2,4-Diacetylphloroglucinol Modulates Candida albicans Virulence

Abstract

The dimorphic fungus Candida albicans is one of the most important opportunistic pathogens for humankind. The use of fungicides against Candida could be associated with sub-inhibitory effects, which are referred to as fungal stress responses and are undesirable for the host. In this work, we investigated the antifungal action of 2,4-diacetylphloroglucinol (2,4-DAPG) against Candida albicans ATCC 10231 with a focus on their biofilm-forming ability. We found that 2,4-DAPG was able to reduce the ability of Candida cells to form biofilms, but complete inhibition and eradication effects were not achieved. Furthermore, C. albicans cells in the adherent state were characterized by reduced susceptibility to 2,4-DAPG compared to planktonic cells. The investigation of the mechanisms that could explain the antibiofilm action of 2,4-DAPG revealed a reduction in the cell`s surface hydrophobicity and the inhibition of the yeast-to-hyphae transition. The inhibition of the Candida cells filamentation was accompanied by an increase in the expression of the NRG1 gene, which is a negative regulator of hyphal development. In addition, we microscopically visualized the treated biofilms and revealed numerous channels that were decorated with particles and localized on the hyphae. We assumed that these hyphal structures could be associated with the secretion of aspartyl proteases (Sap). The performed assessments revealed an increase in the activity of Sap, which was accompanied by an increase in the expression of the sap2 and sap4 genes. The antifungal action of 2,4-DAPG is known to be associated with affecting the permeability of cellular structures, which leads to H⁺ATPase malfunction and the disruption of mitochondrial respiration. The subsequent cytosol acidification and generation of ROS trigger the inhibition of Candida filamentation and activation of Sap production. The introduction of antioxidant Trolox simultaneously with 2,4-DAPG leads to a reduction in Sap production. Collectively, the obtained data indicate new aspects of the interaction of fungal cells with 2,4-DAPG, an antimicrobial metabolite of Pseudomonas spp.

Keywords: 2,4-diacetylphloroglucinol; Candida albicans; aspartyl protease; biofilm; hyphal channels; virulence.

Figure 1

Influence of 2,4-DAPG on the C. albicans ATCC 10231 biofilm development (a,b) and on the mature biofilm (c,d). 2,4-DAPG action was estimated by measuring biofilm biomass (a,c) and metabolic activity of the biofilm-embedded cells (b,d). The y-axis represents percentages calculated with respect to the control sample. The means plus standard deviations of results from two independent experiments with three technical replicates are shown. Values that are significantly different from the values for “DMSO 5%” are indicated * p < 0.05, ** p < 0.01 (pair-sample Student’s t-test).

Figure 2

Microscopy investigations of C. albicans biofilm. Representative SEM images of the intact biofilm structure (a,b). SEM images of the biofilm formed in the presence of 2,4-DAPG (250 µg/mL) (c,d). Representative AFM images (phase shift mode) of hyphae treated with 2,4-DAPG (250 µg/mL) (e) and enlarged area of hyphae surface with protrusions (f). Scale bars—50 µm (a,c); 2 µm (b,d,e); 200 nm (f).

Figure 3

Protein (a) and carbohydrate content (b) in EPS of C.albicans biofilms which were formed under the 2,4-DAPG treatment for 24 h. The y-axis represents absorbance units. The means plus standard deviations of results from two independent experiments with three technical replicates are shown. Values that are significantly different from the values for “control” are indicated ** p < 0.01 (pair-sample Student’s t-test).

Figure 4

Total biomass quantification (crystal violet) assay was applied for estimation of the adhesion efficacy within 1.5 h (a). Adhesion to polystyrene and cellular surface hydrophobicity (CSH) of C. albicans ATCC 10231 cells (b). The means plus standard deviations of results from two independent experiments with three technical replicates are shown. Values that are significantly different from the values for “DMSO 5%” or “control” are indicated * p < 0.05, ** p < 0.01 (pair-sample Student’s t-test).

Figure 5

The protease activity of planktonic cells (Sap1–3) (a) and biofilms (Sap4–6) (b) of C. albicans ATCC 10231 under the influence of different concentrations of 2,4-DAPG. Trolox addition (50 µM) reduced Sap production (c). Lipase production was not affected by the action of 2,4-DAPG (d). The means plus standard deviations of results from two independent experiments with three technical replicates are shown. Values that are significantly different from the values for “control” are indicated ** p < 0.01, ns—values are not significantly different (pair-sample Student’s t-test).

Figure 6

Yeast-to-hyphae transition of C. albicans ATCC 10231 on spider agar in the presence of sub-inhibitory concentrations of various antifungals. 2,4-DAPG was added at 31 µg/mL (1/4 MIC); 15.5 µg/mL (1/8 MIC); 7.5 µg/mL (1/16 MIC); and 0 µg/mL.

Figure 7

The analysis of the expression level of genes responsible for protease production and hyphae filamentation. Transcript levels of the analyzed genes were determined by RT-qPCR in relation to the ACT1 expression. Sap2—the gene responsible for the synthesis of Sap 1–3 proteases; Sap6—the gene responsible for the synthesis of Sap4–6 proteases; NRG1—the negative regulator of filamentous growth; PMA1—the gene of H⁺ATPase. The means plus standard deviations of results from three technical replicates are shown. Values that are significantly different from the values for “control” are indicated **—p < 0.01; ***—p < 0.001 (pair-sample Student’s t-test).

Figure 8

The amount of tyrosol which was produced by planktonic cells of C. albicans (a) and biofilms (b). The means plus standard deviations of results from two independent experiments with three technical replicates are shown. Values that are significantly different from the values for “Control” are indicated *—p < 0.05; **—p < 0.01 (pair-sample Student’s t-test).