A food color based colorimetric assay forCryptococcus neoformanslaccase activity

Abstract

AbstractCryptococcus neoformansis a fungal pathogen that causes cryptococcosis mostly in immune compromised patients, such as those with HIV/AIDS. One survival mechanism ofC. neoformansduring infection is melanin production, which is catalyzed by laccase. Hence comparative assessment of laccase activity is useful for characterizing cryptococcal strains. After a serendipitous observation that culturingC. neoformanswith food coloring resulted in the degradation of some dyes with phenolic structures, we associated this phenomenon with catalytic activity by cryptococcal laccase. Consequently, we investigated the color changes for the food dyes metabolized byC. neoformanslaccase and explored using this effect for the development of colorimetric assays to measure laccase activity. We developed several versions of a food dye based colorimetric laccase assay that can be used to compare the relative laccase activities between differentC. neoformansstrains. We found that phenolic color degradation was glucose dependent, which may reflect changes in the reduction properties of the media. Our food color based colorimetric assay has several advantages over the commonly used 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for laccase activity, including lower cost, no reversibility and easier application since it does not require constant monitoring. Our method has potential applications in environmental fields, since laccases can be used to degrade pollutants in bodies of water, and this is an efficient test to determine laccase activity. Additionally, it is useful for comparing laccase activity across differentC. neoformansstrains, which can provide insight into the expression of this virulence factor across strains.ImportanceCryptococcus neoformansis present in the environment and while infection is common, disease occurs mostly in immunocompromised individuals.C. neoformansinfection in the lungs results in symptoms like pneumonia, and consequently cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor ofC. neoformans. Developing a simple and less costly assay to determine levels of laccase activity ofC. neoformans, as compared to the commonly used 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, is useful as a quick, permanent measure of laccase activity that could provide insight as to the relative virulence of the fungal cells tested. Environmental applications of this assay include determining which fungal strains would be best to degrade dye-related pollutants in wastewater.