Melanization of Cryptococcus neoformans and Histoplasma capsulatum Reduces Their Susceptibilities to Amphotericin B and Caspofungin

Chemicals.Glucose, MgSO₄, and KH₂PO₄ were purchased from J. T. Baker Inc. (Philipsburg, N.J.). Glycine, NaCl, RPMI 1640 medium (with l-glutamine and without sodium bicarbonate), vitamin B₁, synthetic melanin (prepared by the oxidation of tyrosine with hydrogen peroxide), morpholinepropanesulfonic acid (MOPS), and l-dopa were purchased from Sigma Chemical Co. (Cleveland, Ohio). Amphotericin B was purchased from Bristol-Myers Squibb (New York, N.Y.), fluconazole was purchased from Pfizer (New York, N.Y.), flucytosine (5FC) was purchased from Roche (Nutley, N.J.), itraconazole was purchased from Janssen (Spring House, Pa.), and caspofungin was purchased from Merck (Whitehouse Station, N.J.). Caspofungin was used in these experiments, even though it has limited clinical efficacy against these pathogens, to establish proof of principle for the effect of melanin on antifungal drugs.

C. neoformans and H. capsulatum.C. neoformans strain 24067 (serotype D) and H. capsulatum strain G217B were obtained from the American Type Culture Collection (Manassas, Va.). C. neoformans H99 (serotype A) was obtained from J. Perfect (Durham, N.C.). A pair of congenic strains, 2E-TUC (laccase positive) and 2E-TU (laccase deficient), were gifts from P. R. Williamson (Chicago, Ill.) (32). H. capsulatum strain CIB 1980 was a gift from A. Restrepo (Medellín, Colombia). Melanization was induced by growing yeast cells on defined minimal medium agar plates (15 mM glucose, 10 mM MgSO₄, 29.4 KH₂PO₄, 13 mM glycine, 3.0 μM vitamin B₁, 2% agar; Difco) with the addition of 1 mM l-dopa for 10 days. Nonmelanized controls were obtained by growing yeast cells on defined minimal medium agar plates without l-dopa for 10 days. C. neoformans and H. capsulatum cells were grown at 30 and 37°C, respectively. Growth of C. neoformans strain 2E-TU in agar with l-dopa resulted in albino colonies.

MIC determination.MICs were determined by the standardized protocol (the M27A protocol) for yeasts developed by the National Committee for Clinical Laboratory Standards (NCCLS) (24). Briefly, melanized or nonmelanized yeast cells were suspended in sterile normal saline and diluted to a concentration of 2 × 10⁶ cells/ml. Cell counts were determined with a hemacytometer. The suspensions were diluted 1:1,000 in RPMI 1640 medium with l-glutamine, without bicarbonate, and buffered to pH 7.0 with 0.165 M MOPS. Polystyrene tubes containing 0.1-ml aliquots of an antifungal at 10 times the final drug concentration were inoculated with 0.9 ml of the diluted suspensions. Final drug concentrations ranged from 0.0625 to 4 μg/ml for amphotericin B, 0.125 to 16 μg/ml for fluconazole and 5FC, 0.0625 to 4 μg/ml for itraconazole, and 0.125 to 64 μg/ml for caspofungin. The MICs were recorded after incubation at 35°C (C. neoformans strains 24067 and H99) or 37°C (H. capsulatum strain CIB 1980) for 72 h. The MICs of amphotericin B and caspofungin were defined as the lowest concentrations at which there was an absence of growth. The MICs of fluconazole, itraconazole, and 5FC were defined as the lowest drug concentrations which achieved 80% growth inhibition compared to the growth of the drug-free control.

