title: Synergistic Activity of the Plant Defensin HsAFP1 and Caspofungin against Candida albicans Biofilms and Planktonic Cultures.
abstract: Plant defensins are small, cysteine-rich peptides with antifungal activity against a broad range of yeast and fungi. In this study we investigated the antibiofilm activity of a plant defensin from coral bells (Heuchera sanguinea), i.e. HsAFP1. To this end, HsAFP1 was heterologously produced using Pichia pastoris as a host. The recombinant peptide rHsAFP1 showed a similar antifungal activity against the plant pathogen Fusarium culmorum as native HsAFP1 purified from seeds. NMR analysis revealed that rHsAFP1 consists of an α-helix and a triple-stranded antiparallel β-sheet stabilised by four intramolecular disulfide bonds. We found that rHsAFP1 can inhibit growth of the human pathogen Candida albicans as well as prevent C. albicans biofilm formation with a BIC50 (i.e. the minimum rHsAFP1 concentration required to inhibit biofilm formation by 50% as compared to control treatment) of 11.00 ± 1.70 μM. As such, this is the first report of a plant defensin exhibiting inhibitory activity against fungal biofilms. We further analysed the potential of rHsAFP1 to increase the activity of the conventional antimycotics caspofungin and amphotericin B towards C. albicans. Synergistic effects were observed between rHsAFP1 and these compounds against both planktonic C. albicans cells and biofilms. Most notably, concentrations of rHsAFP1 as low as 0.53 μM resulted in a synergistic activity with caspofungin against pre-grown C. albicans biofilms. rHsAFP1 was found non-toxic towards human HepG2 cells up to 40 μM, thereby supporting the lack of a general cytotoxic activity as previously reported for HsAFP1. A structure-function study with 24-mer synthetic peptides spanning the entire HsAFP1 sequence revealed the importance of the γ-core and its adjacent regions for HsAFP1 antibiofilm activity. These findings point towards broad applications of rHsAFP1 and its derivatives in the field of antifungal and antibiofilm drug development.
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Vriens
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.
authors.forename: Tanne L
authors.initials: TL
authors.lastname: Cools
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
authors.forename: Peta J
authors.initials: PJ
authors.lastname: Harvey
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: Department of Hepatology, University Hospitals Leuven, Leuven, Belgium; Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Spincemaille
authors.affiliation: Department of Hepatology, University Hospitals Leuven, Leuven, Belgium; Metabolic Center, University Hospitals Leuven, Leuven, Belgium.
authors.forename: David
authors.initials: D
authors.lastname: Cassiman
authors.affiliation: Department of Materials Engineering, KU Leuven, Leuven, Belgium.
authors.forename: Annabel
authors.initials: A
authors.lastname: Braem
authors.affiliation: Department of Materials Engineering, KU Leuven, Leuven, Belgium.
authors.forename: Jozef
authors.initials: J
authors.lastname: Vleugels
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Nibbering
authors.affiliation: Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jan Wouter
authors.initials: JW
authors.lastname: Drijfhout
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium; Department of Plant Systems Biology, VIB, Ghent, Belgium.
authors.forename: Barbara
authors.initials: B
authors.lastname: De Coninck
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium; Department of Plant Systems Biology, VIB, Ghent, Belgium.
authors.forename: Bruno P A
authors.initials: BP
authors.lastname: Cammue
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.
authors.forename: Karin
authors.initials: K
authors.lastname: Thevissen
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D002176:Candida albicans / Q000187:drug effects* / Q000254:growth & development; D000077336:Caspofungin; D023082:Defensins / Q000494:pharmacology*; D004357:Drug Synergism; D054714:Echinocandins / Q000494:pharmacology*; D006801:Humans; D055666:Lipopeptides
chemical_list: D000935:Antifungal Agents; D023082:Defensins; D054714:Echinocandins; D055666:Lipopeptides; D000077336:Caspofungin