Detection of Melanin-Like Pigments in the Dimorphic Fungal PathogenParacoccidioides brasiliensisIn Vitro and during Infection

Abstract
Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogenParacoccidioides brasiliensisproduce melanin or melanin-like compounds in vitro and during infection. Growth ofP. brasiliensismycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium withl-3,4-dihydroxyphenylalanine (l-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated 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, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro whenP. brasiliensiswas grown inl-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis froml-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected withP. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology ofP. brasiliensisyeasts. These data strongly suggest thatP. brasiliensispropagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.