in HEK cells, which reached 25% at 500 µg/ml. Thus the toxic concentration (LD25) was 100x the MIC for D-GL13K. In the wax worm G. Mellonella, 50% lethal dose was reached at 33 µg/g body weight. This represents 6 x MIC.
Conclusions: The D-enantiomer of GL13K is bactericidal to Gram-negative and Gram-positive bacteria at low µg/ml concentrations. The differences between L-GL13K and D-GL13K suggest that, in addition to proteolytic resistance, D-GL13K may overcome D-alanylation in Gram-positive bacteria. The peptides showed only low toxicity in a cell culture model while G. mellonella showed higher toxicity. The latter may result from problems with injection technique and volume.
Methods: Minimum inhibitory concentrations (MIC) were determined in a standard 96-well plate assay. Minimum bactericidal concentrations (MBC) were determined by culturing MIC-assay aliquots. In vitro toxicity was determined by quantitating LDH-release from HEK-blue cells after incubation with serial peptide dilutions. In vivo toxicity testing of D-GL13K was conducted using Galleria mellonella (waxworm) as a model organism. Mortality was determined after overnight incubation with the peptides by melanization and lack of response upon probing.
Results: D-GL13K generally shows a lower MIC than the L-enantiomer. In S. gordonii, the difference was abolished by mutation of dltA, which is responsible for D-alanylation of the cell wall. In both S. aureus and P. aeruginosa, the MIC was identical to the MBC, i.e. the peptides are bactericidal. Both peptide enantiomers caused moderate cytotoxicity in HEK cells, which reached 25% at 500 µg/ml. Thus the toxic concentration (LD25) was 100x the MIC for D-GL13K. In the wax worm G. Mellonella, 50% lethal dose was reached at 33 µg/g body weight. This represents 6 x MIC.
Conclusions: The D-enantiomer of GL13K is bactericidal to Gram-negative and Gram-positive bacteria at low µg/ml concentrations. The differences between L-GL13K and D-GL13K suggest that, in addition to proteolytic resistance, D-GL13K may overcome D-alanylation in Gram-positive bacteria. The peptides showed only low toxicity in a cell culture model while G. mellonella showed higher toxicity. The latter may result from problems with injection technique and volume.