The Rhodamine B-encrypted Vipericidin Peptide, RhoB-Ctn[1-9], Displays In vitro Antimicrobial Activity Against Opportunistic Bacteria and Yeasts

Background: Crotalicidin (Ctn), a snake venom cathelicidin-related antimicrobial peptide, is a 34-residue-long linear lysine-rich vipericidin obtained from the South American rattlesnake, Crotalus durissus terrificus. Ctn contains tandem repeats of nine amino acid residues (1KRFKKFFKK9 and 16KRLKKIFKK24; consensus: (1)KRhKKhFKK(9), h = hydrophobic amino acid) as an integral part of its structure. Objective: The aim of this study was to evaluate the antimicrobial activity of the encrypted vipericidin nonapeptide KRFKKFFKK, designated as Ctn[1-9], and its structural analogue, rhodamineB-conjugated Ctn[1-9], designated as RhoB-Ctn[1-9]. Method: The susceptibility of representative pathogenic bacteria and yeasts to antimicrobial agents was determined using the broth microdilution minimum inhibitory concentration (MIC) method. Cytotoxicity was estimated using a hemolytic assay. The accumulation of RhoB-Ctn[1-9] in microbial cells was observed by fluorescence microscopy. The antimicrobial synergism of RhoB-Ctn[1-9] with antimicrobials was evaluated using a checkerboard analysis. Results: RhoB-conjugated Ctn[1-9] displayed selective antimicrobial activity against infectious gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and pathogenic species of Candida with low hemolytic effects on human erythrocytes which were not observed with unconjugated Ctn[1-9]. RhoB-Ctn[1-9] could permeate cell membranes and accumulate intracellularly in microbial cells. RhoB-Ctn[1-9] exhibits synergistic effects when used with antibiotics or antifungal agents and reduced the MICs of the peptide and antimicrobials. Conclusion: These findings indicate the potential of crotalicidin-related short peptides as structural motifs for the diversification of biological functionalities. Further, they set the stage to investigate the molecular mechanisms by which chemically modified vipericidin repeats modulate cell fate.