Membrane Permeabilization and Antimicrobial Activity of Recombinant Defensin-d2 and Actifensin against Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans

Abstract

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.

Recombinant Actifensin and Defensin-d2 Induce Critical Changes in the Proteomes of Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans

“…We postulate that the disruption of membrane integrity or membrane permeability in the treated organism induced dysfunctional transmembrane transport, resulting in the increased leakage of ATP and accumulation of oxidative stress, which in turn altered nucleotide synthesis, affected the mitochondria, or caused DNA/RNA degradation. This correlates with our previous study that showed that both peptides induced oxidative stress in C. albicans within 1 h after exposure ( 20 ). Other studies ( 46 – 48 ) also reported a membrane-targeted mechanism of action of AMPs against C. albicans .…”

“…The method of recombinant production of defensin-d2 and actifensin was described previously ( 20 ). Briefly, an overnight culture of transformed Escherichia coli Shuffle T7 (New England Biolabs, USA) containing recombinant plasmids (pTXB1-defensin-d2 and pTXB1-actifensin) was grown in modified Terrific broth (Sigma-Aldrich, Germany) containing 100 μg/mL ampicillin.…”

“…In an earlier study ( 20 ), we expressed defensin-d2 and actifensin as recombinant peptides and determined their MICs against P. aeruginosa and C. albicans . The study further established that both peptides exhibited membrane permeabilization activities and induced reactive oxygen species (ROS) production in P. aeruginosa and C. albicans .…”

Anti-Pseudomonas aeruginosa activity of natural antimicrobial peptides when used alone or in combination with antibiotics

“…It exhibited weak antibacterial activity against P. aeruginosa strains with MIC of 1448 μg/mL, whereas predominantly inhibited methicillin-resistant S. aureus and Candida albicans with MIC values of 23 μg/mL and 45 μg/mL. Its mechanism of anti- P. aeruginosa may be related to induce reactive oxygen species (ROS) generation, thereby destroyed cellular membrane permeability, but further molecular mechanisms were unclear ( Sugrue et al, 2020 ; Gbala et al, 2022 ). More importantly, it has been found to have a high biosafety, and it causes less than 1.5% hemolysis on mouse erythrocytes in all tested concentrations.…”

“…Plant defensins are small cationic peptides with 5–7 kDa in size that contain multiple cysteine groups to safeguard plants against microbial invasion ( Gbala et al, 2022 ). These peptides have a wide-ranging ability to inhibit the growth of various pathogens including filamentous fungi and bacteria ( Perez-Rodriguez et al, 2022b ), we summarized the data on the inhibitory activity of several plant -derived AMPs against P. aeruginosa in Table 1 .…”