title: Revealing the sequence of interactions of PuroA peptide with Candida albicans cells by live-cell imaging.
abstract: To determine the mechanism(s) of action of antimicrobial peptides (AMPs) it is desirable to provide details of their interaction kinetics with cellular, sub-cellular and molecular targets. The synthetic peptide, PuroA, displays potent antimicrobial activities which have been attributed to peptide-induced membrane destabilization, or intracellular mechanisms of action (DNA-binding) or both. We used time-lapse fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM) to directly monitor the localization and interaction kinetics of a FITC- PuroA peptide on single Candida albicans cells in real time. Our results reveal the sequence of events leading to cell death. Within 1 minute, FITC-PuroA was observed to interact with SYTO-labelled nucleic acids, resulting in a noticeable quenching in the fluorescence lifetime of the peptide label at the nucleus of yeast cells, and cell-cycle arrest. A propidium iodide (PI) influx assay confirmed that peptide translocation itself did not disrupt the cell membrane integrity; however, PI entry occurred 25-45 minutes later, which correlated with an increase in fractional fluorescence of pores and an overall loss of cell size. Our results clarify that membrane disruption appears to be the mechanism by which the C. albicans cells are killed and this occurs after FITC-PuroA translocation and binding to intracellular targets.
authors.affiliation: Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, PO Box 218, Hawthorn, VIC, Australia.
authors.forename: Nadin
authors.initials: N
authors.lastname: Shagaghi
authors.affiliation: Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, PO Box 218, Hawthorn, VIC, Australia.
authors.forename: Mrinal
authors.initials: M
authors.lastname: Bhave
authors.affiliation: Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, PO Box 218, Hawthorn, VIC, Australia.
authors.forename: Enzo A
authors.initials: EA
authors.lastname: Palombo
authors.affiliation: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, PO Box 218, Hawthorn, VIC, Australia.
authors.forename: Andrew H A
authors.initials: AH
authors.lastname: Clayton
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000648:ultrastructure; D050296:Microbial Viability / Q000187:drug effects; D008856:Microscopy, Fluorescence; D013997:Time Factors; D059008:Time-Lapse Imaging
chemical_list: D023181:Antimicrobial Cationic Peptides