Melanins: The dark side of Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a free-living fungus that causes disease predominantly in immunocompromised individuals. The ability of this fungus to synthesize melanins from exogenous substrates has been associated with protection against harsh environment conditions and with virulence. However, it has not been established whether C. neoformans is melanized in the environment or in infected tissues. To address these questions, we undertook two approaches: one involved the generation of monoclonal antibodies (MAbs) to melanin for use in immunofluorescence (IF) analyses of C. neoformans in pigeon excreta (a major environmental niche) and in infected tissues, and the other sought to isolate C. neoformans -derived melanin by enzymatic and chemical digestion of the cells.;IF analyses of C. neoformans in pigeon excreta demonstrated binding of the MAbs to the fungal cell wall. Protease, denaturant, and boiling HCl digestion of C. neoformans in pigeon excreta yielded melanin-like particles. These results strongly suggest that C. neoformans synthesizes melanins in pigeon excreta and imply that infection initially involves exposure to melanized cells. We also demonstrated that in vitro melanization protects C. neoformans against extremes of temperature and enzymatic degradation, which suggests that melanins could play important protective roles for C. neoformans in the environment.;IF analyses of C. neoformans in experimentally infected rodent tissues and in infected human brain tissue demonstrated binding of the MAbs to the fungal cell wall. Digestion of C. neoformans from infected tissues yielded melanin-like particles. Furthermore, immunization with the MAbs to melanin prolonged the survival of and reduced the fungal burden in C. neoformans-infected mice compared to mice immunized with NSO ascites. These results strongly suggest that C. neoformans synthesizes melanins during infection and imply that melanins could be targets for antimicrobial therapy.;We also studied the interaction of melanins with the immune system. Fungal DOPA-melanin elicited intense T cell independent IgM and IgG responses in mice, with predominance of IgG2a and IgG2b. Fungal melanins also induced the activation of the complement system in vitro and in vivo. The finding that melanins are immunologically active has important implications for the role of these compounds in the pathogenesis of several infectious, degenerative, and autoimmune diseases.