Killing assay.Yeast cells were suspended in sterile normal saline at a density of 2.2 × 10³ cells/ml. Cell counts were determined with a hemacytometer. Microcentrifuge tubes containing 0.1-ml aliquots of an antifungal at 10 times the final concentration were inoculated with 0.9 ml of the yeast suspensions. For C. neoformans, final drug concentrations ranged from 0.125 to 0.5 μg/ml for amphotericin B, 16 to 64 μg/ml for caspofungin, 0.25 to 4 μg/ml for 5FC and fluconazole, and 0.125 to 0.5 μg/ml for itraconazole. For H. capsulatum, final drug concentrations ranged from 0.125 to 0.5 μg/ml for amphotericin B, 2 to 8 μg/ml for caspofungin, 0.25 to 4 μg/ml for 5FC and fluconazole, and 0.125 to 0.5 μg/ml for itraconazole. After incubation at 35°C (C. neoformans) or 37°C (H. capsulatum) for 2 h, aliquots were plated on Sabouraud dextrose agar (C. neoformans) or brain heart infusion agar (H. capsulatum) to determine their viabilities, as measured by determination of the numbers of CFU. The rate of survival was compared to that of fungal cells incubated in phosphate-buffered saline (PBS).

Melanin ghosts.The term “melanin ghosts” refers to the melanin structures isolated from melanized C. neoformans. The ghosts were obtained from melanized C. neoformans cells as described previously (31). Briefly, melanized cryptococcal cells were serially treated with 10 mg of cell wall-lysing enzymes (from Trichoderma harzianum; Sigma) per ml, 4.0 M guanidine thiocyanate, and 1.0 mg of proteinase K per ml, extracted three times with chloroform, and boiled in 6.0 M HCl for 1 h. The resulting black debris was then dialyzed against distilled H₂O (dH₂O) for 10 days. Nonmelanized cryptococcal cells that are subjected to this protocol are completely solubilized.

Binding of antifungal drugs to melanin.Two milliliters each of stock solutions of fluconazole (5 mg/ml in dH₂O), amphotericin B (0.25 mg/ml in dimethyl sulfoxide), and caspofungin (5 mg/ml in dimethyl sulfoxide) was incubated with cryptococcal melanin (7.7 × 10⁹ particles/ml) and synthetic melanin (20 mg) for 2 h at 30°C. Itraconazole and 5FC (5 mg/ml in dH₂O) were incubated with synthetic melanin. The melanins were removed by centrifugation. The supernatants of the amphotericin B and caspofungin solutions were used in the killing assay with C. neoformans strain 24067 and H. capsulatum strain CIB 1980. The supernatants of the fluconazole, itraconazole, and 5FC solutions were used for MIC determinations with C. neoformans strain 24067 and H. capsulatum strain CIB 1980. The melanin pellets from the incubated solutions were washed extensively with dH₂O and lyophilized with a Flexi-Dry microprocessor (FTS Systems, Inc., Stone Ridge, N.Y.). As controls, cryptococcal melanin and synthetic melanin in dH₂O were lyophilized. Quantitative elemental analysis of the melanins was performed by Quantitative Technologies Inc. (Whitehouse, N.J.). Briefly, the melanin samples were converted into gases by combustion, and the product gases were separated under steady-state conditions. The percentage of each element was measured as a function of thermal conductivity. Elemental ratios were calculated by dividing the percentage of each element measured by its respective atomic weight.

Statistics.Microsoft Excel 97 software was used to determine the standard deviations, standard errors of the means, and P values (Student's t test).

MICs.Table 1 summarizes the in vitro susceptibilities of melanized versus nonmelanized cells of C. neoformans strains 24067 and H99 and H. capsulatum strain CIB 1980 to amphotericin B, caspofungin, fluconazole, itraconazole, and 5FC. The MICs were within the range of values previously reported for these fungi (9, 16, 18, 21, 36), except for the relatively low MIC of caspofungin for H. capsulatum (16, 18). There were no significant differences in the MICs for melanized and nonmelanized cells of the same strain for any of the drugs tested. For H. capsulatum, an extended incubation time for MIC testing has been described (29, 36). Even though there was sufficient growth to determine the MICs at 72 h, we also measured the MICs for H. capsulatum at 96 to 144 h. These tests also showed no differences in the MICs for the melanized and the nonmelanized cells (data not shown).

