Bactericidal activity of cyclotides where phosphatidylethanolamine-lipid selectivity determines antimicrobial spectra

Abstract

Cyclotides are a family of plant peptides characterized by a cystine knot embedded in a macrocyclic backbone. They bind to and disrupt phospholipid membranes, which explain their lytic activity on cells. In this study, we expose the full antibacterial potency of cyclotides by avoiding its inhibition by rich growth media assay conditions. For that purpose a two-step microdilution assay protocol was developed, using non-growing conditions during initial peptide incubation. A diverse set of cyclotides was tested for antibacterial and antifungal activity, and the results show that most cyclotides are active under these conditions, especially against Gram-negative bacteria. Activity was observed at sub-micromolar concentrations for three of the cyclotides tested, surpassing that of the control peptides LL-37 and melittin. Noteworthy, two anionic cyclotides were active on Pseudomonas aeruginosa at low micromolar concentrations. Broad-spectrum activity was pronounced among cycloviolacin cyclotides, which included activity on Staphylococcus aureus and Candida albicans. The factors influencing their bactericidal spectrum were revealed by correlating antimicrobial activity with membrane permeabilization on various liposome systems and with the physiochemical properties of the cyclotides. Whereas general electrostatic and hydrophobic parameters are more important for broad-spectrum cyclotides; a phospholipid-specific mechanism of membrane permeabilization, through interaction with phosphatidylethanolamine-lipids, is essential for cyclotides active primarily on Gram-negative bacteria.

A biofilm resistance surface yielded by grafting of antimicrobial peptides on stainless steel surface

“…The PD values of S. aureus and E. coli biofilm on MAG II modified sample surfaces were 71.4% and 53.85%, respectively, which is due to the presence of MAG II immobilization (Figure A). The results illustrated that MAG II modified samples possessed stronger anti‐ S. aureus biofilm formation ability than E. coli , which could be due to the influence of LB medium . MAG I and MAG II modified surfaces possessed equivalent antibacterial efficiency .…”

Alanine and Lysine Scans of the LL‐37‐Derived Peptide Fragment KR‐12 Reveal Key Residues for Antimicrobial Activity

“…Each peptide sequence, net charge, hydropathy index, and molecular weight are given in Table S1. The peptide‐adapted two‐step microdilution assay was used to determine the MIC against four clinical strains of human pathogens, Gram‐negative E. coli and Pseudomonas aeruginosa , Gram‐positive Staphylococcus aureus , and the fungus Candida albicans . The assay determines antimicrobial activity in buffer conditions, to avoid AMP inhibition by factors associated with rich growth media .…”

“…However, both LL‐37 and the KR‐12 analogues show similar leakage kinetics that resemble a steep logarithmic function with an immediate effect, which gradually subsides within minutes until leakage reaches a plateau (for leakage kinetics of LL‐37, see ref. ). The liposomes used in the current assay were based on an E. coli polar lipid extract, and are comparable in terms of charge and phospholipid composition to the outer membranes of both P. aeruginosa and E. coli .…”

“…Antimicrobial assay : Antibacterial and antifungal activities of the peptides were evaluated by means of the two‐step microdilution assay optimised for AMP‐activity testing . The method assessed the MIC under buffer conditions.…”

Natural and grafted cyclotides in cancer therapy: An insight

“…More recently, a number of cyclotides from Viola ignobilis including vigno 5, an acyclotide has been proven to induce apoptosis dependent cytotoxicity against HeLa cell lines . A large population of other cyclotides from diverse plant families, ie, cycloviolacin O2, cycloviolacin O3, cycloviolacin O19, CterB, CterE, CterG, CterR, tricyclon A, varv A, kalata B1, kalata B2, kalata B13, kalata B7, vibiG, vibiH, vibiD, vibiE, psyle A, psyle C, psyle E, vaby A, and vaby D, cycloviolacin O4 exhibited cytotoxic activity against lymphoma cell line (U‐937 GTB) . Apart from cyclotides, acyclic peptides also have been reported to show cytotoxic action against cancer cell lines …”