Department of Pathology and Legal Medicine, School of Medicine, Specialized Medical Mycology Center, Postgraduate Program in Medical Microbiology, Federal University of Ceará, Fortaleza, CE, Brazil.
School of Veterinary Medicine, Postgraduate Program in Veterinary Sciences, State University of Ceará, Fortaleza, CE, Brazil.
Mycoses. 2017 Oct;60(10):697-702. doi: 10.1111/myc.12650. Epub 2017 Jul 12.
Melanin is an important virulence factor for several microorganisms, including Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato, thus, the assessment of melanin production and its quantification may contribute to the understanding of microbial pathogenesis. The objective of this study was to standardise an alternative method for the production and indirect quantification of melanin in C. neoformans sensu lato and C. gattii sensu lato. Eight C. neoformans sensu lato and three C. gattii sensu lato, identified through URA5 methodology, Candida parapsilosis ATCC 22019 (negative control) and one Hortaea werneckii (positive control) were inoculated on minimal medium agar with or without L-DOPA, in duplicate, and incubated at 35°C, for 7 days. Pictures were taken from the third to the seventh day, under standardised conditions in a photographic chamber. Then, photographs were analysed using grayscale images. All Cryptococcus spp. strains produced melanin after growth on minimal medium agar containing L-DOPA. C. parapsilosis ATCC 22019 did not produce melanin on medium containing L-DOPA, while H. werneckii presented the strongest pigmentation. This new method allows the indirect analysis of melanin production through pixel quantification in grayscale images, enabling the study of substances that can modulate melanin production.
黑色素是包括新型隐球菌复合体和格特隐球菌复合体在内的几种微生物的重要毒力因子，因此，评估黑色素的产生及其定量可能有助于了解微生物的发病机制。本研究的目的是标准化一种用于新型隐球菌复合体和格特隐球菌复合体黑色素产生和间接定量的替代方法。通过 URA5 方法鉴定了 8 株新型隐球菌复合体和 3 株格特隐球菌复合体、假丝酵母 ATCC 22019（阴性对照）和 1 株 Werneckii 拟青霉（阳性对照），在含有或不含有 L-DOPA 的最小培养基琼脂上进行了双重接种，并在 35°C 下孵育 7 天。在摄影室内的标准条件下，从第 3 天到第 7 天拍摄照片。然后，使用灰度图像分析照片。所有新型隐球菌复合体菌株在含有 L-DOPA 的最小培养基琼脂上生长后产生黑色素。假丝酵母 ATCC 22019 在含有 L-DOPA 的培养基上不产生黑色素，而 Werneckii 拟青霉呈现出最强的色素沉着。这种新方法可以通过灰度图像中的像素定量来间接分析黑色素的产生，从而研究可以调节黑色素产生的物质。
