Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE Cristiane B. Pereira, 1 Frank L. Bueno, 1 Amanda L. T. Dias, 1 Maı ´sa R. P. L. Brigaga˜o, 2 Claudete R. Paula 3 and Antonio M. Siqueira 1 Correspondence Amanda L. T. Dias amanda_unifal2008 @yahoo.com.br 1 Departamento de Cieˆ ncias Biolo´ gicas, Laborato´ rio de Microbiologia e Imunologia, Universidade Federal de Alfenas, Alfenas, MG, Brazil 2 Departamento de Cieˆ ncias Biolo´ gicas, Laborato´ rio de Bioquı ´mica, Universidade Federal de Alfenas, Alfenas, MG, Brazil 3 Departamento de Microbiologia, Laborato´ rio de Micologia, Instituto de Cieˆ ncias Biome´ dicas II, Universidade de Sa˜ o Paulo (USP), Sa˜ o Paulo, Brazil Received 5 November 2008 Accepted 26 January 2009 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. INTRODUCTION Cryptococcus neoformans is a cosmopolitan fungus and a major opportunistic pathogen in immunocompromised hosts, accounting for a significant proportion of AIDS- related infections (Pinner et al., 1995; Casadevall & Perfect, 1998). Cryptococcosis is responsible for high morbidity and mortality rates among patients with AIDS (Kovacs et al., 1985; Rozenbaum & Gonc¸alves, 1994; Kozel, 1995; Fernandes et al., 2000). However, cryptococcosis also can occur among immunocompetent hosts (Kokturk et al., 2005; Chen et al., 2008). Due to its increasing incidence, additional studies on the pathogenesis of this micro-organism are necessary for a better understanding of the aetiological agent of cryptococ- cosis and its virulence factors. C. neoformans strains have been grouped into three varieties: C. neoformans var. grubii (serotype A), C. neoformans var. gattii (serotypes B and C; now named Cryptococcus gattii) and C. neoformans var. neoformans (serotypes D and AD). We have found C. neoformans var. neoformans and var. grubii in clinical material, but var. grubii is the predominant aetiological agent in 99 % of cases of infections in AIDS patients and other immuno- compromised individuals. C. gattii can be isolated from environmental samples, but C. gattii serotype B has also been found to cause disease in immunocompetent individuals (Mitchell & Perfect, 1995; Casadevall & Perfect, 1998; Steenbergen & Casadevall, 2003). Among the possible virulence factors of C. neoformans, melanin is thought to be a major factor and is thought to be involved in adaptation to environmental stress condi- tions. It is synthesized by laccase enzymes, a group of multifunctional enzymes, in medium containing substrates such as L-dopa, and the colonies become brown or black as result of pigmentation of the cell (Williamson, 1994; Wang et al., 1995; Williamson et al., 1998; Hamilton & Gomez, 2002; Nosanchuk & Casadevall, 2003; Plonka & Grabacka, 2006). Journal of Medical Microbiology (2009), 58, 563–566 DOI 10.1099/jmm.0.007716-0 007716 G 2009 SGM Printed in Great Britain 563