Melanin particles isolated from the fungus Fonsecaea pedrosoi activates the human complement system.

Lysianne Pinto,Sonia Rozental,Luiz Fernando Zmetek Granja,Celuta Sales Alviano,Daniela Sales Alviano,Maria Helena Da Silva,Regina Ejzemberg,Mariana Amorim De Almeida

doi:10.1590/0074-02760180120

Lysianne Pinto, Sonia Rozental + Show 6 more

Open Access

https://doi.org/10.1590/0074-02760180120

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Abstract

BACKGROUNDMelanin production has been associated with virulence in various pathogenic fungi, including Fonsecaea pedrosoi, the major etiological agent for chromoblastomycosis, a subcutaneous fungal disease that occurs in South America.OBJECTIVEThe aim of this study was to evaluate the effects of acid-basic extracted F. pedrosoi melanin particles and fungal cell ghosts obtained by Novozym 234 treatment on their ability to activate the human complement system.METHODSThe ability of melanin particles and fungal cell ghosts to activate the human complement system was evaluated by complement consumption, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA).FINDINGSUnsensitised melanin particles and melanin ghosts presented complement consumption of 82.67 ± 2.08% and 96.04 ± 1.13%, respectively. Immunofluorescence assays revealed intense deposition of the C3 and C4 fragments on the surface of melanin particles and ghosts extracted from F. pedrosoi. Deposition of the C3, C4, and C5 fragments onto melanin samples and zymosan was confirmed by ELISA. Deposition of small amounts of C1q and C9 onto melanin samples and zymosan was detected by ELISA.CONCLUSION Fonsecaea pedrosoi melanin particles and fungal cell ghosts activated the complement system mainly through an alternative pathway.

Highlights

Melanin production has been associated with virulence in various pathogenic fungi, including Fonsecaea pedrosoi, the major etiological agent for chromoblastomycosis, a subcutaneous fungal disease that occurs in South America
The ability of melanin particles and fungal cell ghosts to activate the human complement system was evaluated by complement consumption, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA)
Chromoblastomycosis is a chronic progressive fungal infection of the mammalian skin and subcutaneous tissue caused by transcutaneous inoculation of a group of melanised fungi that belong to the Dematiaceae family (Torres-Guerrero et al 2012)

Summary

Methods

The ability of melanin particles and fungal cell ghosts to activate the human complement system was evaluated by complement consumption, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Reagents and buffers - The following buffers were used: PBS (10 mM phosphate, 15 mM saline, pH 7.2), PBS-Tween (PBS containing 0.05% of Tween 20), VBS (5 mM Veronal-buffered saline, pH 7.35-5 mM Veronal, 142 mM), GVB (VBS containing 0,1% gelatine), GVB2+. Tetraacetic acid) and 10 mM MgCl2), and GVB-EDTA (GVB containing 10 mM EDTA), 10 mM sodium citrate-phosphate, pH 5.1. The following antibodies were used: rabbit serum anti-human C3c conjugated with fluorescein isothiocyanate and rabbit serum anti-human C4 purchased from Dako (Carpinteria, CA, USA) and goat serum anti-human C1q, goat serum anti-human C3c, rabbit serum anti-human C4, rabbit serum anti-human. C5, rabbit serum anti-human C9, goat serum anti-rabbit IgG and rabbit serum anti-goat IgG conjugated with peroxidase and rabbit serum anti-sheep erythrocytes (haemolysin), and rabbit anti-guinea pig immunoglobulins (g-1 and/or g-2) purchased from Sigma Chemical. A sample of F. pedrosoi was grown in Czapeck-Dox medium for 14 days at room temperature

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