Synthesis of Polymerized Melanin by Cryptococcus neoformans in Infected Rodents

The ability of Cryptococcus neoformans to synthesize polymerized melanin in vitro has been associated with virulence, but it is unclear whether this fungus synthesizes polymerized melanin during infection. To study this question, we used two approaches: one involved the generation of monoclonal antibodies (MAbs) to melanin for use in immunohistochemical studies of C. neoformans-infected rodents, and the other sought to isolate fungal melanin from infected tissues. Digestion of in vitro-melanized C. neoformans cells with proteases, denaturant, and hot concentrated acid yields melanin particles that retain the shape of fungal cells and are therefore called melanin ghosts. BALB/c mice were immunized with melanin ghosts, and two immunoglobulin M MAbs to melanin were generated from the spleen of one mouse. Immunofluorescence analyses of lung and brain tissues of rodents infected with wild-type melanin-producing (Mel(+)) C. neoformans strains demonstrated binding of the MAbs to the fungal cell wall. No binding was observed when infections were performed with mutant albino (Mel(−)) C. neoformans strains. Particles with striking similarity to melanin ghosts were recovered after digestion of lung and brain tissues from Mel(+) C. neoformans-infected rodents and were reactive with the MAbs to melanin. No particles were recovered from tissues infected with Mel(−) C. neoformans. A Mel(+) C. neoformans strain grown on lung or brain homogenate agar became lightly pigmented and also yielded particles similar to melanin ghosts upon digestion, providing additional evidence that lung and brain tissues contain substrate for C. neoformans melanization. These results demonstrate that C. neoformans synthesizes polymerized melanin during infection, which has important implications for pathogenesis and antifungal drug development.