title: The radish defensins RsAFP1 and RsAFP2 act synergistically with caspofungin against Candida albicans biofilms.
abstract: The radish defensin RsAFP2 was previously characterized as a peptide with potent antifungal activity against several plant pathogenic fungi and human pathogens, including Candida albicans. RsAFP2 induces apoptosis and impairs the yeast-to-hypha transition in C. albicans. As the yeast-to-hypha transition is considered important for progression to mature biofilms, we analyzed the potential antibiofilm activity of recombinant (r)RsAFP2, heterologously expressed in Pichia pastoris, against C. albicans biofilms. We found that rRsAFP2 prevents C. albicans biofilm formation with a BIC-2 (i.e., the minimal rRsAFP2 concentration that inhibits biofilm formation by 50% as compared to control treatment) of 1.65 ± 0.40 mg/mL. Moreover, biofilm-specific synergistic effects were observed between rRsAFP2 doses as low as 2.5 μg/mL to 10 μg/mL and the antimycotics caspofungin and amphotericin B, pointing to the potential of RsAFP2 as a novel antibiofilm compound. In addition, we characterized the solution structure of rRsAFP2 and compared it to that of RsAFP1, another defensin present in radish seeds. These peptides have similar amino acid sequences, except for two amino acids, but rRsAFP2 is more potent than RsAFP1 against planktonic and biofilm cultures. Interestingly, as in case of rRsAFP2, also RsAFP1 acts synergistically with caspofungin against C. albicans biofilms in a comparable low dose range as rRsAFP2. A structural comparison of both defensins via NMR analysis revealed that also rRsAFP2 adopts the typical cysteine-stabilized αβ-motif of plant defensins, however, no structural differences were found between these peptides that might result in their differential antifungal/antibiofilm potency. This further suggests that the conserved structure of RsAFP1 and rRsAFP2 bears the potential to synergize with antimycotics against C. albicans biofilms.
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Vriens
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, 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 Materials Engineering, KU Leuven, Kasteelpark Arenberg 44, 3001 Heverlee, Belgium.
authors.forename: Annabel
authors.initials: A
authors.lastname: Braem
authors.affiliation: Department of Materials Engineering, KU Leuven, Kasteelpark Arenberg 44, 3001 Heverlee, Belgium.
authors.forename: Jozef
authors.initials: J
authors.lastname: Vleugels
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium; Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Ghent, Belgium.
authors.forename: Barbara
authors.initials: B
authors.lastname: De Coninck
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium; Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Ghent, Belgium. Electronic address: bruno.cammue@biw.kuleuven.be.
authors.forename: Bruno P A
authors.initials: BP
authors.lastname: Cammue
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium.
authors.forename: Karin
authors.initials: K
authors.lastname: Thevissen
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects*; D002176:Candida albicans / Q000187:drug effects* / Q000502:physiology; D000077336:Caspofungin; D023082:Defensins / Q000737:chemistry / Q000494:pharmacology*; D004357:Drug Synergism; D054714:Echinocandins / Q000494:pharmacology*; D055666:Lipopeptides / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010940:Plant Proteins / Q000737:chemistry / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D031224:Raphanus / Q000737:chemistry
chemical_list: D000935:Antifungal Agents; D023082:Defensins; D054714:Echinocandins; D055666:Lipopeptides; D010940:Plant Proteins; D000077336:Caspofungin
keywords: Candida albicans; NMR analysis; RsAFP1; RsAFP2; fungal biofilm; plant defensin; recombinant protein production