Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection

Melanins are implicated in the pathogenesis of several human diseases, incl uding some microbial infections. In this study, we analyzed whether the con idia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidio ides brasiliensis produce melanin or melanin-like compounds in vitro and du ring infection. Growth of P. brasiliensis mycelia on water agar alone produ ced pigmented conidia, and growth of yeasts in minimal medium with L-3,4-di hydroxyphenylalanine (L-DOPA) produced pigmented cells. Digestion of the pi gmented conidia and yeasts with proteolytic enzymes, denaturant, and hot co ncentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeas ts, and particles. Electron spin resonance spectroscopy identified the yeas t-derived particles produced in vitro when P. brasiliensis was grown in L-D OPA medium as a melanin-like compound. Nonreducing polyacrylamide gel elect rophoresis of cytoplasmic yeast extract revealed a protein that catalyzed m elanin synthesis from L-DOPA. The melanin binding MAb reacted with yeast ce lls in tissue from mice infected with A brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb tha t had the typical morphology of A brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on wh at is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.