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 f ungus synthesizes polymerized melanin during infection. To study this quest ion, we used two approaches: one involved the generation of monoclonal anti bodies (MAbs) to melanin for use in immunohistochemical studies of C. neofo rmans-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 ghos ts. BALB/c mice were immunized with melanin ghosts, and two immunoglobulin M MAbs to melanin were generated from the spleen of one mouse. Immunofluore scence analyses of lung and brain tissues of rodents infected with wild-typ e melanin-producing (Mel(+)) C. neoformans strains demonstrated binding of the MAbs to the fungal cell wall. No binding was observed when infections w ere performed with mutant albino (Mel(-)) C. neoformans strains. Particles with striking similarity to melanin ghosts were recovered after digestion o f lung and brain tissues from Mel(+) C. neoformans-infected rodents and wer e reactive with the MAbs to melanin. No particles were recovered from tissu es infected with Mel- C. neoformans. A Mel(+) C. neoformans strain grown on lung or brain homogenate agar became lightly pigmented and also yielded pa rticles similar to melanin ghosts upon digestion, providing additional evid ence that lung and brain tissues contain substrate for C. neoformans melani zation. These results demonstrate that C. neoformans synthesizes polymerize d melanin during infection, which has important implications for pathogenes is and antifungal drug development.