Antimicrobial peptide plectasin recombinantly produced in Escherichia coli disintegrates cell walls of gram-positive bacteria, as proven by transmission electron and atomic force microscopy.

Abstract

Plectasin, an antimicrobial peptide, was initially isolated from the saprophytic fungus Pseudoplectania nigrella. This peptide, a member of the cysteine-stabilized α-helix and β-sheet family, has demonstrated potent antimicrobial activity against gram-positive pathogens, including strains resistant to conventional antibiotics. Our CASPON platform process enables the production of substantial quantities of plectasin, facilitating investigations on the activity and the mode of action of this recombinantly produced peptide. To this end, we developed an activity assay that reflects the growth inhibition of selected model bacteria, allowing for statistical analysis and evaluation of reproducibility. The mode of action was investigated using transmission electron microscopy and atomic force microscopy. The latter provided new insights into alterations in the cell surface of gram-positive bacteria treated with plectasin at the single-cell level. While the cell diameter remained unaltered, the roughness increased by up to twofold, and the cell stiffness decreased by approximately one-third in the four gram-positive bacterial strains tested. Statistical analysis of these morphological changes provides further insights into the effects and efficiency of antimicrobial peptides targeting pathogen cell walls.

Importance: The rise of antibiotic-resistant bacteria is a major threat to global health. Antimicrobial peptides (AMPs) offer a promising way to combat this. With the CASPON technology, we produced the AMP plectasin comprising three disulfide bonds using Escherichia coli. The activity of purified plectasin with and without a CASPON fusion tag was determined for four gram-positive and four gram-negative bacteria. As anticipated, only gram-positive bacteria showed a growth inhibition response to un-tagged plectasin. Plectasin treatment on gram-positive bacteria was visualized via electron microscopy. Evaluation of atomic force microscopy indicated that plectasin treatment led to increased roughness but maintained thickness. Based on our study, we assume that the CASPON technology can be employed in the future for the production and characterization of medical-grade AMPs.