RESUMEN
Drug-resistant strains of Pseudomonas aeruginosa and Candida albicans pose serious threats to human health because of their propensity to cause fatal infections. Defensin and defensin-like antimicrobial peptides (AMPs) are being explored as new lines of antimicrobials, due to their broad range of activity, low toxicity, and low pathogen resistance. Defensin-d2 and actifensin are AMPs from spinach and Actinomyces ruminicola, respectively, whose mechanisms of action are yet to be clearly elucidated. This study investigated the mechanisms of action of the recombinant AMPs through label-free quantitative proteomics. The data are available at PRIDE with accession number PXD034169. A total of 28 and 9 differentially expressed proteins (DEPs) were identified in the treated P. aeruginosa and C. albicans, respectively, with a 2-fold change threshold and P values of <0.05. Functional analysis revealed that the DEPs were involved in DNA replication and repair, translation, and membrane transport in P. aeruginosa, while they were related mainly to oxidative phosphorylation, RNA degradation, and energy metabolism in C. albicans. Protein-protein interactions showed that the DEPs formed linear or interdependent complexes with one another, indicative of functional interaction. Subcellular localization indicated that the majority of DEPs were cytoplasmic proteins in P. aeruginosa, while they were of nuclear or mitochondrial origin in C. albicans. These results show that recombinant defensin-d2 and actifensin can elicit complex multiple organism responses that cause cell death in P. aeruginosa and C. albicans. IMPORTANCE AMPs are considered essential alternatives to conventional antimicrobials because of their broad-spectrum efficacy and low potential for resistance by target cells. In this study, we established that the recombinant AMPs defensin-d2 and actifensin exert proteomic changes in P. aeruginosa and C. albicans within 1 h after treatment. We also found that the DEPs in peptide-treated P. aeruginosa are related to ion transport and homeostasis, molecular functions including nucleic and amino acid metabolism, and structural biogenesis and activity, while the DEPs in treated C. albicans are mainly involved in membrane synthesis and mitochondrial metabolism. Our results also highlight ATP synthase as a potential drug target for multidrug-resistant P. aeruginosa and C. albicans.
Asunto(s)
Antiinfecciosos , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Candida albicans/genética , Proteoma , Pruebas de Sensibilidad Microbiana , Proteómica , Defensinas/farmacología , Péptidos , Aminoácidos , Adenosina TrifosfatoRESUMEN
Antimicrobial resistance requires urgent efforts towards the discovery of active antimicrobials, and the development of strategies to sustainably produce them. Defensin and defensin-like antimicrobial peptides (AMPs) are increasingly gaining pharmacological interest because of their potency against pathogens. In this study, we expressed two AMPs: defensin-d2 derived from spinach, and defensin-like actifensin from Actinomyces ruminicola. Recombinant pTXB1 plasmids carrying the target genes encoding defensin-d2 and actifensin were generated by the MEGAWHOP cloning strategy. Each AMP was first expressed as a fusion protein in Escherichia coli, purified by affinity chromatography, and was thereafter assayed for antimicrobial activity against multidrug-resistant (MDR) pathogens. Approximately 985 µg/mL and 2895 µg/mL of recombinant defensin-d2 and actifensin, respectively, were recovered with high purity. An analysis by MALDI-TOF MS showed distinct peaks corresponding to molecular weights of approximately 4.1 kDa for actifensin and 5.8 kDa for defensin-d2. An in vitro antimicrobial assay showed that MDR Pseudomonas aeruginosa and Candida albicans were inhibited at minimum concentrations of 7.5 µg/mL and 23 µg/mL for recombinant defensin-d2 and actifensin, respectively. The inhibitory kinetics of the peptides revealed cidal activity within 4 h of the contact time. Furthermore, both peptides exhibited an antagonistic interaction, which could be attributed to their affinities for similar ligands, as deduced by peptide-ligand profiling. Moreover, both peptides inhibited biofilm formation, and they exhibited no resistance potential and low hemolytic activity. The peptides also possess the ability to permeate and disrupt the cell membranes of MDR P. aeruginosa and C. albicans. Therefore, recombinant actifensin and defensin-d2 exhibit broad-spectrum antimicrobial activity and have the potential to be used as therapy against MDR pathogens.