A food color-based colorimetric assay for Cryptococcus neoformans laccase activity
Lia Sanchez Ramirez, Quigly Dragotakes, Arturo Casadevall
TL;DR
Researchers developed a low-cost, color-based test to measure laccase activity in Cryptococcus neoformans, a fungus that causes disease in immunocompromised individuals.
Contribution
A novel, cost-effective colorimetric assay using food dyes to assess laccase activity in Cryptococcus neoformans strains.
Findings
Phenolic color degradation by C. neoformans laccase was found to be glucose-dependent.
The food dye-based assay offers advantages such as lower cost and irreversibility compared to the ABTS assay.
The method has potential applications in bioremediation and virulence factor studies.
Abstract
Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis primarily in immunocompromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase and protects fungal cells against immune attack. Hence, the comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and by using this effect explored the development of a colorimetric assay 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…
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Taxonomy
TopicsFungal Infections and Studies · Antifungal resistance and susceptibility · Plant Pathogens and Fungal Diseases
INTRODUCTION
C. neoformans causes cryptococcosis and is a public health concern due to its high mortality rate in immunocompromised individuals, especially those with HIV/AIDS. Consequently, C. neoformans was recently designated as a critical priority pathogen by the World Health Organization (WHO). Most cases of cryptococcosis occur in Sub-Saharan Africa, where cryptococcal meningitis has become a major cause of death in HIV/AIDS patients, surpassing the mortality rate of tuberculosis (1). Therefore, C. neoformans infections are particularly dangerous in low-resource countries where these is less access to treatment for immunocompromised individuals. Studying the ways in which C. neoformans protects itself against an immune response allows us to further understand the immune mechanisms that combat C. neoformans infection.
Laccases catalyze a broad spectrum of reactions and serve as an important virulence factor for C. neoformans that is expressed in its cell wall (2). Since laccases are versatile, in the sense that they can catalyze the oxidation of various substrates, this makes them useful for a range of environmental applications. Laccases can oxidize polyphenolic compounds and iron, and the functions of the enzyme are regulated through signal transduction pathways (2). Laccase has even more diverse functions since a broad range of organisms utilize the laccase enzyme, including fungi, plants, and insects (2).
Specifically, in C. neoformans, the laccase enzyme converts Iron (II) to Iron (III), which lowers the susceptibility of C. neoformans cells to hydroxyl radicals that are generated in vitro (3). Regulation of laccase expression has been found to possibly be strain-dependent because disruptions of a G-protein subunit homolog affect laccase activity in wild-type C. neoformans, but this was not observed in a serotype A H99 strain (2). More research studies are needed to understand the molecular complexities of the laccase enzyme serving as a virulence factor of C. neoformans. Vacuolar proton pump ATPases were found to play a role in laccase activity by testing strains with mutated genes that encode for ATPases, so targeting these proton pumps could be useful for drugs that aim to control laccase activity (2).
Laccase plays an important role in virulence by catalyzing the generation of melanin in C. neoformans, which protects the fungus from reactive oxygen species and nitrogen oxidants produced by phagocytic cells (3). Fungal laccase may also contribute to virulence by reducing the formation of antimicrobial hydroxyl radicals (3). Laccase is also involved in the generation of prostaglandins by fungal cells that can affect local inflammatory responses (4). Specifically, laccase is induced during glucose starvation and increased temperature (30°C) and helps moderate fungal stress resistance (2). Studying laccase can provide insights into C. neoformans pathogenesis and its mechanisms of defense in the phagolysosome. The C. neoformans laccase is known to have broad structural activity, producing pigments of a spectrum of colors, including those similar to melanin, following the oxidation of phenols and catechols (5).
Laccases are known to destroy dyes and are used in food preparation, industry, and environmental applications such as reducing the environmental impact of synthetic dyes (6, 7). Factory production of non-biodegradable synthetic dyes results in contaminated wastewater, and fungal laccases can be used to degrade the colors in these bodies of water (6). The by-products of laccase catalysis are lower in toxicity than those from alternative chemical methods for wastewater purification, and laccases have broad specificity, which allows them to break down a variety of synthetic dyes (6). Additionally, a common substrate for laccase is phenol, a toxic pollutant which is dangerous to humans (6). The use of laccases as versatile agents of bioremediation is emerging as a sustainable option to degrade chemical pollutants, and there has been interest in investigating the catalytic mechanisms of laccase to optimize its function in bioremediation (8).
