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.
Highlights
Antimicrobial peptides (AMPs) are natural defence molecules produced by the majority of living organisms, from microorganisms to humans
When antimicrobial peptides (AMPs) have transmembrane pore-forming mechanisms combined with intracellular targets, it is believed that the AMPs form those pores to reach their intracellular targets[19,20,21,22]
The major findings in this work are how PuroA attacks C. albicans cells in real time and determining the main target that causes cell death
Summary
Antimicrobial peptides (AMPs) are natural defence molecules produced by the majority of living organisms, from microorganisms to humans. Many studies aimed to understand their mode of action have shown that AMPs attach to and insert into membrane bilayers to form stable transmembrane pores such as barrel-stave pores[2] or toroidal pores[3] or micellization in a detergent-like way (carpet mechanism)[4]. It has been believed that the killing mechanism of AMPs depend on the membrane composition, the peptide concentration, and the final peptide ⁄ lipid ratio. At low peptide ⁄ lipid ratios, AMPs can translocate across cell membranes, disturbing their structure in a transient, non-lethal manner, and reach their intracellular target[7]. Some AMPs, like Arasin 1, used a dual mechanism of action which was concentration-dependent; at concentrations above the MIC, Arasin 1 lysed membranes in a detergent-like manner, while at concentrations below MIC, Arasin 1 penetrated membranes and bound to intracellular targets[17,18]. When AMPs have transmembrane pore-forming mechanisms combined with intracellular targets, it is believed that the AMPs form those pores to reach their intracellular targets[19,20,21,22]
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