Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE.
The increased incidence of infections caused by the opportunistic pathogen Cryptococcus neoformans, which mainly affects immunocompromised patients but can also infect immunocompetent individuals, has needed additional studies on this micro-organism's pathogenicity and factors related to virulence, such as enzyme production, for a better understanding of the aetiology of cryptococcosis. The aim of this study was to verify the applicability of non-denaturing PAGE for analysis of laccases by quantification of the amount of melanin pigment produced by clinical and environmental strains of C. neoformans. After incubation of the gel with the substrate L-dopa, strains produced melanin spots of a bright brown to black colour. Quantification of these spots was performed by densitometry analysis and the amount of melanin produced was calculated and compared among the strains. All strains showed laccase activity. Serotype B strains showed a higher melanin intensity than serotype A strains. Over half of the clinical strains (56.2%) showed the lowest melanin intensities, suggesting that melanin production may not be the main virulence factor against host defence. The clinical strain ICB 88 revealed two melanin spots on the gel, indicating the presence of two laccase isoforms. The environmental strains showed the highest values of melanin intensity, which may be related to previous exposure to environmental stress conditions.
Departamento de Ciências Biológicas, Laboratório de Microbiologia e Imunologia, Universidade Federal de Alfenas, Alfenas, MG, Brazil.
Cristiane B
CB
Pereira
Departamento de Ciências Biológicas, Laboratório de Microbiologia e Imunologia, Universidade Federal de Alfenas, Alfenas, MG, Brazil.
Frank L
FL
Bueno
Departamento de Ciências Biológicas, Laboratório de Microbiologia e Imunologia, Universidade Federal de Alfenas, Alfenas, MG, Brazil.
Amanda L T
ALT
Dias
Departamento de Ciências Biológicas, Laboratório de Bioquímica, Universidade Federal de Alfenas, Alfenas, MG, Brazil.
Maísa R P L
MRPL
Brigagão
Departamento de Microbiologia, Laboratório de Micologia, Instituto de Ciências Biomédicas II, Universidade de São Paulo (USP), São Paulo, Brazil.
Claudete R
CR
Paula
Departamento de Ciências Biológicas, Laboratório de Microbiologia e Imunologia, Universidade Federal de Alfenas, Alfenas, MG, Brazil.
Antonio M
AM
Siqueira
D001938:Brazil; D003453:Cryptococcosis / Q000276:immunology / Q000382:microbiology*; D003455:Cryptococcus neoformans / Q000145:classification / Q000201:enzymology / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D016867:Immunocompromised Host; D042845:Laccase / Q000378:metabolism*; D008543:Melanins / Q000096:biosynthesis*; D012703:Serotyping
D008543:Melanins; D042845:Laccase