Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE

Abstract

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.

Comparative Genomics of Serial Isolates of Cryptococcus neoformans Reveals Gene Associated With Carbon Utilization and Virulence

“…However, phenotypic analysis revealed decreased production of two key virulence traits, capsule and melanin, and an overall decrease in virulence was observed in avc1 Δ. Clinical isolates of C. neoformans produce inconsistent virulence profiles, however, and melanin in particular is frequently variable in these isolates (Clancy et al 2006; Pereira et al 2009). Mutants lacking melanin production due to mutation in the laccase gene LAC1 have been shown to have compromised ability to escape from the lungs, whereas growth in the brain is unaffected (Noverr et al 2004).…”

An alternative method for the analysis of melanin production in Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato

“…Under this circumstance, melanin extraction was not necessary, similar to what was described by Fernanades et al. (2016), who applied microscopic techniques to quantify cellular melanin, which reduces costs, accelerates the process of analysis and allows other types of studies with these cells, once melanin extraction procedures completely destroy fungal cells . Moreover, this methodology allowed the detection of differences in melanin production per strain and per day of analysis.…”

“…Different methods for melanin quantification have been widely used and are necessary to evaluate microbial biology and test new drugs for the inhibition of melanin production. As previously mentioned, the current methods for melanin quantification are difficult to apply, time consuming and expensive, such as the use of SEM, which requires costly equipment and reagents .…”