In this study, we carried out a serendipitous observation to investigate the use of mangosteen-colored food dye in a fast, efficient, low-cost colorimetric assay for determining laccase activity in C. neoformans cultures. Using absorbance spectroscopy, we could compare several strains of C. neoformans in various culture conditions to both detect and quantify relative laccase activity. Low-cost and facile assays for laccase activity have many potential applications in environmental industries, including purification of wastewater. These assays are also applicable when comparing the virulence of different strains by measuring relative laccase activity, an important fungal virulence factor.
MATERIALS AND METHODS
Yeast strains and culture conditions
C. neoformans species complex serotype A strain H99 was obtained from John Perfect in Durham, NC (9). The lac1Δ mutant is from the 2007 Lodge library (Fungal Genetics Stock Center) (10). The H99 GFP strain was obtained from the laboratory of Dr. Robin May at the University of Birmingham, United Kingdom (11). The CNAG 01373Δ, CNAG 06646Δ, and CNAG 01029Δ strains are KN99α mutants obtained from a previously published gene knockout library (12). The KN99α strain was obtained from Heitman Lab at Duke University Medical Center Durham, North Carolina (13). Additional C. neoformans strains used are 24067, Mu-1, and B3501. Other yeast strains used for comparisons were Candida albicans 90067, Saccharomyces cerevisiae S188C, C. gatti R265, and C. gatti WM179.
Medium preparation
Minimal media (MM) was prepared with 15 mM dextrose, 10 mM MgSO_4_, 29.3 mM KH_2_PO_4_, 13 mM glycine, and 3 µM thymine-HCL dissolved in water supplemented with either 2.7 g/L or 20 g/L glucose, vacuum-sterilized via SteriCup Quick Release filter, and stored at room temperature. Yeast extract peptone dextrose (YPD), manufactured by Difco Laboratories, was prepared according to the manufacturer’s protocol.
Comparison of color change in H99 and lac1Δ mutant cultures
H99 and lac1Δ mutant strains were seeded at a concentration of 10^4^ cells /mL in 3 mL of MM in 12-well tissue culture plates. The following colors of Limino brand food coloring (Amazon Corp. Limino Baiyun, Guangzhou, China) were used: Strawberry, Tangerine, Lemon, Lime, Purple Cabbage, Blueberry, and Mangosteen. These food colorings contained various food dyes and sorbitol (CAS No. 50–70-4), water (CAS No. 7732–18-5), glycerin (CAS No. 56–81-5), carboxymethylcellulose sodium (CAS No. 9004–32-4), and potassium sorbate (CAS No. 590–00-1). The food dyes present in the food coloring were Acid Red 27, Food Red 7, Food Blue No. 1, Food Yellow 3, and Food Yellow No. 4. No lot numbers were provided for the food coloring. Ten wells contained MM supplemented with 10 µL food coloring, one well with uncolored MM, and one well with uncolored YPD. Color-change observations were recorded 7 days after the plate was either left on the bench at room temperature or placed on a 120 rpm shaker in a 30°C incubator and measured either by the unaided eye or via Spectramax iD5.
Different concentrations of glucose were tested with high-glucose minimal media conditions
Two H99 cultures were seeded in MM supplemented with mangosteen color in Falcon 14-mL polypropylene round-bottom tubes (REF 352059). One tube was prepared with MM at its regular glucose concentration of 2.7 g/L, while the second tube was prepared with MM at an elevated glucose concentration of 20 g/L. Culture tubes were placed in the 30°C incubator with shaking, and color change observations were recorded after 7 days.
Kinetic assay for laccase activity
Twenty-well tissue culture plates were seeded with 10^6^ C. neoformans cells in 1 mL of MM with 10 g / L glucose in each well. Three wells were immediately treated with 1X stock of the NuPAGE antioxidant reagent (Product Number: NP0005), and then the plate was incubated at 30°C shaking overnight. This antioxidant has a proprietary formulation of N,N-dimethylformamide. The next morning, an antioxidant reagent was added to the last three wells and incubated for another hour to observe a possible color change of blue to green or back to purple.
