A Linear 19-Mer Plant Defensin-Derived Peptide Acts Synergistically with Caspofungin against Candida albicans Biofilms

Abstract

Public health problems are associated with device-associated biofilm infections, with Candida albicans being the major fungal pathogen. We previously identified potent antibiofilm combination treatment in which the antifungal plant defensin HsAFP1 is co-administered with caspofungin, the preferred antimycotic to treat such infections. In this study, we identified the smallest linear HsAFP1-derived peptide that acts synergistically with caspofungin or anidulafungin against C. albicans as HsLin06_18, a 19-mer peptide derived from the C-terminal part of HsAFP1. The [caspofungin + HsLin06_18] combination significantly reduced in vitro biofilm formation of Candida glabrata and C. albicans on catheters, as well as biofilm formation of a caspofungin-resistant C. albicans strain. The [caspofungin + HsLin06_18] combination was not cytotoxic and reduced biofilm formation of C. albicans in vivo using a subcutaneous rat catheter model, as compared to control treatment. Mode of action research on the [caspofungin + HsLin06_18] combination pointed to caspofungin-facilitated HsLin06_18 internalization and immediate membrane permeabilization. All these findings point to broad-spectrum antibiofilm activity of a combination of HsLin06_18 and caspofungin.

Keywords: Candida; antimicrobial peptides; biofilms; catheters; fungal infections.

Figures

Figure 1

HsAFP1 core regions responsible for prevention of Candida albicans biofilm formation (red box) and synergy with caspofungin (blue box), identified by screening HsLin06-variants (Table 1) for both activities. Both regions are situated within the γ-core of plant defensins (yellow box). Gray regions represent conserved amino acids among plant defensins.

Figure 2

The [echinocandin + HsLin06_18] combination affects Candida albicans biofilms, determined in an in vitro microtiter plate assay. Dose-response curves of the echinocandin [caspofungin (A), anidulafungin (B) and micafungin (C)] are presented with different colors for different HsLin06_18 concentrations; orange, 1.25 μM, red, 0.313 μM, blue, 0.156 μM and black 0 μM HsLin06_18. Black arrows represent the HsLin06_18's potentiation effect on echinocandin's action, i.e., the shift of the echinocandin concentration needed for 50% reduction of metabolic activity between the echinocandin and the [echinocandin + 1.25 μM HsLin06_18] treatment. Data are means ± SEM for n = 3 independent experiments.

Figure 3

HsLin06_18-FITC uptake and membrane permeabilization in Candida albicans biofilms treated with the [caspofungin + HsLin06_18-FITC] combination. Confocal microscope images of 24 h treated C. albicans biofilms with 0.625 μM caspofungin (CASPO) and/or 0.5 μM HsLin06_18-FITC and 30 μg/mL propidium iode (PI). Bar: 5 μm.

Figure 4

Kinetics of HsLin06_18-FITC uptake and membrane permeabilization in planktonic Candida albicans cells treated with (A) 4.6 μM HsLin06_18-FITC (HsL-F), (B) 0.01 μM caspofungin (CASPO) or (C) its combination as well as 2 μg/mL propidium iodide (PI), determined via flow cytometry. For all treatments, the % of cells is presented that: only have permeabilized membranes (gray), both have permeabilized membranes and HsLin06_18-FITC associated to their surface or internalized (orange) or only HsLin06_18-FITC associated to their surface or internalized (black). Data are means ± SEM in presented for n = 3 independent experiments. To analyse significant differences in the size of the subpopulations between the t0 and other time points, one-way ANOVA followed by Dunnett multiple comparison was performed, with brackets (in the color of the corresponding subpopulation) representing significance for that subpopulation. Only the primary time point that is significantly different from t0 is presented, with *representing P < 0.05.

Figure 5

The [caspofungin + HsLin06_18] affects (A)Candida albicans, (B)Candida dubliniensis, (C)Candida krusei and (D)Candida glabrata biofilms in an in vitro microtiter plate assay, using XTT quantification. Dose-response curves of caspofungin are presented, with different colors for different HsLin06_18 concentrations; orange, 1.25 μM, red, 0.313 μM, blue, 0.156 μM, and black, 0 μM HsLin06_18. Black arrows represent the HsLin06_18's potentiation effect on caspofungin's action, i.e., the shift of the caspofungin concentration needed for 50% reduction of metabolic activity between the caspofungin and the [caspofungin + 1.25 μM HsLin06_18] treatment. Data are means ± SEM for n = 3 independent experiments.

Figure 6

The [caspofungin (CASPO) + HsLin06_18] affects Candida albicans(A) and C. glabrata(B) biofilms in an in vitro catheter assay. The amount of the viable biofilm cells on the catheters treated with 0.4 μM CASPO, 0.5 μM HsLin06_18, its combination or the control (0.5% DMSO) was determined via CFU counting. Horizontal lines indicate the median values for log10 numbers of CFU ± SEM obtained per catheter piece, for n = 3 independent experiments. Significant differences between all treatments were determined via one-way ANOVA followed by Tukey multiple comparison, with *representing P < 0.05.

Figure 7

Effect of intravenous administration of caspofungin (CASPO) and HsLin06_18 on Candida albicans biofilm formation inhibition, using catheter pieces in a subcutaneous rat catheter model. Rats, containing nine catheter pieces each, were treated with 0.25 mg/kg/day CASPO, 2.5 mg/kg/day HsLin06_18, its combination or the control (0.9% NaCl and 0.2% DMSO) for 7 days, after which survival of viable biofilm cells on the catheters was determined via CFU counting. Horizontal lines indicate the median values for log10 numbers of CFU and 95% CI obtained per catheter piece, for n = 27 catheter pieces. Significant differences between all treatments were determined via one-way ANOVA followed by Tukey multiple comparison, with *representing P < 0.05.