Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibilities to amphotericin B and caspofungin

Abstract

The fungal pathogens Cryptococcus neoformans and Histoplasma capsulatum produce melanin-like pigments in the presence of L-dopa in vitro and during mammalian infection. We investigated whether melanization affected the susceptibilities of the fungi to amphotericin B, caspofungin, fluconazole, itraconazole, or flucytosine (5FC). Using the standard macrodilution MIC protocol (the M27A protocol) of the National Committee for Clinical Laboratory Standards for yeast, we found no difference in the susceptibilities of melanized and nonmelanized C. neoformans and H. capsulatum isolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficient C. neoformans cells grown with L-dopa were significantly more susceptible than congenic melanin-producing yeast to killing by amphotericin B or caspofungin. Preincubation of amphotericin B or caspofungin with melanins decreased their antifungal activities. Elemental analysis of melanins incubated with amphotericin B or caspofungin revealed an alteration in the C:N ratios of the melanins, which indicated binding of these drugs by the melanins. In contrast, incubation of fluconazole, itraconazole, or 5FC with melanins did not significantly affect the antifungal efficacies of the drugs or the chemical composition of the melanins. The results suggest a potential explanation for the inefficacy of caspofungin against C. neoformans in vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protect C. neoformans and H. capsulatum from the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.

FIG. 1.

Amphotericin B killing assay. The rates of survival of melanized versus nonmelanized yeast cells (C. neoformans strains H99 [CN H99] and 24067 [CN 24067] and H. capsulatum strains CIB 1980 [HC CIB 1980] and G217B [HC G217B]) after exposure to various concentrations of amphotericin B for 2 h were compared to those of fungal cells incubated in PBS. Values are averages ± standard errors of the means for four measurements. P values were calculated by comparing nonmelaninzed and melanized cells. P values were <0.01 unless noted otherwise (∗, P = 0.06; ∗∗, P = 0.1) (Student's t test). Similar results were obtained in two separate experiments.

FIG. 2.

Caspofungin killing assay. The rates of survival of melanized versus nonmelanized yeast cells (C. neoformans strains H99 [CN H99] and 24067 [CN 24067] and H. capsulatum strains CIB 1980 [HC CIB 1980] and G217B [HC G217B]) after exposure to various concentrations of caspofungin for 2 h were compared those of fungal cells incubated in PBS. Values are averages ± standard errors of the means for four measurements. P values were calculated by comparing nonmelaninzed and melanized cells. ∗, P < 0.001; ∗∗, P < 0.05 (Student's t test). Similar results were obtained in two separate experiments.

FIG. 3.

Killing assay with C. neoformans congenic strains 2E-TUC (laccase positive) and 2E-TU (laccase deficient). Yeast cells were grown with or without l-dopa for 10 days and then exposed to various concentrations of amphotericin B or caspofungin for 2 h, and the rates of killing were compared to those for fungal cells incubated in PBS. Values are averages ± standard errors of the means for three measurements. Melanized 2E-TUC yeast cells were significantly (P < 0.001) less susceptible to killing under each condition examined.

FIG. 4.

Killing assay with melanins. The rates of survival of nonmelanized yeast cells (C. neoformans strain H99 [CN H99] and H. capsulatum strain CIB 1980 [HC CIB 1980]) exposed to amphotericin B or caspofungin with or without preincubation of the antifungals with C. neoformans or synthetic melanin were compared. Values are averages ± standard errors of the means for four measurements. P values refer to the difference between the rates of survival for nonmelanized yeast cells after preincubation of the antifungals with C. neoformans or synthetic melanin and those for cells not preincubated with synthetic melanin (control). All P values were <0.001 unless noted otherwise (∗, P < 0.01) (Student's t test).