Addition of antioxidants to colorimetric assay
96-well tissue culture plates were seeded with 10^4^ C. neoformans cells in 100 µL volumes of MM. The 1X stock of commercial antioxidant was added either at 0 hour or after 24 hours of observing the color change.
24-hour assay for laccase activity
Every culture of interest was grown in regular MM and left in a 30°C incubator with rotation at 120 rotations per minute. A 1.5 mL volume of each culture was centrifuged at 2,300 g for 5 minutes in microcentrifuge tubes and then resuspended in 1.5 mL of 10 g/L MM. A volume of 150 µL of the resuspended culture was pipetted into 10 wells of a 96-well plate, so each culture was measured with 10 replicates of the well. Then, 7.5 µL of a 1–10 dilution of Mangosteen food coloring in water (100 µL food coloring in 1 mL of H_2_O) was added to each well. At 0 hour, 50 µL of the supernatant was placed in another 96-well plate for the initial 520 nm absorbance measurement with the Spectramax iD5 (Baltimore, Maryland), and at 24 hours, another 50 µL of the supernatant was placed in another 96-well plate for the 24-h measurement with the Spectramax iD5. For 24 hours, the plate was incubated in a 30°C incubator on a 120 rpm shaker. We compared the laccase activity of the following C. neoformans strains: H99, H99 GFP, KN99α, CNAG 01373Δ, CNAG 06646Δ, and CNAG 01029Δ. Additional trials were conducted for comparing the following C. neoformans strains: H99, 24067, Mu-1, and B3501 to the following other yeast strains: C. albicans 90067, S. cerevisiae S188C, C. gatti R265, and C. gatti WM179.
2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay
The H99 strain was seeded in MM cultures. Each culture used was washed twice with phosphate-buffered saline (PBS). Then, a 1:100 dilution, or 1:1 dilution for some wells, was prepared with MM. A 1 mL volume of 20 mM of the ABTS solution was prepared and filter-sterilized. ABTS was added for a final concentration of 1 mM ABTS in the cultures. Incubate for 24 hours in a 30°C incubator on a 120 rpm shaker. Initial and 24-h absorbance measurements were taken at 734 nm with the Spectramax iD5.
Statistical analysis
The statistical tests conducted for each absorbance measurement experiment are denoted in their respective figure descriptions with tests for multiple hypotheses. Two-way ANOVA with Tukey’s comparison analyses were conducted using RStudio Version 2023.09.1+494 and GraphPad Prism Version 10.0.2 (171). Statistical comparisons were made both with all the data for each individual experiment replicate and for data from all trials pooled.
RESULTS
Phenolic dyes are degraded in the C. neoformans culture
The observation that C. neoformans degrade some food dyes was made serendipitously. While searching for conditions to study the growth of C. neoformans, we noted difficulty in the measurement of fungal growth caused by turbidity when comparing cultures grown in MM and YPD. We hypothesized that the problem arose because MM was clear, while YPD had a yellowish color. We thought that adding food coloring to MM wells would facilitate the interpretation of growth curve absorbance data. However, after 7 days of culture growth, we noticed a color change occurred in multiple wells. Upon closer inspection, we noted color changes only in dyes containing a red component, whose chemical structures contained phenolic groups, and that the resulting colors resembled the original dye if only the phenolic (red) components were removed ([Fig. 1 and 2C](#F1 F2)). When the absorbance was measured, we observed a loss of the red color in wells with the C. neoformans culture that had degraded the red dye (Fig. 2A). Specifically, these wells contained the phenolic dyes Food Red 7 and Acid Red 27 (Table S1). We hypothesized that the dye degradation was a result of laccase activity, due to broad substrate specificity in cryptococcal laccases, and established this by replicating the experiment with ΔLac1-H99 and found no color change after 7 days (14, 15). Taken together, these data suggest that fungal laccases can degrade phenolic food dyes, and we hypothesized this phenomenon could be harnessed for a colorimetric laccase activity assay.
