Mycoses. 2017;1–6. wileyonlinelibrary.com/journal/myc © 2017 Blackwell Verlag GmbH
|
1 DOI: 10.1111/myc.12650O R I G I N A L A R T I C L E
An alternative method for the analysis of melanin production
in
Cryptococcus neoformans sensu lato
and
Cryptococcus gattii
sensu lato
Raimunda S. N. Brilhante
1|
Jaime D. A. España
1|
Lucas P. de Alencar
1|
Vandbergue S. Pereira
1|
Débora de S. C. M. Castelo-Branco
1|
Waldemiro de A. Pereira-Neto
1|
Rossana de A. Cordeiro
1|
José J. C. Sidrim
1|
Marcos F. G. Rocha
1,21Department of Pathology and Legal Medicine, School of Medicine, Specialized Medical Mycology Center, Postgraduate Program in Medical Microbiology, Federal University of Ceará, Fortaleza, CE, Brazil 2School of Veterinary Medicine, Postgraduate Program in Veterinary Sciences, State University of Ceará, Fortaleza, CE, Brazil
Correspondence
Raimunda S. N. Brilhante, Department of Pathology and Legal Medicine, School of Medicine, Specialized Medical Mycology Center, Postgraduate Program in Medical Microbiology, Federal University of Ceará, Fortaleza, CE, Brazil.
Email: [email protected]
Funding information
National Council for Scientific and Technological Development, Grant/Award Number: 445670/2014-2; Coordination for the Improvement of Higher Education Personnel (CAPES)
Summary
Melanin is an important virulence factor for several microorganisms, including Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato, thus, the
assess-ment of melanin production and its quantification may contribute to the understand-ing of microbial pathogenesis. The objective of this study was to standardise an alternative method for the production and indirect quantification of melanin in C. neo-formans sensu lato and C. gattii sensu lato. Eight C. neoformans sensu lato and three
C. gattii sensu lato, identified through URA5 methodology, Candida parapsilosis ATCC
22019 (negative control) and one Hortaea werneckii (positive control) were inoculated on minimal medium agar with or without L- DOPA, in duplicate, and incubated at 35°C, for 7 days. Pictures were taken from the third to the seventh day, under standardised conditions in a photographic chamber. Then, photographs were analysed using gray-scale images. All Cryptococcus spp. strains produced melanin after growth on minimal
medium agar containing L- DOPA. C. parapsilosis ATCC 22019 did not produce melanin on medium containing L- DOPA, while H. werneckii presented the strongest pigmenta-tion. This new method allows the indirect analysis of melanin production through pixel quantification in grayscale images, enabling the study of substances that can modulate melanin production.
K E Y W O R D S
Cryptococcus, grayscale images, melanin, pigmentation measurement, yeast
1
|
INTRODUCTION
Melanin is a negatively charged pigment, usually brown to black, polymerised from phenolic and/or indolic compounds. It is a hydro-phobic pigment of high molecular weight, usually associated with proteins, and often with carbohydrates.1,2 Melanin is one of the main natural pigments and it is synthesised by members of all biological kingdoms, including a wide array of fungi, bacteria, and helminths that causes diseases in humans.3 It is not essential for normal growth
and development, but it seems to contribute for the protection of mi-crobes against environmental stress, such as ultraviolet (UV) light, ox-idising agents and ionising radiation.2,4 Fungi may synthesise melanin from endogenous substrates, via a 1,8- dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from L- 3,4- dihydroxyphenylalanine (L- DOPA), like Cryptococccus neoformans sensu lato and Cryptococcus gattii sensu lato.
