Comparisons of the Laccase Gene among Serotypes and Melanin-Deficient Variants ofCryptococcus neoformans

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Abstract
Cryptococcus neoformans is a fungus causing life-threatening infections in immunocompromised hosts. Melanin production is a major virulence factor of this fungus and the initial steps of dihydroxyphenylalanine (DOPA)-melanin biosynthesis pathways are catalyzed by laccase. To understand phylogenetic relationships among serotypes of three varieties, partial sequences (about 600 bases) of the laccase gene (CNLAC1) were determined in a total of 64 strains, including 10 melanin-deficient variants. The phylogenetic tree constructed from the nucleotide sequence grouped the 64 strains into the clusters corresponding to the three varieties. The diversity of the fragment sequences was very minor among strains of each of var. grubii and var. neoformans. Strains in var. gattii, however, were subdivided into two groups, although differences between serotypes B and C were not large. The sequences of the melanin-deficient variants were almost completely homologous to those of the melanin-producing strains in the same serotype. Results of laccase assay and northern blot analysis suggested that the lower melanin production in the variants was associated with lower transcription of the laccase gene.