Chemical structures of food dyes used and melanization mechanism. Panels A and E drawn from World dye variety (16, 17) with ChemDraw software (Version 20.1.0.112) (18). Panels B, C, and D adapted from NIH PubChem (19–21). Panel F adapted from Chandrakant and Shwetha (22) and redrawn using ChemDraw software (18). (A). Food Red 7 (B). Acid Red 27 (C). Food Blue 1 (D). Food Yellow 3 (E). Food Yellow 4 (F). Oxidation by the laccase mechanism
Observed degradation of red color after 7 days in different temperature, agitation, and culture conditions. (A). Absorbance measurements showing loss of red color peak in wells with C. neoformans culture compared to wells with the undegraded food dye. Experiments were repeated in triplicate with similar results. (B). Sample 12-well plate setup showing the location of the various compounds in the assay. Wells shown in panels A to C. Wells with a red circle designation are colors that commonly showed red color degradation. Image created with Biorender.com. (C). Left: Plate with the wild-type H99 culture. Right: After 7 days of agitation at room temperature. (D). Left: Plate with the lac1Δ mutant culture. Right: After 7 days of agitation at 30°C. (E). Left: Wild-type H99 plate after 7 days of agitation at 30°C. Right: Wild-type H99 plate after 7 days left at the bench.
Glucose concentration, culture agitation, and cell density affect laccase activity
We sought to optimize culture conditions for detecting laccase expression and activity, attempting to determine conditions which would provide robust results while remaining cost- and time-efficient for any basic laboratory. We found that a 7-d incubation with agitation at 30°C was required to observe color changes with a standard MM preparation (Fig. 2C).
We observed that high glucose concentrations (10–20 g/L) induced color change even at room temperature and without agitation at high initial cell densities. Additionally, using glucose concentrations of 10 g/L and 20 g/L MM allowed us to observe a visible color change quicker than by using regular glucose MM with any cell concentration from 10^3^ to 10^7^ cells / mL in a plate with agitation at 30°C (Fig. 3B). We found that high glucose concentrations repressed laccase expression (Fig. 3A). Through an examination of glucose-dependence, we observed increased dye degradation in wells with cultures resuspended in MM with higher glucose concentrations (Fig. 4A). Using a higher glucose concentration allowed us to view color change effects quicker as compared to wells with lower glucose concentrations, and with a 24-h assay, we were not able to see any color change in MM glucose conditions (Fig. 4B).
(A). Absorbance measurements at 520 nm wavelength for wells with varying cell densities and MM glucose concentrations. (B). Color change from Mangosteen (purple) to blue observed in wells with varying cell densities with regular 2.7 g/L MM, MM with 10 g/L of glucose, and MM with 20 g/L of glucose. Experiment conducted for two trials.
(A). Absorbance measurements of the supernatant at 520 nm at 0 and 24 ours for different glucose concentrations. Each point represents an individual measurement. The following significant differences were determined when analyzing differences in absorbance across different glucose conditions over 24 hours: 10 g/L and 0 g/L condition (P = 0.0002), 20 g/L and 0 g/L condition (P < 0.00001), 20 g/L and 10 g/L condition (P < 0.00001), and 20 g/L and 2.7 g/L condition (P < 0.00001). Experiments conducted in triplicate. (B). Observations of clor change from Mangosteen (purple) to blue. No color change observed with 0 g/L glucose MM in the 24-h colorimetric assay. Rows 1–3 are replicates of the given glucose condition with the H99 strain. Each experiment is conducted in triplicate: vertical columns.
With the combined data containing all individual trials of the glucose dependence experiments measuring the absorbance at 520 nm, a statistically significant difference was found between the 20 g/L glucose MM condition when compared to the 0 g/L glucose MM condition (P = 0.002). An ANOVA test for glucose dependence was also conducted with the change in absorbance measurements between 24 hours and 0 hour of exposure to food coloring, which revealed significant differences between the 10 g/L and 0 g/L conditions (P = 0.0002), 20 g/L and 0 g/L conditions (P < 0.00001), 20 g/L and 10 g/L conditions (P < 0.00001), and 20 g/L and 2.7 g/L conditions (P < 0.00001). Although not many significant differences were observed when analyzing the absorbance measurements themselves, analyzing the change in the absorbance throughout the 24-hour timeframe showed differences between the glucose conditions. Overall, it was easier qualitatively to observe differences in laccase activity across different glucose conditions than it was to establish the quantitative differences, which we suspect could be due to the sensitivity of the equipment used.