Cryptococcus neoformas sensu lato currently comprises the old var. grubii, now known as the species Cryptococcus neoformans sensu stricto (AFLP1/VNI, AFLP1A/VNB/VNII), the former var. neofor-mans, now known as the species Cryptococcus deneoformans (AFLP2/ VNIV), and the C. neoformans intervariety hybrid (AFLP3/VNIII). Cryptococcus gatii sensu lato, on the other hand, was separated into five species: Cryptococcus gattii sensu stricto (serotype B, genotype AFLP4/VGI), Cryptococcus deuterogattii (serotype B, genotype AFLP6/ VGII), Cryptococcus bacillisporus (serotype C, genotype AFLP5/VGIII), Cryptococcus tetragattii (serotype C, genotype AFLP7/VGIV) and Cryptococcus decagattii (serotype B, genotype AFLP10).5
Cryptococcus neoformans is found in soils containing pigeon faeces,6 whereas C. gattii is mainly isolated from decaying wood and
contaminated soils.7 Infection with C. neoformans is most commonly
presented as meningitis, especially in areas of the world where high HIV seroprevalence is reported. In addition, the risk of infection with C. neoformans is increased in transplant recipients and other individu-als with impaired cell- mediated immunity. C. gattii, on the other hand, had previously been reported to mainly infect immunocompetent individuals,8,9 but has currently been described to affect individuals with underlying lung diseases or history of therapy with systemic cor-ticosteroids, causing pulmonary and central nervous system (CNS) infections.10 Moreover, two species of C. gattii sensu lato, ie C.
bacillis-pora (VIII) and C. tetragattii (VGIV), have mainly been found in HIV in-fected patients as major cause of cryptococcal meningitis in southern California and Zimbabwe respectively.11,12
Melanin is an important virulence factor for C. neoformans and C. gattii, while other species of the genus Cryptococcus, such as
Cryptococcus albidus and Cryptococcus uniguttulatus,13 either present weak melanin production or do not produce melanin. At present, two species have been recognised for C. neoformans sensu lato and five for the C. gattii sensu lato, which are all able to produce melanin.14,15 In vitro assays have shown that C. neoformans/C. gattii melanised cells are more resistant to phagocytosis and less susceptible to the anti-fungal agent amphotericin B than non- melanised cells.16,17 In addition, melanised cells of Cryptococcus spp. are less susceptible to free- radical killing, suggesting that melanin- like pigments protect against oxidative agents produced by host effector cells.18 Finally, it has been shown that C. neoformans strains with disruption of the CNLAC1 gene for laccase production was associated with a significant reduction in virulence.19
Therefore, the assessment of melanin production and its quanti-fication in pathogens is important. Minimal medium (MM) with dif-ferent substrates for melanogenesis has been widely used, but this liquid medium does not allow direct melanin quantification, requiring an extraction process of pure melanin from microbes, which involves harsh chemical methods, probably, modifying melanin structure.2 In
addition, other culture media have been described to induce melanin production by C. neoformans, such as cowitch (Mucuna pruriens) seed agar.20 These seeds induce fungal melanisation because they contain
L- DOPA, an important precursor for melanin production.10
In melanin- producing fungi, this pigment can be measured, after chemical extraction from the cells, by Scanning Electron Microscopy (SEM).1 This methodology has also been widely used, despite the
difficulties in the extraction process and costs of quantification by SEM. In some studies, such as that of Nosanchuk et al. (2001), [3] the supernatant was read by spectrophotometry, after stimulation of melanogenesis in liquid minimal medium with L- epinephrine. These authors reported difficulties associated with the oxidation of the used compound, which interfered with the spectrophotometric reading.
Based on the above, the objective of this study was to standardise a new alternative method for the production and indirect quantifica-tion of melanin in C. neoformans and C. gattii, on minimal medium agar through the analysis of grayscale images.
2
|
MATERIALS AND METHODS
2.1
|
Fungal strains
Eight C. neoformans sensu stricto (genotype VNI) and three C. gat-tii sensu lato, two Cryptococcus gattii sensu stricto (VGI) and one Cryptococcus deuterogattii (VGII), were used in this study. The genotype of the strains was determined through restriction fragment length pol-ymorphism (RFLP) assay, as recommended by Velegraki et al. [21] One Candida parapsilosis ATCC 22019 and one clinical isolate of Hortaea werneckii were included as negative and positive control for melanisa-tion, respectively. The strains were obtained from the fungal collection of the Specialized Medical Mycology Center of the Federal University of Ceará, Brazil. All strains were preserved on potato agar at room tem-perature of 28°C, with frequent subculture to maintain their viability.
