Youngchim Sirida, Morris-Jones Rachael, Hay Roderick J, Hamilton Andrew J
Dermatology Department, St Johns Institute of Dermatology, Guy's Hospital, Kings and St Thomas' Medical Schools, London, UK 2Microbiology Department, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand 3Faculty of Medicine and Health Sciences, Queen University, Belfast, Northern Ireland, UK.
J Med Microbiol. 2004 Mar;53(Pt 3):175-181. doi: 10.1099/jmm.0.05421-0.
Melanins, or melanin-like compounds, may play a role in the pathogenesis of a number of human fungal infections. This study investigated the production of melanin by the important opportunistic pathogen Aspergillus fumigatus. Conidia from A. fumigatus were harvested and treated with proteolytic enzymes, denaturant and hot, concentrated acid; this yielded dark particles which were similar in size and shape to the original propagules. Electron spin resonance spectroscopy revealed that the conidial-derived particles were stable free radicals consistent with an identification as melanin. Melanin particles were used to immunize BALB/c mice in order to produce a total of five anti-melanin monoclonal antibodies (mAbs). The latter mAbs were strongly reactive both with intact conidia and with extracted melanin particles by ELISA and immunofluorescence reactivity. Immunofluorescence labelling with the novel mAbs was used to examine the temporal expression of melanin during in vitro culture of A. fumigatus--melanization was confined to conidial structures and was absent from hyphae. SDS-PAGE L-3,4-dihydroxyphenylalanine (L-DOPA) substrate analysis confirmed the presence of a laccase-type activity in conidial extracts, but not in hyphae. Melanin-binding mAbs were used to detect the presence of melanized conidia in three patients with nasal aspergilloma, indicating that in vivo melanization may occur during infection.
黑色素或类黑色素化合物可能在多种人类真菌感染的发病机制中起作用。本研究调查了重要的机会致病菌烟曲霉产生黑色素的情况。收集烟曲霉的分生孢子，并用蛋白水解酶、变性剂和热的浓酸处理；这产生了大小和形状与原始繁殖体相似的深色颗粒。电子自旋共振光谱显示，分生孢子衍生的颗粒是稳定的自由基，与黑色素的鉴定结果一致。黑色素颗粒用于免疫BALB/c小鼠，以产生总共五种抗黑色素单克隆抗体（mAb）。通过ELISA和免疫荧光反应性，后一种单克隆抗体与完整的分生孢子和提取的黑色素颗粒都有强烈反应。用新型单克隆抗体进行免疫荧光标记，以检测烟曲霉体外培养过程中黑色素的时间表达——黑色素化仅限于分生孢子结构，菌丝中不存在。SDS-PAGE L-3,4-二羟基苯丙氨酸（L-DOPA）底物分析证实，分生孢子提取物中存在漆酶型活性，但菌丝中不存在。黑色素结合单克隆抗体用于检测三名鼻曲霉菌瘤患者中黑色素化分生孢子的存在，表明在感染过程中可能发生体内黑色素化。