Laccase dye degradation is irreversible
One potential disadvantage of current laccase activity assays, and specifically with the commonly used ABTS assay, is that the observed color change is not permanent and must be measured within a specific timeframe. To investigate whether our colorimetric assay was permanent, we treated wells with commercially available ThermoFisher antioxidant before and after the observed color change. We found that adding antioxidants after the reaction did not revert the color, nor did leaving the sample on a benchtop long term over a span of 3 weeks (Fig. 5B). This suggests that the reaction was irreversible, allowing samples to be read at the investigator’s convenience. Interestingly, when treating the culture with antioxidants at the start of the experiment, we observed a new green color. A spectrum scan revealed a new peak at ~420 nm, suggesting the formation of an alternative product (Fig. 5A).
(A). Absorbance measurements at different wavelengths for wells that either degraded the food coloring to produce color change or did not degrade the coloring. Note that formation of a new peak at ~420 nm was observed in the absorbance measurements of wells with color degradation. (B). ThermoFisher NuPAGE antioxidant-treated wells. The top row is the control row without addition of antioxidants, the middle row is the addition of the antioxidant before color change occurs, and the bottom row is the addition of the antioxidant after color change occurs. Each column is a replicate of the given condition. All wells were initially mangosteen (purple) color. The pre-oxidant row was given antioxidant before any color change occurred, while the control and post-oxidant rows were given antioxidants after they turned blue due to red color degradation. Color change is not reversible since the wells turned green and not back to purple.
Developing cheap and available laccase activity assay
Our next aim was to minimize the necessary reagents and instruments in this assay (Fig. S1). The optimized assay protocol uses a concentration of 10^6^ cells / mL yeast seeded in MM with 7 uL of 1:100 diluted food coloring in a 96-well plate. The plate is then incubated at 30°C with 120 rpm agitation to observe color change in around 3–7 days.
To quantify the extent of red food coloring degradation in 24 hours, we designed an assay using culture resuspended in 10 g/L MM. This quicker assay involves centrifuging the culture of interest and resuspending cells in 10 g/L glucose MM, with 100 µL of the culture in each well in a 96-well plate and utilizing 7.5 µL of a 1–10 dilution of food coloring in water for each well. Absorbance measurements at 520 nm at 0 and 24 hours can be used to compare differences between strains or other conditions to quantify changes in laccase activity (Fig. 6). Linear regression analysis allowed us to compare rates of dye degradation across various C. neoformans strains, with increased rates of degradation in an H99 GFP strain ([Fig. 6B and 7](#F6 F7)). Differences between multiple C. neoformans strains and other yeast strains with varying levels of laccase activity were compared with the 24-h assay (Fig. 8).
*Optimized 24-h colorimetric assay with comparisons between H99, H99 GFP, KN99α, and KN99α mutant strains. Complete color change from purple to light blue observed for every strain tested. (A). Graphical results of comparison between different C. neoformans strains with the 24-h assay. Each point represents an individual measurement. Overall significant differences between KN99α and 01029 (P = 0.0006), KN99α and 01373 (P = 0.00005), KN99α and 06646 (P = 0.000007), and H99 and 06646 (P = 0.02). Experiments conducted for four trials. *, **, **, and **** denote P < 0.05, 0.01, 0.001, and 0.0001 respectively. (B). Linear regression analysis of change in 520 nm absorbance over time across C. neoformans strains with the 24-h laccase assay method. Each point represents an average of measurements. As such, the following comparisons were statistically significant: H99 and H99 GFP (P < 0.00001), H99 and 01373 (P < 0.05), H99 and 06646 (P < 0.00001), H99 GFP and KN99α (P < 0.00001), H99 GFP and 01029 (P < 0.00001), KN99α and 06646 (P < 0.00001), and 01029 and 06646 (P < 0.01). This statistical analysis was corrected for multiple comparisons.
Laccase activity observed in the H99 GFP strain after 3 days with various food dye colors. Color change observed with red color degradation: orange to lighter orange hues, mangosteen (purple) to blue, and indigo to lighter blue. (A). Left: 0-h photo of the plate with H99 and H99 GFP wells with different colors. Right: 3-d photo of the plate after being left on the shaker in a 30°C incubator. Experiment conducted in triplicate. (B). Diagram of the 12-well plate set-up diagram for comparison between H99 and H99 GFP strains with the food coloring colors used; official names given by the Limino brand in parentheses. Wells with a red circle designation show the red color degradation.