2.2
|
Stimulation of melanin production
Initially, the 11 strains of Cryptococcus spp. were cultured on birdseed (Guizotia abyssinica) agar,22,23 at 35°C for 5 days, to assure their ability of producing melanin, as this medium is well known for its phenolic com-pounds that stimulate melanogenesis. All tested strains presented colonies with a characteristic dark- brown colour. Then, the strains were cultured on potato dextrose agar, at 35°C, for 3 days, where the fungus presents its hyaline phenotype. The fungal inocula used to perform the additional assays of this study were obtained from colonies grown on potato agar.
2.3
|
Preparation of minimal medium agar
The medium used for melanin production was the minimal medium (MM) agar. This medium was adapted from its liquid formulation,1
which is a chemically known medium, composed by 0.015 mol/L glu-cose, 0.01 mol/L MgSO4, 0.0294 mol/L K2HPO4, 0.013 mol/L glycine and 0.003 mol/L thiamine, pH 5.5. As it cannot be autoclaved, the agar was prepared separately, and, after reaching a temperature of 60°C, it was mixed with the MM, reaching a final concentration of 1.5%.
2.4
|
Preparation and inoculation of the
24- well plates
control, and (B) the same described for condition A, supplemented with 0.001 mol/L of L- DOPA (Sigma Aldrich, St. Louis, MO, USA), to induce melanogenesis. Conditions A and B were added to different wells of a 24- well plate, with a final volume of 2 mL per well. Then, 100 μL of the fungal inoculum for each tested strain, at a turbidity of 3 on McFarland scale, containing approximately 9.0 × 108 cells/mL, was added to four different wells containing both growth media, in duplicate. The plates were incubated at 35°C, for 7 days.
2.5
|
Standardised photographic record
A photograph of each plate was taken daily, from the third until the seventh day of incubation, within a photographic chamber prepared for this experiment, which had an uniform indirect illumination by LED 15W. The photographic camera was placed within the chamber, at a distance of 25 cm from the 24- well plate. Nikon D3300 camera was used in standardised conditions (F/8, exposure time 1/250, ISO 200, maximum aperture 4.6, focal length 40 mm). It is important to empha-sise that the pictures were taken under the same conditions with the same camera, by the same photographer.
2.6
|
Pigment quantification in grayscale images
The obtained photographs were converted into grayscale images and analysed by ImAgEj, version 1.51g (https://imagej.nih.gov/ij/index. html), which has previously been applied for similar purposes.24 In our study, the area of measurement was standardised, ranging from 245 000 to 246 000 pixels per well, where the pixels of each well were quantified in grayscale. The grayscale is a range of grey shades from white to black, which is measured from 0 to 250, where 0 is the darkest value and 250 the whitest.
2.7
|
Analysis of data
The values obtained through pixel quantification on grayscale were converted into percentage of black, in order to facilitate the under-standing and interpretation of the data, by using the formula: [(250 subtracted by the mean grayscale value for each strain per day)/250] × 100. Then, the obtained percentage of black was used to quanti-tatively compare the pigmentation of each strain, per day, through Friedman’s and Kruskal- Wallis’ one- way analysis of variance, followed by Dunn’s post hoc test, performed with the software gRApHpAdpRIsm 7, version 1.51 g (La Jolla, CA, USA). Data are expressed as mean ± standard deviation of the percentage of black obtained for each spe-cies, per day of analysis. P- values lower than .05 indicated significant conclusions.
3
|
RESULTS
Days 1 and 2 were not analysed because fungal growth and mela-nin production were not adequate, hence, photographic analyses
were performed from the third to the seventh day. Cryptococcus spp. TAB
LE 1 M elanisat ion of C ry p toc oc cu s sp
p. (n=11) grow
n on m
inim
al m
ediu
m
agar w
ith and w
ithou t L - D O PA f rom the t hird t o t he sevent h day of grow th S tra ins Percenta ge o f b la ck (m ea
n ± S
D
) per d
strains on condition A presented the lowest measurements of the tested fungal species for this condition, with significantly lower values of percentage of black (P<.05), ranging from 36.1±4.0 to 39.2±2.2, throughout the analysed days. Under condition B, Cryptococcus spp. colonies were significantly darker (P<.05) than those grown under condition A, starting as soon as day 3. The percentage of black of these fungal strains ranged from 62.9±8.4 to 80.8±2.8, throughout the tested days (Table 1). Melanin production varied according to each strain, but all strains produced this pigment (Figure 1). Cryptococcus spp. strains presented a significant increase in pigmentation through-out the days of analysis (P<.05), when grown under condition B, as shown in Table 1 and Figure 1.
