MELANOGENESIS IN CRYPTOCOCCUS NEOFORMANS

Melanin synthesis has been associated with virulence for a variety of microbial pathogens. However, little is known about either melanogenesis or the structure of melanin. The experimental system that Dr. Nosanchuk will study is that of Cryptococcus neoformans, a pathogenic fungus which causes life-threatening meningitis in 5-10 percent of patients with AIDS. This system has several unique advantages for the study of melanin and virulence, including the fact that melanized and non-melanized cells can be easily generated by simply growing the organism with or without a variety of phenoloxidase substrates. In addition, melanogenesis in C. neoformans is catalyzed by a single enzyme. Dr. Nosanchuk proposes a novel approach to the study of melanin and melanogenesis C. neoformans by applying the technology of phage display libraries to identify melanin-binding peptides that will comprise unique reagents for the study of melanin. Furthermore, murine monoclonal antibodies (mAbs) will be generated that bind melanin. Melanin-binding peptides and mAbs will be used to study melanization in vitro and in vivo. The objectives are to determine whether melanogenesis occurs in vivo and to obtain information on the structure of melanin. Four specific aims are proposed: 1. To identify peptides which bind melanin; 2. To study the antibody response to melanin and generate melanin-binding mAbs; 3. To explore the structure of melanin using biochemistry, phage display peptide libraries and mAbs; and 4. To use peptides and mAbs to study C. neoformans melanogenesis in vivo and in vitro.