*Optimized 24-h colorimetric assay with comparisons between C. neoformans and other fungal strains. (A). Graphical results of comparison between different fungal strains with the 24-h assay. Each point represents an individual measurement. Statistical analyses were conducted to compare the absorbance measurements across 24 hours. Overall significant differences between H99 and C. gatti R265 (P = 0.00004), H99 and C. gatti WM179 (P = 0.02), 24067 and C. albicans 90067 (P = 0.04), 24067 and C. gatti R265 (P = 0.0000000), 24067 and S. cerevisiae S188C (P = 0.006), 24067 and C. gatti WM179 (0.000004), B3501 and C. albicans 90067 (P = 0.02), B3501 and C. gatti R265 (P = 0.0000000), B3501 and S. cerevisiae S188C (P = 0.002), B3501 and C. gatti WM179 (P = 0.000001), Mu-1 and C. gatti R265 (P = 0.0001), and C. gatti R265 and C. albicans 90067 (P = 0.005). Experiments conducted for four trials. *, **, **, and **** denote P < 0.05, 0.01, 0.001, and 0.0001, respectively. Parentheses compare strains and not individual data points. (B). Mangosteen (purple) to blue color change observations. Images of plates comparing the degradation of Mangosteen color over 48 hours across fungal strains listed in the figure. Experiment conducted in triplicate.
With the combined data containing all individual trials of the first set of experiments comparing the absorbance at 520 nm across different C. neoformans strains, we observed overall significant differences in absorbance between KN99α and 01029 (P = 0.0006), KN99α and 01373 (P = 0.00005), KN99α and 06646 (P = 0.000007), and H99 and 06646 (P = 0.02). In one of our trials, we observed significant differences in the absorbance between the KN99α strain and its mutant strains 01029 (P = 0.009), 01373 (P = 0.003), and 06646 (P = 0.03). Significant differences between the strains tested within another trial are described in the figure legend of Fig. 6B. While we observed a more noticeable difference in color change between the H99 and H99 GFP strains (Fig. 7), this was not always reflected in the statistical analyses, particularly with the combination of multiple trials, possibly due to a loss of power with the analysis.
For the next group of experiments comparing the absorbance at 520 nm across both C. neoformans strains and different yeast strains, we observed significant differences in absorbance measurements between H99 and C. gatti R265 (P = 0.00004), H99 and C. gatti WM179 (P = 0.02), 24067 and C. albicans 90067 (P = 0.04), 24067 and C. gatti R265 (P = 0.0000000), 24067 and S. cerevisiae S188C (P = 0.006), 24067 and C. gatti WM179 (0.000004), B3501 and C. albicans 90067 (P = 0.02), B3501 and C. gatti R265 (P = 0.0000000), B3501 and S. cerevisiae S188C (P = 0.002), B3501 and C. gatti WM179 (P = 0.000001), Mu-1 and C. gatti R265 (P = 0.0001), and C. gatti R265 and C. albicans 90067 (P = 0.005).
Advantages of food dye-based colorimetric assay relative to the ABTS assay
We compared the standard ABTS assay to our colorimetric assay. The H99 GFP well with the higher concentration of the culture showed a color change within 5 minutes of adding the ABTS solution to the well, so our initial photograph shows this color change already (Fig. 9A). However, this color change was impermanent since it was not clearly evident after 24 hours. This places a limitation on the ABTS experiment because it is possible to miss the window of color change and not obtain the absorbance measurements or photographs needed. Additionally, with our ABTS assay, we observed the clearest color change mostly within the H99 GFP strain and not the H99 strain (Fig. 9A). With the 24-h colorimetric assay, we can observe a clear color change within 24 hours for multiple C. neoformans strains, and this color change exhibits permanence, allowing for absorbance measurements or photographs to be taken post-color change at the investigator’s convenience. The hues of the color change in the ABTS assay were relatively faint and more difficult to see with the unaided eye as compared to the colorimetric assay. Additionally, there is a significant difference in cost of materials for the ABTS assay compared to the food coloring assay. The ABTS solution was sold by Roche Life Science Products in a quantity of 300 mL for 349.00 USD at the time of this study ([23](#B23)). The food coloring used in this experiment is available on Amazon.com, costing 2.66/Fl oz (24). Purchasing an equivalent amount of about 300 mL of food coloring would cost about 26.99, showing that the food coloring method is a significantly cheaper way to confirm laccase activity ([25](#B25)). The ABTS assay costs about 1.16 per assay, while the colorimetric assay costs about $0.02 per assay, or even less than that if using higher dilutions for the optimized 24-h assay.
