Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15-34), antimicrobial peptides from rattlesnake venom.
Clara Pérez-Peinado, Susana Almeida Dias, Marco M Domingues, Aurélie H Benfield, João Miguel Freire, Gandhi Rádis-Baptista, Diana Gaspar, Miguel A R B Castanho, David J Craik, Sónia Troeira Henriques, Ana Salomé Veiga, David Andreu
Author Information
Clara Pérez-Peinado: From the Department of Experimental and Health Science, Universitat Pompeu Fabra, Barcelona Biomedical Research Park, 08003 Barcelona, Spain.
Susana Almeida Dias: the Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Marco M Domingues: the Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Aurélie H Benfield: the Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.
João Miguel Freire: the Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Gandhi Rádis-Baptista: From the Department of Experimental and Health Science, Universitat Pompeu Fabra, Barcelona Biomedical Research Park, 08003 Barcelona, Spain.
Diana Gaspar: the Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Miguel A R B Castanho: the Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
David J Craik: the Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.
Sónia Troeira Henriques: the Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia, s.henriques@imb.uq.edu.au.
Ana Salomé Veiga: the Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal, aveiga@medicina.ulisboa.pt.
David Andreu: From the Department of Experimental and Health Science, Universitat Pompeu Fabra, Barcelona Biomedical Research Park, 08003 Barcelona, Spain, david.andreu@upf.edu.
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.
