Basidiomycete metabolites attenuate virulence properties of Candida albicans in vitro

Abstract

Secreted aspartic proteases (Saps) represent an important virulence factor facilitating fungal adherence. Several protease inhibitors (PIs), including HIV PIs, have been shown to reduce Candida adhesion. The aim of this study was to ascertain whether or not the recently discovered PIs Aureoquinone and Laccaridiones A and B, isolated from Basidiomycete cultures, or Bestatin, act as Sap-inhibitors and/or inhibitors of fungal adhesion. Drug effects on candidial Sap-production were determined by Sap-ELISA. Control tubes, in the absence of drug, served as positive controls, while tubes excluding both drug and proteinase induction medium were used as negative controls. Aureoquinone as well as Laccaridiones A and B, but not Bestatin, significantly inhibited Candida albicans adhesion to both epithelial and endothelial cells in a dose dependent manner and also reduced Sap-release (effects were not because of a direct interaction of the Basidiomycete metabolites with secreted Saps). Laccaridione B was consistently found to be the most effective PI tested. Interestingly, these drugs are neither fungistatic nor fungicidal at the concentrations applied. Laccaridione B may represent a promising novel type of antimycotic drug--targeting virulence factors without killing the yeast.

Figure 1

(a) Effect of Aureoquinone on CBS 5982 adherence to HeLa S3. The light grey bars mark controls, containing solely the same percentage of methanol as used for dissolving the drug at that concentration (namely, 5% for 50 μg ml⁻¹, 1% for 10 μg ml⁻¹ and 0.2% for 2 μg ml⁻¹). Data shown represent mean ± SD of five experiments. **: highly significant (P ≤ 0.01); statistical significance was determined using Student’s t-test analysis. (b) Effect of Laccaridione A on CBS 5982 adherence to HeLa S3. Methanol was 10% at a drug concentration of 50 μg ml⁻¹, 2% at 10 μg ml⁻¹ and 0.4% for 2 μg ml⁻¹ respectively. Data shown represent mean ± SD of three experiments. **: highly significant (P ≤ 0.01); *: significant (P ≤ 0.05). (c) Effect of Laccaridione B on CBS 5982 adherence to HeLa S3. Details as described in (b). (d) Effect of Laccaridione B on CBS 5982 adherence to EAhy 926. Details as described in (b).

Figure 2

(a) Effect of Aureoquinone on Sap-release of CBS 5982. Drug addition was performed every 48 h. The percentage of methanol used for dissolving the drug was 5% for a Aureoquinone concentration of 50 μg ml⁻¹ and 3% for 30 μg ml⁻¹. Pos. Co. denotes positive control of CBS 5982 in Sap-induction medium; Co. + NaCl denotes control + NaCl and served as negative control along with PBS-T. Extinction was determined at 405 nm with a reference wave length of 492 nm. Data shown represent mean ± SD of four experiments. **: highly significant (P ≤ 0.01); *: significant (P ≤ 0.05); statistical significance was determined using Student’s t-test analysis. (b) Effect of Laccaridione B on Sap-release of CBS 5982. The percentage of methanol used for dissolving the drug was 10% for a Laccaridione B concentration of 50 μg ml⁻¹, 6% for 30 μg ml⁻¹ and 2% for 10 μg ml⁻¹. Further details are as described in (a). (c) Effect of Laccaridione B on Sap-release of CBS 5982. Drug addition was performed once only at the start of the experiment.

Figure 3

(a). Sap expression and PI activity determined by BSA cleavage of supernatants derived from CBS 5982 incubated for 8 days in PIM supplemented with BSA and vitamins in the absence or presence of PI or methanol (SDS-PAGE). Lanes 1 and 2, plus 2% and 10% methanol, respectively; 3, plus Indinavir [0.1 mmol l⁻¹]; 4 and 5, plus Laccaridione B at concentrations of 50 μg ml⁻¹ and 10 μg ml⁻¹, respectively; 6, molecular weight standards (kDa); 7 and 8, plus freshly added Laccaridione B applied to the 8-day-incubation samples at concentrations of 10 μg ml⁻¹ and 50 μg ml⁻¹, respectively; 9, just BSA with supernatant; 10, just BSA; + denotes visible BSA-cleavage; − denotes inhibition or absence of BSA-cleavage; M denotes molecular weight standards; C denotes control. (b) Sap expression and PI-activity determined by BSA cleavage of supernatants derived from CBS 5982 incubated for seven days in PIM supplemented with BSA and vitamins in the absence or presence of PI or methanol (SDS-PAGE and western blot). Lane 1, molecular weight standard (kDa); 2 and 3, plus freshly added Laccaridione B applied to the 8-day-incubation samples at drug concentrations of 50 μg ml⁻¹ and 10 μg ml⁻¹, respectively; 4 and 5, plus Laccaridione B at concentrations of 50 μg ml⁻¹ and 10 μg ml⁻¹, respectively; 6, BSA only; 7, plus freshly added Indinavir [0.1 mmol l⁻¹]; 8, BSA with CBS 5982 and PIM (=control); 9 and 10, plus 10% and 2% methanol, respectively; + denotes visible BSA-cleavage; − denotes inhibition or absence of BSA-cleavage; M denotes molecular weight standards; C denotes control.