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Infection and Immunity, September 2001, p. 5760-5767, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5760-5767.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Detection of Melanin-Like Pigments in the Dimorphic Fungal Pathogen Paracoccidioides brasiliensis In Vitro and during Infection

Beatriz L. Gómez,1,2 Joshua D. Nosanchuk,3 Soraya Díez,1,2 Sirida Youngchim,1,4 Philip Aisen,5 Luz E. Cano,2 Angela Restrepo,2 Arturo Casadevall,3,6 and Andrew J. Hamilton1,*

Dermatology Department, St. Johns Institute of Dermatology, Guy's Hospital, Guy's, Kings and St. Thomas Medical Schools, London, United Kingdom1; Corporación para Investigaciones Biológicas, Medellín, Colombia2; Department of Medicine,3 Department of Physiology and Biophysics,5 and Department of Microbiology and Immunology,6 Albert Einstein College of Medicine, Bronx, New York; and Microbiology Department, Chiang-Mai Medical School, Chiang-Mai, Thailand4

Received 13 February 2001/Accepted 28 May 2001

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 pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with L-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 when P. brasiliensis was grown in L-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from L-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. 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 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.

* Corresponding author. Mailing address: Dermatology Laboratory, 5th Floor, Thomas Guy House, Guys Hospital, London, SE1 9RT, United Kingdom. Phone: (44) (0) 20 7955 4663. Fax: (44) (0) 20 7955 2103. E-mail: andrew.j.hamilton{at}kcl.ac.uk.

Infection and Immunity, September 2001, p. 5760-5767, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5760-5767.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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