Vesicle inhibition reduces Candida biofilm resistance

Abstract

Candida biofilm matrix components are delivered to the extracellular space by vesicles where they deposit and confer biofilm-associated drug resistance. Here, we present evidence that drugs designed to inhibit mammalian exosome production exhibit similar effects on C. albicans extracellular vesicles, ultimately eliminating biofilm matrix assembly. We find that vesicle reduction renders biofilm communities susceptible to the antifungal fluconazole. Our findings argue that vesicle trafficking pathways represent a promising target to optimize for recalcitrant fungal biofilms.

Keywords: Candida albicans; antifungal resistance; biofilms; extracellular matrix; vesicles.

Fig 1

Impact of EV inhibitors on C. albicans biofilms. (A) The percent reduction in biofilm formation was measured using the XTT assay following treatment with either fluconazole (1,000  µg/mL) or a human EV inhibitor (8 µg/mL) [simvastatin, omeprazole, imipramine, indomethacin, or glyburide] alone or combined, compared to untreated biofilms. Each dot represents an independent biological replicate and reflects the mean of three technical replicates. Error bars denote standard deviation. A nonparametric Kruskal–Wallis one-way analysis of variance with an uncorrected Dunn’s multiple comparison test was performed. Indicated P values: *, P <0.05; **, P <0.01; ***, P <0.005; ****. (B) Quantification of in vivo biofilms using the rat central venous catheter model following antifungal therapy with either fluconazole (250  µg/mL) or a human EV inhibitor (32  µg/mL) [simvastatin or omeprazole] alone or in combination, compared to 0.9 M NaCl followed by the CFU analysis. Three animals and culture replicates per condition, n = 3. Error bars represent standard deviation. Nonparametric Kruskal–Wallis one-way analysis of variance with uncorrected Dunn’s multiple comparison test was performed. Indicated P values, *, P, 0.05; **, P, 0.01; ***, P, 0.005; ****. (C) SEM of C. albicans biofilms grown on coverslips in control and human EV inhibitor-treated biofilms (8  µg/mL) [omeprazole, simvastatin, or imipramine]. Arrows highlight the blue pseudocolored extracellular matrix. Scale bars: 40 µm. (D) Quantitative analysis of EV concentration in C. albicans biofilms in a 6-well biofilm assay using NTA following treatment with a human EV inhibitor (8 µg/mL) [simvastatin, omeprazole, or imipramine]. Each dot represents an independent biological replicate and reflects the mean of three technical replicates. Error bars denote standard deviation. A nonparametric Kruskal–Wallis one-way analysis of variance with an uncorrected Dunn’s multiple comparison test was performed, with a significant P value indicated *, P, 0.05; **, P, 0.01; ***, P, 0.005; ****. (E) The percent reduction in biofilm formation was measured using the XTT assay following treatment with fluconazole (1,000  µg/mL) plus a human EV inhibitor (8 µg/mL) [simvastatin or omeprazole] alone or combined with C. albicans biofilm EVs (4.3 × 10⁶ ± 1.4 × 10⁵ particles/mL) after 48 h of growth. Each dot represents an independent biological replicate and reflects the mean of three technical replicates. Error bars denote standard deviation. A nonparametric Kruskal–Wallis one-way analysis of variance with an uncorrected Dunn’s multiple comparison test was performed, with a significant P value indicated *, P, 0.05; **, P, 0.01; ***, P, 0.005; ****.