Candida parapsilosis ATCC 22019 was used as negative control for pigmentation, reaching values of percentage of black ranging from 46.3±1.4 to 49.3±1.0 and from 53.3±2.1 to 55.7±3.2 for growth on conditions A and B, respectively. H. werneckii, the positive control for pigmentation, did not show satisfactory growth on condition A, pre-senting a percentage of black ranging from 52.4±2.0 to 60±0.4, for the studied days. Under condition B, colonies were well grown, obtain-ing a percentage of black rangobtain-ing from 73.4±1.6 to 86.0±0.6. When data on percentage of black were analysed for the complete period of analysis, the pigmentation of Cryptococcus spp. under condition B was
significantly higher (P<.05) than that obtained for C. parapsilosis ATCC 22019, but similar to that obtained for H. werneckii.
4
|
DISCUSSION
As previously mentioned, melanin is an important virulence factor for several microorganisms, including C. neoformans and C. gattii.2,4,25 For instance, it has been shown that the presence of melanin on fungal cell wall protects Cryptococcus cells from antifungal drugs,26 opsonising
antibodies27 and oxidative compounds produced by macrophages.17
Therefore, considering all the protective effects of melanin on C. neo-formans sensu lato and C. gattii sensu lato, it seems important to evalu-ate drugs that modulevalu-ate fungal melanin production.
Different methods for melanin quantification have been widely used1,24,28–30 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 diffi-cult to apply, time consuming and expensive, such as the use of SEM, which requires costly equipment and reagents.1,29 Furthermore, mela-nin extraction can also be performed, but this process may modify its composition.1,2 Another method used to indirectly evaluate melanin
F I G U R E 1 Analysis of the melanisation of Cryptococcus neoformans sensu lato and C. gattii sensu lato on minimal medium agar with and without L- DOPA. (A, B, C) Representative grayscale image of fungal colonies grown under conditions A, minimal medium (MM) agar without L- DOPA, and condition B, MM agar with L- DOPA, on the fourth day of growth. Similar images were obtained for all tested strains, from the third to the seventh day of growth in order to analyse colony pigmentation, through grayscale pixel quantification by the software ImAgEj™; (D and E) Percentage of black obtained for C. neoformans sensu lato (n=8) and C. gattii sensu lato (n=3), Candida parapsilosis ATCC 22019 and Hortaea werneckii, after growth under condition A (D) or condition B (E), from the third to the seventh day of growth. Percentage of black, expressed as mean ± standard deviation, was calculated from the quantification of pixels obtained from grayscale images of the fungal colonies. C. parapsilosis ATCC 22019: negative control for melanisation; H. werneckii: positive control for melanisation
(A)
(D) (E)
production is the spectrophotometric quantification of laccase activ-ity.30 It is an inexpensive method that gives an estimate of melanin
production, however, it is important to emphasise that the activity of this enzyme is variable and influenced by temperature and other ex-perimental conditions, which may affect the reliability of the results. In addition, minimal medium broth has been widely used in assays to in-duce melanin production,1 but it requires melanin extraction to
quan-tify this pigment, once it does not allow to visually evaluate differences in cell pigmentation, as fungal colonies are not formed in liquid media. Hence, as previously described, the proposal of a new methodology in this field may be very beneficial.29
In the present work, instead of using minimal medium broth, minimal medium agar was proposed, which allowed proper growth of Cryptococcus strains, as well as C. parapsilosis ATCC 22019, when tested under condition A, ie without L- DOPA. Under this condition, the strain of H. werneckii formed a thin layer of growth with weak mel-anisation, probably due to the limited nutritional resources of this poor culture medium, corroborating the findings of Rani et al., [31] who ob-served that melanin production by H. werneckii varies according to the nutritional conditions provided for fungal growth. Despite this scarcity of resources, differences in strain pigmentation were observed in con-dition A, and H. werneckii obtained the highest percentage of black.
