Synergic Effect of the Antimicrobial Peptide ToAP2 and Fluconazole on Candida albicans Biofilms

Abstract

Candida albicans is one of the agents of invasive candidiasis, a life-threatening disease strongly associated with hospitalization, particularly among patients in intensive care units with central venous catheters. This study aimed to evaluate the synergistic activity of the antifungal peptide ToAP2 combined with fluconazole against C. albicans biofilms grown on various materials. We tested combinations of different concentrations of the peptide ToAP2 with fluconazole on C. albicans biofilms. These biofilms were generated on 96-well plates, intravenous catheters, and infusion tubes in RPMI medium at two maturation stages. Scanning electron microscopy and atomic force microscopy were employed to assess the biofilm structure. We also evaluated the expression of genes previously proven to be involved in C. albicans biofilm formation in planktonic and biofilm cells after treatment with the peptide ToAP2 using qPCR. ToAP2 demonstrated a synergistic effect with fluconazole at concentrations up to 25 µM during both the early and mature stages of biofilm formation in 96-well plates and on medical devices. Combinations of 50, 25, and 12.5 µM of ToAP2 with 52 µM of fluconazole significantly reduced the biofilm viability compared to individual treatments and untreated controls. These results were supported by substantial structural changes in the biofilms observed through both scanning and atomic force microscopy. The gene expression analysis of C. albicans cells treated with 25 µM of ToAP2 revealed a decrease in the expression of genes associated with membrane synthesis, along with an increase in the expression of genes involved in efflux pumps, adhesins, and filamentation. Our results highlight the efficacy of the combined ToAP2 and fluconazole treatment against C. albicans biofilms. This combination not only shows therapeutic potential but also suggests its utility in developing preventive biofilm tools for intravenous catheters.

Keywords: Candida albicans; antifungal drugs; antimicrobial peptides; biofilms; fluconazole; synergism.

Figure 1

Biofilm inhibition dose–response matrix after treatment with ToAP2 and conventional antifungals. (A,B) Inhibition of early-phase (4 h) and mature (24 h) C. albicans biofilms after 24 h treatment with ToAP2 and/or Amphotericin B, respectively. (C,D) Inhibition of early-phase (4 h) and mature (24 h) C. albicans biofilms after 24 h treatment with ToAP2 and/or fluconazole, respectively. Cell viability was measured by fluorescence with Alamar blue reagent after 2 h of incubation. The viability data were transformed to inhibition using the SynergyFinder Plus R package 3.10.3. The axes represent the concentrations for ToAP2 (rows) and each antifungal (columns). The heatmap scales vary from red, indicating no growth, to green, indicating maximum growth. Data are presented as mean ± standard error of the mean of three independent assays.

Figure 2

SEM images of C. albicans mature biofilms after different treatments. (A) Treatment with 100 µM ToAP2, (B) 100 µM NDBP-5.7, (C) 52 µM Fluconazole, (D) 1.08 µM Amphotericin B, (E) combination of ToAP2 and Amphotericin, (F) combination of ToAP2 and fluconazole, (G) untreated control. Scale bar: 10 µm. White arrows highlight morphological changes in biofilms after treatment in comparison to the control.

Figure 3

AFM amplitude images of C. albicans cells on mature biofilm after different treatments. (A) Treatment with 100 µM ToAP2, (B) 100 µM NDBP-5.7, (C) 52 µM Fluconazole, (D) 1.08 µM Amphotericin B, (E) combination of ToAP2 and Amphotericin, (F) combination of ToAP2 and fluconazole, (G) untreated control, (H) quantification of biofilm surface roughness after different treatments. Data were analyzed with one-way ANOVA and T-test (p < 0.05) Scale bar: 1 µm. Bars represent standard deviation. White arrows highlight morphological changes in biofilms after treatment in comparison to the control.

Figure 4

Viability of C. albicans cells on mature biofilms treated with different concentrations of ToAP2 or ToAP2 associated with Fluconazole. (A) Viability of C. albicans on mature biofilms formed in infusion tubes. (B) Viability of C. albicans mature biofilms formed in PU catheters. The data were analyzed by ANOVA (p < 0.05) and Tukey’s post-test. p-values of statistically significant comparisons are shown in the figure. Mean ± SEM.

Figure 5

Effects of ToAP2 on the expression of genes related to C. albicans virulence factors. (A) Planktonic cells of C. albicans were treated with concentrations of 25 µM and 50 µM of ToAP2. (B) C. albicans biofilms were treated with concentrations of 25 µM and 50 µM of ToAP2. Data are presented as mean ± upper and lower limits. Statistical significance was calculated by Student’s t-test. * p-value < 0.05.