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

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• dc.contributor.author Pérez-Peinado, Clara, 1991-
• dc.contributor.author Almeida Dias, Susana
• dc.contributor.author Domingues, Marco M.
• dc.contributor.author Benfield, Aurélie H.
• dc.contributor.author Miguel Freire, João
• dc.contributor.author Rádis-Baptista, Gandhi
• dc.contributor.author Gaspar, Diana
• dc.contributor.author Castanho, Miguel A.R.B.
• dc.contributor.author Craik, David J.
• dc.contributor.author Troeira Henriques, Sónia
• dc.contributor.author Salomé Veiga, Ana
• dc.contributor.author Andreu Martínez, David
• dc.date.accessioned 2022-10-18T08:48:49Z
• dc.date.available 2022-10-18T08:48:49Z
• dc.date.issued 2018
• dc.description.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 Escherichia coli and Pseudomonas aeruginosa 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.
• dc.description.sponsorship This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) Grants SAF2011-24899 and AGL2014-52395-C2, by Fundação para a Ciência e a Tecnologia (FCT, Portugal) Grants PTDC/QEQ-MED/4412/2014, and by EU Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) program Grant 644167, 2015–2019. The authors declare that they have no conflicts of interest with the contents of this article.
• dc.format.mimetype application/pdf
• dc.identifier.citation Pérez-Peinado C, Dias SA, Domingues MM, Benfield AH, Freire JM, Rádis-Baptista G, et al. Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15–34), antimicrobial peptides from rattlesnake venom. J. Biol. Chem. 2018 Feb;293(5):1536-49. DOI: 10.1074/jbc.ra117.000125
• dc.identifier.doi http://dx.doi.org/10.1074/jbc.ra117.000125
• dc.identifier.issn 0021-9258
• dc.identifier.uri http://hdl.handle.net/10230/54461
• dc.language.iso eng
• dc.publisher American Society for Biochemistry and Molecular Biology (ASBMB)
• dc.relation.ispartof Journal of Biological Chemistry. 2018 Feb;293(5):1536-49
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/644167
• dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/SAF2011-24899
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/AGL2014-52395-C2
• dc.rights This is an Open Access article under the CC BY license.
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.keyword Gram-negative bacteria
• dc.subject.keyword Antimicrobial peptide (AMP)
• dc.subject.keyword Atomic force microscopy (AFM)
• dc.subject.keyword Bacterial membrane disruption
• dc.subject.keyword Bactericidal mechanism
• dc.subject.keyword Confocal microscopy
• dc.subject.keyword Surface plasmon resonance (SPR)
• dc.subject.keyword Time-resolved flow cytometry
• dc.title Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15–34), antimicrobial peptides from rattlesnake venom
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion