Melanization ofCryptococcus neoformansandHistoplasma capsulatumReduces Their Susceptibilities to Amphotericin B and Caspofungin

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Abstract
The fungal pathogensCryptococcus neoformansandHistoplasma capsulatumproduce melanin-like pigments in the presence ofl-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 nonmelanizedC. neoformansandH. capsulatumisolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficientC. neoformanscells grown withl-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 againstC. neoformansin vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protectC. neoformansandH. capsulatumfrom the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.