van Duin David, Casadevall Arturo, Nosanchuk Joshua D
Department of Medicine, Division of Infectious Diseases. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Antimicrob Agents Chemother. 2002 Nov;46(11):3394-400. doi: 10.1128/AAC.46.11.3394-3400.2002.
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.
新型隐球菌和荚膜组织胞浆菌这两种真菌病原体在体外存在左旋多巴的情况下以及在哺乳动物感染期间会产生类黑色素。我们研究了黑色素化是否会影响这些真菌对两性霉素B、卡泊芬净、氟康唑、伊曲康唑或氟胞嘧啶（5FC）的敏感性。使用美国国家临床实验室标准委员会针对酵母的标准宏稀释MIC方案（M27A方案），我们发现黑色素化和未黑色素化的新型隐球菌及荚膜组织胞浆菌分离株在敏感性上没有差异。杀菌试验表明，黑色素化降低了这两种真菌对两性霉素B和卡泊芬净的敏感性。用左旋多巴培养的漆酶缺陷型新型隐球菌细胞比同基因产生黑色素的酵母对两性霉素B或卡泊芬净的杀伤作用明显更敏感。将两性霉素B或卡泊芬净与黑色素预孵育会降低它们的抗真菌活性。对与两性霉素B或卡泊芬净一起孵育的黑色素进行元素分析发现，黑色素的C:N比发生了变化，这表明这些药物与黑色素结合。相比之下，将氟康唑、伊曲康唑或5FC与黑色素一起孵育不会显著影响药物的抗真菌效力或黑色素的化学成分。这些结果为卡泊芬净在体内对新型隐球菌无效（尽管在体外有活性）提供了一个潜在的解释。此外，结果表明真菌黑色素可保护新型隐球菌和荚膜组织胞浆菌免受两性霉素B和卡泊芬净的作用，并且这种保护作用在标准肉汤宏稀释试验中无法体现。
