Secondary shift analysis of rHsAFP1, pH 4.0 at 298 K.

<p>Regions of α-helix and β-strand are indicated at the top of the figure.</p

Synergy between caspofungin and HsLin06 for biofilm inhibition.

<p>Metabolic activity was measured using CTB. Sigmoidal curves were generated using data of at least three independent experiments (n ≥ 3), using the model <i>Y = Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)</i>) in GraphPad Prism. Dose response curves of caspofungin in the presence of synergistic concentrations of HsLin06 are presented. Black arrows represent synergy. Coloured lines represent different HsLin doses, as follows: brown: 43.75 μM; orange: 21.88 μM; dark yellow: 10.94 μM; green: 5.47 μM; blue: 1.5; purple: 0.75 μM μM and black: 0 μM.</p

Scanning electron microscopy images of 4 hours-old biofilms, grown in the presence or absence (untreated) of 11.8 μM rHsAFP1.

<p>Images at multiple magnifications (500x, 1000x and 2000x) are presented.</p

Three-dimensional structure of rHsAFP1.

<p>(<b>A</b>) A family of 20 lowest energy structures superimposed over all backbone heavy atoms; (<b>B</b>) A ribbon representation with disulfide bonds shown in yellow. The termini are labeled as N and C. Diagrams were generated using MOLMOL.</p

Synergy between rHsAFP1 and caspofungin or amphotericin B, for (A) biofilm inhibition, as determined by CTB assay; (B) biofilm eradication, as determined by CTB assay; and (C) growth inhibition of planktonic cultures.

<p>Growth was analysed by measuring the OD₄₉₀. Sigmoidal curves were generated using data of at least three independent experiments (n ≥ 3), using the model <i>Y = Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)</i>) in GraphPad Prism. Dose response curves of caspofungin in the presence of synergistic concentrations of rHsAFP1 are presented. Black arrows represent synergy. Coloured lines represent different rHsAFP1 doses, as follows: brown: 16.8 μM; red: 8.4 μM; orange: 4.2 μM; dark yellow: 2.1 μM; green: 1.05 μM; turquois: 0.53 μM; blue: 0.26 μM and black: 0 μM.</p

Synergistic Activity of the Plant Defensin HsAFP1 and Caspofungin against <i>Candida albicans</i> Biofilms and Planktonic Cultures

<div><p>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 (<i>Heuchera sanguinea</i>), <i>i</i>.<i>e</i>. HsAFP1. To this end, HsAFP1 was heterologously produced using <i>Pichia pastoris</i> as a host. The recombinant peptide rHsAFP1 showed a similar antifungal activity against the plant pathogen <i>Fusarium culmorum</i> 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 <i>Candida albicans</i> as well as prevent <i>C</i>. <i>albicans</i> biofilm formation with a BIC50 (<i>i</i>.<i>e</i>. 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 <i>C</i>. <i>albicans</i>. Synergistic effects were observed between rHsAFP1 and these compounds against both planktonic <i>C</i>. <i>albicans</i> cells and biofilms. Most notably, concentrations of rHsAFP1 as low as 0.53 μM resulted in a synergistic activity with caspofungin against pre-grown <i>C</i>. <i>albicans</i> 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.</p></div

Structure-function relationship study of HsAFP1-derived fragments against <i>C</i>. <i>albicans</i> biofilms*.

<p>* BIC50 values were determined by CTB assay; mean ± SEM for n ≥ 3 independent experiments is presented; BIC50, minimum inhibitory concentration that is required to inhibit biofilm formation by 50% as compared to control treatment. Unpaired Student t-tests were performed to analyse significant differences between the effect of the linear fragments and rHsAFP1; the significance level is presented (*, ** and *** represent <i>P</i><0.05, <i>P</i><0.01 and <i>P</i><0.001, respectively; NS, no significant difference).</p><p>Structure-function relationship study of HsAFP1-derived fragments against <i>C</i>. <i>albicans</i> biofilms*.</p

Representation of the HsLin peptides imposed on the rHsAFP1 structure, according to the amino acid sequence.

<p>HsLin peptides are shown as a thick blue line in the same orientation as rHsAFP1; other residues of rHsAFP1, not present in the HsLin peptide, are shown as a thin blue line. Note that (i) the cysteine residues are replaced by α-aminobutyric acid to avoid formation of disulfide bonds and that (ii) the CSαβ scaffold is not present in the HsLin peptides, and therefore, the peptides do not adopt the same conformation as the mature rHsAFP1.</p

Sequence alignment of HsAFP1 with other plant defensins.

<p>(<b>A</b>) Amino acid sequence alignment of NaD1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref006" target="_blank">6</a>], Psd1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref005" target="_blank">5</a>], MtDef4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref061" target="_blank">61</a>], RsAFP1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref062" target="_blank">62</a>], RsAFP2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref062" target="_blank">62</a>] and HsAFP1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref032" target="_blank">32</a>], matching their cysteine residues (numbered I-VIII). Multiple alignment was performed using the COBALT alignment tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref063" target="_blank">63</a>]. Cysteine-pairing is shown at the top of the figure. Highly conserved residues are shown in grey; (-) denote gaps in the alignment. Blue boxes represent peptide fragments that exhibit antifungal activity similar to the parental peptide, and hence, are important for antifungal activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref004" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref064" target="_blank">64</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref066" target="_blank">66</a>]. The orange box indicates the position of the γ-core. (<b>B</b>) Amino acid sequence alignment of HsAFP1 and the HsAFP1 linear peptide fragments (HsLin01-HsLin06). Multiple alignment was performed using the COBALT alignment tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref063" target="_blank">63</a>]. Highly conserved residues are shown in grey; (-) denote gaps in the alignment. The orange box indicates the position of the γ-core.</p

Integrity of <i>S</i>. <i>aureus</i> liposomes after treatment with SPI031 at 1x MIC and 4x MIC.

<p>SDS (5%) and tetracycline (TET) (4x MIC) were used as positive and negative controls, respectively. Means and standard deviation (SD) of 3 independent experiments are shown (*p < 0.05; **p < 0.01; ***p < 0.001 compared to treatment with tetracycline).</p