Search

© 2018 Elsevier B.V. All rights reserved., Antimicrobial peptides (AMPs) are promising candidates for the development of future antibiotics. In an attempt to increase the efficacy of therapeutic AMPs, computer-based design methods appear as a reliable strategy. In this study, we evaluated the antimicrobial efficiency and mechanism of action of a novel designed AMP named PaDBS1R1, previously designed by means of the Joker algorithm, using a fragment of the ribosomal protein L39E from the archaeon Pyrobaculum aerophilum as a template. PaDBS1R1 displayed low micromolar broad-spectrum antimicrobial activity against Gram-negative (MIC of 1.5 μM) and Gram-positive (MIC of 3 μM) bacteria, including carbapenem-resistant Klebsiella pneumoniae (MIC of 6.25 μM) and methicillin-resistant Staphylococcus aureus (MIC of 12.5 μM), without cytotoxicity towards HEK-293 cells. In addition, membrane permeabilization and depolarization assays, combined with time-kill studies and FEG-SEM imaging, indicated a fast membrane permeation and further leakage of intracellular content. Biophysical studies with lipid vesicles show a preference of PaDBS1R1 for Gram-negative bacteria-like membranes. We investigated the three-dimensional structure of PaDBS1R1 by CD and NMR analyses. Our results suggest that PaDBS1R1 adopts an amphipathic α-helix upon interacting with hydrophobic environments, after an initial electrostatic interaction with negative charges, suggesting a membrane lytic effect. This study reveals that PaDBS1R1 has potential application in antibiotic therapy., The authors acknowledge Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal) for funding, including fellowship SFRH/BD/100517/2014 to M.R.F., Marie Sklodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167), IMPC (Institut des Matériaux de Paris Centre, FR 2482) and the C'Nano projects of the Region Ile-de-France for SEM-FEG funding. Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq for financial support and researcher fellowship (Processes: 141327/2012-0; 407181/2013-0; 303181/20150), Coordenação de Aperfeiçoamento de Pessoal do Nível Superior – CAPES, for scholarship (Process: BEX 2758/15-6), and Fundação de Amparo à Pesquisa do Estado de Goiás – FAPEG (Process: 201710267000062). This work was also supported by Fundação de Amparo a Pesquisa do Mato Grosso do Sul (FUNDECT) and Fundação de Amapro a Pesquisa do Distrito Federal (FAPDF).

National audience

International audience; The emergence of antibiotic-resistant clinical isolates and the decreased rate of development of new antibiotics are a constant threat to human health. In this context, the therapeutic value of mastoparan (MP), a toxin from wasp venom, has been extensively studied. However, since MP shows significant cytotoxic activities, further optimization is needed. Here we evaluated the antimicrobial and cytolytic activities of an MP analog created by Ala-substitution in positions 5 and 8, named [I-5, R-8] mastoparan ([I-5, R-8] MP). We found that [I-5, R-5] MP displayed a broad-spectrum antimicrobial activity against bacteria and fungi (MIC in the range 3-25 mu M), without being hemolytic or cytotoxic toward HEK-293 cells. In addition, [I-5, R-8] MP-amide was highly potent (MIC = 3 mu M) against antibiotic-resistant bacteria. The interaction with microbial membranes was investigated revealing that [I-5, R-8] MP is able to form an active amphipathic ct-helix conformation and to disturb membranes causing lysis and cell death. Based on our findings, we hypothesize that [I-5, R-8] MP follows a mechanism of action similar to that proposed for MP, where the pore-forming activity leads to cell death. Our results indicate that hydrophobic moment modified by amino acid substitution may enhance MP selectivity.