ABTS assay. Color change observed from clear to shades of blue–green. (A). Left: Initial photo of the ABTS assay plate at 0 hour. Right: Photo of the ABTS assay plate after 24 hours in a 30°C incubator on a shaker. Note that well 3C turned blue rapidly, but by 24 hours, the color was largely gone. (B). 12-well plate set-up diagram of the ABTS experiment, created with Biorender.com. Wells with a red circle designation show color change from clear to shades of blue–green.
DISCUSSION
Laccase enzymes are involved in survival strategies of C. neoformans through the process of melanization. Melanization protects fungal cells from reactive oxidative stress, among other stressors, and investigation of the role of the laccase enzyme in C. neoformans pathogenesis can provide insights into the fungal survival strategy. Laccases are also relevant in many aspects of the food industry and may be useful in reducing the environmental impact of synthetic dyes from factory production. Synthetic food dyes may be used as an efficient and cheaper assay for determining laccase activity in C. neoformans cultures, and methods to quantify laccase activity are of interest in a variety of fields ranging from environmental concerns from accumulation of dyes in bodies of water and the defense mechanisms of the pathogenic fungi (26, 27). Since laccase is involved in catalyzing the melanization defense mechanism of C. neoformans, the relative comparisons of laccase activity across C. neoformans strains could be valuable in understanding differences across different strains.
Our results suggest that laccase irreversibly breaks down the red pigment known as Food Red 7 and Acid Red 27, as color changes were not observed with the lac1Δ mutant strain. We hypothesize this phenomenon is due to the enzyme’s ability to catalyze formation of free radicals through “removal of a hydrogen atom from the hydroxyl group of ortho- and para-substituted mono- and polyphenolic substrates” (26). We initially considered that agitation was necessary to promote red color degradation in the wild-type H99 strain since the cryptococcal laccase reaction uses oxygen but found that it was not necessary since plates at room temperature without agitation still showed color change when exposed to elevated glucose conditions, albeit requiring a few more days to exhibit color degradation.
We observed more colorimetric activity in cultures with higher concentrations of glucose. This result was unexpected, as previous literature reported increased melanization at lower glucose concentrations, which we expected to correlate with laccase activity (28). In fact, a review of laccase activity cites glucose as a repressor (2). However, those observations refer to melanization and not to direct laccase activity (28). However, melanization may not always be a reliable proxy measurement for laccase activity (29). Other reports have noted that laccase expression in C. neoformans is induced during glucose starvation, but also “stimulated by copper, iron, and calcium and repressed at elevated temperatures.” (30). The glucose-dependency of color degradation shown in these experiments leads us to hypothesize that the phenomenon we observed could require energy. Alternatively, the glucose effect could reflect changes in the reduction potential of the solution that potentiate the dye-destroying reaction of laccase. The laccase reaction is heavily influenced by the reduction potential of the solution, and glucose has reducing properties, as illustrated by the blue bottle experiment carried out in high school chemistry courses (31, 32).
When comparing different C. neoformans strains, we found that the H99 GFP strain showed signs of increased laccase activity with an earlier visible color change and a greater change in the absorbance at 520 nm over time. We do not have an explanation for this phenomenon but suspect that association of the GFP construct to the actin promoter of this C. neoformans laboratory strain could cause secondary metabolic effects. Additionally, we note that GFP can generate free radicals and affect the oxidative state of the cell (33). Given the importance of reduction potential in the laccase reaction, it is possible that the enhanced color associated with GFP-expressing C. neoformans reflects altered oxidative conditions in the cells (31).
The 24-hour color change method requires absorbance measurements to quantify differences in the extent of red color degradation by the fungi. Therefore, we have determined that the different variations of the assay could be used for different purposes depending on what is needed for the experiment. Larger wells and higher cell densities can be used to observe color change over the course of 3–7 days to provide a positive or negative conclusion on presence of laccase activity, whereas resuspending smaller volume cultures in high-glucose MM can determine reaction differences over the course of 24 hours. Strain differences did not always reach statistical significance, even though color changes were visible to the human eye, and this could reflect variability sensitivity of the plate used by the reader and a need to further optimize the assay to improve the quantification of results. While significant differences were observed when comparing C. neoformans strains to other fungal strains, the relevance of these results in terms of laccase expression is unknown. As of now, this assay is preferentially suited for determining whether a strain produces laccase rather than comparing strain activities.
