Análise da inibição de melanina em Cryptococcus e filogenia de genes e proteínas envolvidas na melanização

Abstract

Cryptococcus spp. is a pathogen that causes cryptococcosis, an opportunistic fungal disease with greater clinical manifestation in immunocompromised patients. Pathogenic fungi produce virulence factors that aid in their pathogenicity, such as the polysaccharide capsule, antiphagocytic protein and melanin. Cryptococcus spp. A phenoloxidase enzyme, called laccase, which participates in the production of melanin, is elaborated from L-dopa. The objective of this study was to analyze the inhibition of melanin in strains of Cryptococcus neoformans and Cryptococcus gattii as a virulence factor against some drugs. The strains were isolated in Petri dishes with Niger Agar, observing the intensity of melanization until the 8th day and the data were analyzed using ImageJ software. For the melanin inhibition test, the following inhibitors were used with niger agar in Petri dishes: Omeprazole, Nimesulide, Glyphosate and Cashew bark (Anacardium occidentale) at the following concentrations: 125 µg, 250 µg and 500 µg. Genomic and proteomic data were obtained from NCBI and Uniprot and the protein profiles were sequenced and detailed for construction of similarity trees using platforms such as MEGA and Uniprot. The environmental and clinical strains obtained low melanization rates on the third day, being present by the coloration of the colonies, in white or light brown. However, on the eighth day, the clinical strains remained without high visible melization, but the environmental strains produced high melanization by the dark brown coloration present in the colonies. This coloration of the colonies present in petri dishes containing the following inhibitors and possible concentrations was observed: nimesulide 125 µg/mL, omeprazole 250 µg/mL and 500 µg/mL, cashew bark 250 µg/mL and 500 µg/mL, glyphosate 125 µg/mL, 250 µg/mL and 500 µg/mL. Furthermore, in petri dishes with environmental strains containing other concentrations of the inhibitors used in this study, the coloration of the colonies remained light brown. Therefore, the inhibitors initially have potential effects against melanization, inhibiting enzymes important for fungal growth and colonization. However, Cryptococcus was relatively resistant to inhibitors requiring high concentrations. Furthermore, the strains with the highest melanization rates were the environmental ones, and this may be due to a greater pathogenicity present in these strains.