As for condition B, ie with the supplementation of L- DOPA, C. parapsilosis ATCC 22019 did not present melanisation, obtaining the lowest percentage of black, while C. neoformans sensu lato and C. gat-tii sensu lato and H. werneckii presented good pigmentation, which started to increase from the fourth day. In this study, no significant differences in melanin production were observed between C. neofor-mans sensu stricto (VNI), C. gattii sensu stricto (VGI) and C. deuterogattii (VGII), corroborating the research of Thompson et al. 2014 [15].
It is important to mention that for the last day of analysis, H. wer-neckii presented the highest percentage of black (86%), followed by the strains of Cryptococcus spp. (81%). Colony morphology for the two genera was different and growth of H. werneckii was visibly better under condition B. Melanin production and colony pigmentation in-creased over time for the strains of Cryptococcus spp. and H. werneckii. In addition, the proposed methodology promoted a stable and repro-ducible melanisation of C. neoformans sensu lato and C. gattii sensu lato strains.
In this research, the use of minimal medium in a solid presenta-tion allowed studying cell pigmentapresenta-tion through image analysis with a widely available software. Under this circumstance, melanin extraction was not necessary, similar to what was described by Fernanades et al. (2016), [28] 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.1 Moreover, this
method-ology allowed the detection of differences in melanin production per strain and per day of analysis. Finally, the present research proposed a fast and low- cost methodology for the indirect quantification of mel-anin in C. neoformans sensu lato and C. gattii sensu lato, which led to reliable and reproducible results, when performed under standardised conditions.
ACKNOWLEDGEMENTS
This work was supported by the National Council for Scientific and Technological Development (CNPq; Brazil; process: 445670/2014- 2) and Coordination for the Improvement of Higher Education Personnel (CAPES).
CONFLICT OF INTEREST
None to declare.
REFERENCES
1. Rosas AL, Nosanchuk JD, Gomez BL, Edens WA, Henson JM, Casadevall A. Isolation and serological analyses of fungal melanins. J Immunol Methods. 2000;244:69-80.
2. Butler MJ, Day AW. Fungal melanins: a review. Can J Microbiol. 1998;44:1115-1136.
3. Nosanchuk JD, Stark RE, Casadevall A. Fungal melanin: what do we know about structure? Front Microbiol. 2015;6:1-7.
4. Eisenman HC, Casadevall A. Synthesis and assembly of fungal
mela-nin. Appl Microbiol Biotechnol. 2012;93:931-940.
5. Hagen F, Khayhan K, Theelen B, et al. Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet Biol. 2015;78:16-48.
6. Oh KS, Hwang SM. Isolation and characterization of Cryptococcus neoformans from environmental sources in Busan. Mycobiology. 2005;33:188-193.
7. Kidd SE, Chow Y, Mak S, et al. Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States. Appl Environ Microbiol. 2007;73:1433-1443.
8. Chayakulkeeree M, Perfect JR. Cryptococcosis. Infect Dis Clin North Am. 2006;20:507-544.
9. Galanis E, Hoang L, Kibsey P, Morshed M, Phillips P. Clinical presen-tation, diagnosis and management of Cryptococcus gattii cases: les-sons learned from British Columbia. Can J Infect Dis Med Microbiol. 2009;20:23-28.
10. Chen SCA, Slavin MA, Heath CH, et al. Clinical manifestations of Cryptococcus gattii infection: determinants of neurological sequelae and death. Clin Infect Dis. 2012;55:789-798.
11. Springer DJ, Billmyre RB, Filler EE, et al. Cryptococcus gattii VGIII iso-lates causing infections in HIV/AIDS patients in Southern California: identification of the local environmental source as arboreal. PLoS Pathog. 2014;10:e1004285.