The absorbance spectrum revealed a new peak at ~420 nm in wells containing C. neoformans that degraded the food coloring, suggesting the formation of a new product when pretreated with antioxidants. This new product was chemically stable. Filamentous fungi have been found to degrade a red diazo dye, using laccases to transfer the azo dye to nontoxic products (34). On the other hand, synthetic dyes that are made up of aromatic compounds, such as azo dyes, when degraded produce amines, which are mutagenic to humans (35). These studies suggest that these products could have been produced in our experiments, but further investigation is needed to determine what product was produced by the fungal cells when degrading the food coloring.
Currently, the most widely used laccase activity screen in the cryptococcal field is the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay in which blue-colored ABTS is oxidized to green-colored ABTS+. A disadvantage of this assay is that the ABTS reduction is impermanent, and samples must be measured before the oxidized ABTS + is reduced. In contrast, the observed color change in our assay was permanent with no time-sensitive window required for performing the measurement. Samples may be left for long periods of time without special preservation before measurement. We also observed differences in the degree of visible color change across strains in the ABTS assay, which so far have been mitigated by the 24-hour colorimetric assay that shows complete color change across the strains we tested. The colorimetric assay with food coloring materials is also significantly less costly relative to the cost of the ABTS solution (23).
The C. neoformans laccase enzyme has other functions in addition to melanin production. For example, C. neoformans laccase is involved in the production of fungal prostaglandins (4). It is possible that laccase deactivates molecules toxic to the fungi and that its effect on food dye color is a reflection of its nonspecific chemical activity. In this study, we have used this effect to develop a new assay for laccase activity that can be used to study fungal laccases.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1C. neoformans infection statistics | fungal diseases | CDC. 2022. Available from: https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html. Retrieved 5 Apr 2024.
- 2Zhu X, Williamson PR. 2004. Role of laccase in the biology and virulence of Cryptococcus neoformans. FEMS Yeast Res 5:1–10. doi:10.1016/j.femsyr.2004.04.00415381117 · doi ↗ · pubmed ↗
- 3Casadevall A, Rosas AL, Nosanchuk JD. 2000. Melanin and virulence in Cryptococcus neoformans. Curr Opin Microbiol 3:354–358. doi:10.1016/s 1369-5274(00)00103-x 10972493 · doi ↗ · pubmed ↗
- 4Erb-Downward JR, Noggle RM, Williamson PR, Huffnagle GB. 2008. The role of laccase in prostaglandin production by Cryptococcus neoformans. Mol Microbiol 68:1428–1437. doi:10.1111/j.1365-2958.2008.06245.x 18410494 PMC 3973538 · doi ↗ · pubmed ↗
- 5Chaskes S, Tyndall RL. 1978. Pigment production by Cryptococcus neoformans and other Cryptococcus species from aminophenols and diaminobenzenes. J Clin Microbiol 7:146–152. doi:10.1128/jcm.7.2.146-152.1978344335 PMC 274883 · doi ↗ · pubmed ↗
- 6Ardila-Leal LD, Poutou-Piñales RA, Pedroza-Rodríguez AM, Quevedo-Hidalgo BE. 2021. A brief history of colour, the environmental impact of synthetic dyes and removal by using laccases. Molecules 26:3813. doi:10.3390/molecules 2613381334206669 PMC 8270347 · doi ↗ · pubmed ↗
- 7Paz A, Carballo J, Pérez MJ, Domínguez JM. 2017. Biological treatment of model dyes and textile wastewaters. Chemosphere 181:168–177. doi:10.1016/j.chemosphere.2017.04.04628437742 · doi ↗ · pubmed ↗
- 8Jeyabalan J, Veluchamy A, V VP, Kumar A, Chandrasekar R, Narayanasamy S. 2023. A review on the laccase assisted decolourization of dyes: recent trends and research progress. J Taiwan Inst Chem Eng 151:105081. doi:10.1016/j.jtice.2023.105081 · doi ↗