In an attempt to determine whether the inability to demonstrate differences between melanized and nonmelanized cells was due to the absence of substrate for further melanin formation in the melanized cells and their progeny (J. Nosanchuk, A. L. Rosas, and A. Casadevall, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. F13, 1999), we added l-dopa to the RPMI medium. However, this resulted in the formation of black precipitates when the antifungals were added to the medium, whether or not yeast cells were present. We also tried a modification of the NCCLS protocol using yeast nitrogen base medium buffered at a pH of 7.0 instead of RPMI medium (10, 37). Unfortunately, this modification was not successful since precipitates also formed when antifungals and l-dopa were added to this medium in the presence or absence of cells. The precipitates could be a complex of drug and l-dopa or could represent the induction of auto-oxidation of l-dopa by the antifungals. Other precursors, such as epinephrine, caused similar precipitates. Hence, we were unable to modify the assay to investigate this question.

Killing assay.Melanized cells were significantly less susceptible to amphotericin B or caspofungin than nonmelanized cells (Fig. 1 and 2). The differences were statistically significant for all concentrations tested with C. neoformans (Fig. 1). Melanized cells of H. capsulatum were significantly protected against amphotericin B compared to the susceptibilities of nonmelanized cells at concentrations <0.25 and <0.5 μg/ml for strains CIB 1980 and G217B, respectively. Melanized C. neoformans cells were also less susceptible than nonmelanized cells to caspofungin at all concentrations tested, except for strain 24067 with caspofungin at 64 μg/ml (Fig. 2). For H. capsulatum, significant differences in rates of survival between melanized and nonmelanized cells were demonstrated at concentrations >2 and >4 μg/ml for strains CIB 1980 and G217B, respectively. With fluconazole, itraconazole, and 5FC, there were no significant differences in rates of survival for melanized versus nonmelanized cells (data not shown).

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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.

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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.

To further validate these findings, we investigated the effects of antifungal agents on laccase-deficient C. neoformans strain 2E-TU, which does not melanize in the presence of l-dopa. The MICs, determined by the NCCLS M27A protocol, for melanized and nonmelanized cells of strains 2E-TU and 2E-TUC were similar (Table 2). However, melanized C. neoformans strain 2E-TUC was significantly less susceptible to killing by either antifungal than strain 2E-TU (laccase-deficient) incubated with l-dopa or either strain grown without l-dopa (P < 0.001 for all concentrations tested) (Fig. 3). This demonstrates that the protective effect seen in cells grown in the presence of l-dopa occurs only if the yeast cells produce melanin.

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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.

Binding of antifungals to melanin.To evaluate whether the results of the killing assays were due to binding of the drugs to melanin, we incubated the antifungals with melanin before we performed the killing assays or determined the MICs. Incubation of amphotericin B or caspofungin with either C. neoformans or synthetic melanin prior to use significantly reduced the efficacies of the drugs compared to their efficacies in assays in which the antifungals were not preincubated with melanin (Fig. 4). The MICs of fluconazole, itraconazole, and 5FC determined after incubation with melanin were unchanged (data not shown).

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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).

The results of the elemental analysis of the melanins incubated with the antifungals are shown in Table 3. The analysis was performed twice with melanins from different experiments, with similar results obtained in each experiment. Incubation of melanin with amphotericin B or caspofungin resulted in alterations of the C:N ratios of the melanins, which is consistent with the fact these drugs had bound to the melanins. In contrast, incubation of melanin with the azoles or 5FC did not alter the melanin C:N ratios. Additionally, analysis of the melanins revealed no fluorine after incubation with either fluconazole or 5FC and no chloride after incubation with itraconazole (data not shown). These results are consistent with the notion that caspofungin and amphotericin B bind to melanin, whereas fluconazole, itraconazole, and 5FC do not.