12. Nyazika TK, Hagen F, Meis JF, Robertson VJ. Cryptococcus tetragattii as a major cause of cryptococcal meningitis among HIV- infected indi-viduals in Harare, Zimbabwe. J Infect. 2016;72:745-752.
13. Pedroso RDS, Da Costa KRC, Ferreira JC, Candido RC. Evaluation of melanin production by Cryptococcus species in four different culture
media. Rev Soc Bras Med Trop. 2007;40:566-568.
14. Cogliati M. Global molecular epidemiology of Cryptococcus neo-formans and Cryptococcus gattii: an atlas of the molecular types. Scientifica (Cairo). 2013;2013:23.
15. Thompson GR, Albert N, Hodge G, et al. Phenotypic differences of Cryptococcus molecular types and their implications for virulence in a drosophila model of infection. Infect Immun. 2014;82:3058-3065. 16. Wang Y, Aisen P, Casadevall A. Cryptococcus neoformans melanin
and virulence: mechanism of action. Infect Immun.
1995;63:3131-3136.
18. Jacobson ES, Hove E, Emery HS. Antioxidant function of melanin in black fungi. Infect Immun. 1995;63:4944-4945.
19. Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neo-formans. J Exp Med. 1996;184:377-386.
20. Gokulshankar S, Babu K, Valli S, Ranjitsingh AJA, Ranjith MS. Cowitch seed agar medium - a simple new medium for identifica-tion and melanin producidentifica-tion of Cryptococcus neoformans. Mycoses. 2011;54:208-210.
21. Velegraki A, Kiosses VG, Kansouzidou A, Smilakou S, Mitroussia-Ziouva A, Legakis NJ. Prospective use of RFLP analysis on am-plified Cryptococcus neoformans URA5 gene sequences for rapid identification of varieties and serotypes in clinical samples. Med Mycol. 2001;39:409-417.
22. Paliwal DK, Randhawa HS. Evaluation of a simplified Guizotia abyss-inica seed medium for differentiation of Cryptococcus neoformans. J Clin Microbiol. 1978;7:346-348.
23. Denning DW, Stevens DA, Hamilton JR. Comparison of Guizotia abyss-inica seed extract (birdseed) agar with conventional media for selective identification or Cryptococcus neoformans in patients with acquired immunodeficiency syndrome. J Clin Microbiol. 1990;28:2565-2567. 24. Cunha MML, Franzen AJ, Seabra SH, et al. Melanin in Fonsecaea
pe-drosoi: a trap for oxidative radicals. BMC Microbiol. 2010;10:80. 25. Gerstein AC, Nielsen K. It’s not all about us: evolution and
mainte-nance of Cryptococcus virulence requires selection outside the human host. Yeast. 2017;34:143-154.
26. Buchanan KL, Murphy JW. What makes Cryptococcus neoformans a pathogen? Emerg Infect Dis. 1998;4:71-83.
27. Huffnagle GB, Chen GH, Curtis JL, McDonald RA, Strieter RM, Toews GB. Down- regulation of the afferent phase of T- cell- mediated pulmonary inflammation and immunity by a high melanin- producing strain of Cryptococcus neoformans. J Immunol. 1995;155: 3507-3516.
28. Pereira CB, Bueno FL, Dias ALT, Brigagão MRPL, Paula CR, Siqueira AM. Evaluation of laccases and melanization in clinical and environ-mental Cryptococcus neoformans samples by non- denaturing PAGE. J Med Microbiol. 2009;58:563-566.
29. Fernandes B, Matamá T, Guimarães D, Gomes A, Cavaco-paulo A. Fluorescent quantification of melanin. Pigment Cell Melanoma Res. 2016;29:707-712.
30. Singhal A, Choudhary G, Thakur IS. Characterization of laccase ac-tivity produced by Cryptococcus albidus. Prep Biochem Biotechnol. 2012;42:113-124.
31. Rani MHS, Ramesh T, Subramanian J, Kalaiselvam M. Production and characterization of melanin pigment from halophilic black yeast Hortaea werneckii. Int J Pharma Res Rev. 2013;2:9-17.
How to cite this article: Brilhante RSN, España JDA, de
