Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15–34), antimicrobial peptides from rattlesnake venom

Abstract

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of and cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.

Structural determinants conferring unusual long life in human serum to rattlesnake‐derived antimicrobial peptide Ctn[15‐34]

“…For Ctn [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34], the simulations show an N-terminal partial α-helix (residues Lys 1 -Phe 8 /Lys 9 ), in good agreement with previously reported NMR data (α-helical region between Lys 2 -Ile 7 ). 33 For the scrambled peptide, a beta-hairpin motif is predicted in PEP-FOLD3 models 1 [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] and analogs in human serum obtained from chromatogram peak integration as the ones represented in (B). The t 1/2 s estimated by experimental data fitting to an exponential decay model are reported as the mean of three experiments and the corresponding 95% CI.…”

“…Sequences of Ctn [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] and analogs were submitted to the PSIPRED v3.3 (http://bioinf.cs.ucl.ac.uk/psipred/) 38 web server to predict secondary structure propensity. For 3D structure prediction, the PEP-FOLD3 web server (http://bioserv.rpbs.univ-paris-diderot.fr/services/ PEP-FOLD3/#usage) 39 was accessed, and the best five models (lowest OPEP [optimized potential for efficient structure prediction] energy) were superposed and represented using the PyMOL Molecular Graphical System, Version 2.0.…”

“…Experimental mass was calculated from the MS spectra on Figure S1b. Purity was calculated by peak integration of the RP-HPLC chromatograms on Figure S1a. 2.8 | 1-Anilino-8-naphthalenesulfonate (ANS) fluorescence studies ANS at 12.8μM was incubated with Ctn [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] concentrations in the 50-to 500-μM range for 10 minutes. ANS fluorescence emission was detected as previously described.…”

“…After incubation, the minimal inhibitory concentration (MIC) of the peptide-serum mixture against Escherichia coli ATCC 25922 was determined by the broth microdilution method as previously described. 34 3 | RESULTS AND DISCUSSION…”

Membrane Active Peptides and Their Biophysical Characterization

“…Particularly, it can be used to get information on the kinetics of binding. Different studies have used SPR to study interactions of membrane active peptides with model membranes [225][226][227][228].…”