Addition of L-cysteine to the N- or C-terminus of the all-D-enantiomer [(KLAKLAK)] increases antimicrobial activities against multidrug-resistant and .

BACKGROUND

Antimicrobial peptides have a broad spectrum of antimicrobial activities and are attracting attention as promising next-generation antibiotics against multidrug-resistant (MDR) bacteria. The all-d-enantiomer [(KLAKLAK)] has been reported to have antimicrobial activity against and , and to be resistant to protein degradation in bacteria because it is composed of D-enantiomer compounds. In this study, we demonstrated that modification of [(KLAKLAK)] by the addition of an L-cysteine residue to its N- or C- terminus markedly enhanced its antimicrobial activities against Gram-negative bacteria such as MDR , , and .

METHODS

The peptides [(KLAKLAK)] (DP), DP to which L-cysteine was added at the N-terminus C-DP, and DP to which L-cysteine was added at the C-terminus DP-C, were synthesized at >95% purity. The minimum inhibitory concentrations of peptides and antibiotics were determined by the broth microdilution method. The synergistic effects of the peptides and the antibiotics against MDR were evaluated using the checkerboard dilution method. In order to assess how these peptides affect the survival of human cells, cell viability was determined using a Cell Counting Kit-8.

RESULTS

C-DP and DP-C enhanced the antimicrobial activities of the peptide against MDR Gram-negative bacteria, including , and . The antimicrobial activity of DP-C was greater than that of C-DP, with these peptides also having antimicrobial activity against drug-susceptible and drug-resistant overexpressing the efflux pump components. C-DP and DP-C also showed antimicrobial activity against colistin-resistant harboring , which encodes a lipid A modifying enzyme. DP-C showed synergistic antimicrobial activity against MDR when combined with colistin. The LD of DP-C against a human cell line HepG2 was six times higher than the MIC of DP-C against MDR The LD of DP-C was not altered by incubation with low-dose colistin.

CONCLUSION

Attachment of an L-cysteine residue to the N- or C-terminus of [(KLAKLAK)] enhanced its antimicrobial activity against , , and . The combination of C-DP or DP-C and colistin had synergistic effects against MDR . In addition, DP-C and C-DP showed much stronger antimicrobial activity against MDR and than against .