Read Pukkila-Worley, MSIV, Brandy M. Allen, Quincy Gerrald, J. Gray Kimbrough, Joseph Heitman, and J. Andrew Alspaugh
Departments of Medicine, Molecular Genetics and Microbiology, and Pharmacology and Cancer Biology, and the Howard Hughes Medical Institute
Duke University Medical Center, Durham, NC 27710
The Ga protein Gpa1 and cAMP regulate mating, virulence factor production, and pathogenicity in a serotype A (variety grubii) strain of the pathogenic basidiomycete Cryptococcus neoformans. Here we employed divergent congenic serotype D variety neoformans strains of C. neoformans to further elucidate the roles of the Gpa1-cAMP pathway in virulence factor production. The serotype D GPA1 gene was identified, sequenced, and disrupted by homologous recombination. Similar to previous findings in serotype A, serotype D gpa1 mutant strains exhibited defects in mating, melanin biosynthesis and capsule production that were suppressed by exogenous cAMP. The SMG1 gene encoding a novel aryl-alcohol oxidase homolog was identified as a multicopy suppressor that restores melanin production in serotype D gpa1D mutant cells. Specific overexpression of SMG1 increased melanin production in both wild-type and gpa1 mutant cells. Furthermore, smg1D mutants were unable to produce melanin. Genetic epistasis experiments suggest that the Smg1 protein acts with the laccase CnLac1 as one of several redundant mechanisms for melanin biosynthesis in C. neoformans.