Voriconazole inhibits melanization in Cryptococcus neoformans

Abstract

Voriconazole is a triazole antifungal drug that inhibits ergosterol synthesis and has broad activity against yeast and molds. While studying the interaction of voriconazole and Cryptococcus neoformans, we noted that cells grown in the presence of subinhibitory concentrations of voriconazole reduced melanin pigmentation. We investigated this effect systematically by assessing melanin production in the presence of voriconazole, amphotericin B, caspofungin, itraconazole, and fluconazole. Only voriconazole impeded the formation of melanin at subinhibitory concentrations. Voriconazole did not affect the autopolymerization of l-dopa, and 0.5 MIC of voriconazole did affect the gene expression of C. neoformans. However, voriconazole inhibited the capacity of laccase to catalyze the formation of melanin. Hence, voriconazole affects melanization in C. neoformans by interacting directly with laccase, which may increase the efficacy of this potent antifungal against certain pigmented fungi.

FIG. 1.

C. neoformans yeast cells were grown on minimal medium-1 mM l-dopa agar plates for 10 days at 30°C with or without the addition of antifungal drugs. (A) Growth of C. neoformans in the absence of antifungal drugs demonstrating dark pigmentation of the colonies and polymerization of the l-dopa in the agar surrounding the colonies due to the secretion of laccase into the medium. (B to D) C. neoformans grown with voriconazole at 0.125 (B), 0.25 (C), and 0.5 (D) MIC showing increasingly lower amounts of melanin formation in the colonies or within the agar. (E to H) There was no significant reduction of melanin production with 0.5 MIC of amphotericin B (E), caspofungin (F), fluconazole (G), or itraconazole (H). Plates were done in triplicate. This experiment was done twice, with similar results each time.

FIG. 2.

Growth curve of C. neoformans with subinhibitory concentrations of voriconazole or fluconazole used in this study.

FIG. 3.

Voriconazole affects C. neoformans LAC1 gene expression at subinhibitory concentrations. *, P < 0.05 in comparison with results for control or fluconazole groups. Error bars show standard deviations. This experiment was done twice, with similar results.

FIG. 4.

Oxidation of ABTS by C. neoformans laccase from intact cells or from supernatants of yeast cell extracts is suppressed by voriconazole. Recombinant laccase from Rhus vernificera incubated with ABTS represents the positive control, and the negative control is ABTS alone. Error bars show standard deviations. The experiment was done twice, and similar results were obtained.

FIG. 5.

The oxidation of ABTS by laccase is reduced by the presence of voriconazole. (A) Increasing concentrations of voriconazole in the presence of a constant amount of laccase. #, P < 0.05; *, P < 0.001. The P values are in comparison to the activity of laccase in the absence of voriconazole. (B) Decreasing concentrations of laccase occur in the presence of a constant amount of voriconazole. **, P < 0.001 in comparison to the activity of the highest concentration of laccase in the absence of voriconazole. Error bars show standard deviations. The experiments were done twice, with similar results.

FIG. 6.

Voriconazole reduces C. neoformans 24067 melanization and alters yeasts phagocytosis by J774.16 cells. Bars are the averages of the results for three wells, and error bars denote standard deviations. *, P < 0.001 in comparison with the results for the control group; #, P < 0.05 in comparing the voriconazole group with the fluconazole group. This experiment was done twice, with similar results.