2026-04-13T07:24:51Zhttps://recercat.cat/oai/request

oai:recercat.cat:10230/544612025-12-18T01:07:03Zcom_2072_6col_2072_452952

00925njm 22002777a 4500 dc Pérez-Peinado, Clara, 1991- author Almeida Dias, Susana author Domingues, Marco M. author Benfield, Aurélie H. author Miguel Freire, João author Rádis-Baptista, Gandhi author Gaspar, Diana author Castanho, Miguel A.R.B. author Craik, David J. author Troeira Henriques, Sónia author Salomé Veiga, Ana author Andreu Martínez, David author 2022-10-18T08:48:49Z 2022-10-18T08:48:49Z 2018 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. 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. Gram-negative bacteria Antimicrobial peptide (AMP) Atomic force microscopy (AFM) Bacterial membrane disruption Bactericidal mechanism Confocal microscopy Surface plasmon resonance (SPR) Time-resolved flow cytometry Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15–34), antimicrobial peptides from rattlesnake venom