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

2018Journal article Open access
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dc.contributor.authorPeinado, Clara Pérez
dc.contributor.authorDias, Susana
dc.contributor.authorDomingues, Marco André Manso
dc.contributor.authorBenfield, Aurélie H.
dc.contributor.authorFreire, João Miguel
dc.contributor.authorRádis-Baptista, Gandhi
dc.contributor.authorGaspar, Diana
dc.contributor.authorCastanho, Miguel A. R. B.
dc.contributor.authorCraik, David J.
dc.contributor.authorHenriques, Sónia Troeira
dc.contributor.authorVeiga, Ana Salome
dc.contributor.authorAndreu, David
dc.date.accessioned2019-03-22T12:58:27Z
dc.date.available2019-03-22T12:58:27Z
dc.date.issued2018
dc.description© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.pt_PT
dc.description.abstractCrotalicidin (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.pt_PT
dc.description.sponsorshipThis 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.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationJ. Biol. Chem. (2018) 293(5) 1536 –1549pt_PT
dc.identifier.doi10.1074/jbc.RA117.000125pt_PT
dc.identifier.issn0021-9258
dc.identifier.urihttp://hdl.handle.net/10451/37660
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherAmerican Society for Biochemistry and Molecular Biologypt_PT
dc.relation644167pt_PT
dc.relationTowards the development of innovative highly effective dual action anti-HIV peptides
dc.relationAGL2014-52395-C2pt_PT
dc.relation.publisherversionhttp://www.jbc.org/site/misc/about.xhtmlpt_PT
dc.subjectGram-negative bacteriapt_PT
dc.subjectAntimicrobial peptide (AMP)pt_PT
dc.subjectAtomic force microscopy (AFM)pt_PT
dc.subjectBacterial membrane disruptionpt_PT
dc.subjectBactericidal mechanismpt_PT
dc.subjectConfocal microscopypt_PT
dc.subjectSurface plasmon resonance (SPR)pt_PT
dc.subjectTime-resolved flow cytometrypt_PT
dc.titleMechanisms of bacterial membrane permeabilization by crotalicidin Ctn and its fragment Ctn 15–34, antimicrobial peptides from rattlesnake venompt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleTowards the development of innovative highly effective dual action anti-HIV peptides
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FQEQ-MED%2F4412%2F2014/PT
oaire.citation.endPage1549pt_PT
oaire.citation.issue5pt_PT
oaire.citation.startPage1536pt_PT
oaire.citation.titleJournal of Biological Chemistrypt_PT
oaire.citation.volume293pt_PT
oaire.fundingStream3599-PPCDT
person.familyNameDias
person.familyNameMiguel Freire
person.familyNameGaspar
person.familyNameCastanho
person.familyNameVeiga
person.givenNameSusana
person.givenNameJoão
person.givenNameDiana
person.givenNameMiguel
person.givenNameAna Salome
person.identifier103438
person.identifier.ciencia-idF61D-0A28-659A
person.identifier.orcid0000-0001-8910-5404
person.identifier.orcid0000-0002-8674-0813
person.identifier.orcid0000-0002-9602-567X
person.identifier.orcid0000-0001-7891-7562
person.identifier.orcid0000-0002-9892-2243
person.identifier.ridJ-4881-2015
person.identifier.ridM-9562-2015
person.identifier.scopus-author-id55180303000
person.identifier.scopus-author-id56605575600
person.identifier.scopus-author-id56745037100
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsrestrictedAccesspt_PT
rcaap.typearticlept_PT
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