Visualization and Documentation of Capsule and Melanin Production in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen primarily targeting immunosuppressed populations in both resource-rich and resource-limited nations. Successful treatment is limited to a few antifungals that have become compromised by cryptococcal resistance, leading to intensive research seeking new drug candidates. Two distinguishing hallmarks of this species are the ability to develop a polysaccharide capsule and melanization of the fungal cells. These also act as virulence factors, protecting this pathogen in the host as well as in the environment. Here we describe two classic methods to document capsule and melanin. Although initially described and documented several decades ago, these methods remain relevant in spite of the advent of more sophisticated methodology, due in part to their simplicity and cost efficiency.

Distinct pathways of adaptive evolution in Cryptococcus neoformans reveal a mutation in adenylyl cyclase with trade-offs for pathogenicity

“…Melanization of different strains was analyzed following previously established protocols. 87 Briefly, overnight cultures of C. neoformans strains were pelleted by centrifugation (3000xg, 5 minutes), washed twice in sterile water and adjusted to an OD of 0.25. Five microliters of each strain was spotted onto L-DOPA plates (7.6 mM L-asparagine monohydrate, 5.6 mM glucose, 10 mM MgSO 4 , 0.5 mM 3,4-dihydroxy-L-phenylalanine, 0.3 mM thiamine-hydrochloride, and 20 nM biotin) and onto YPD plates and incubated at 30°C or 37°C.…”

Antifungal and Antibiofilm Efficacy of Paeonol Treatment Against Biofilms Comprising Candida albicans and/or Cryptococcus neoformans

“…After 24 h of incubation, yeast-like cells and/or hyphae were assessed microscopically. Moreover, to examine the effect of paeonol on the capsule production of C. neoformans H99, the assay was carried out as previously described ( Nichols, 2021 ). Briefly, the C. neoformans H99 (approximately 1 × 10 6 CFUs ml −1 ) was treated with various concentrations of paeonol (0, 1/2 MIC, MIC, 2 MIC) at 30°C for 24 h. Fungal cells were then collected and washed three times with 10 mM PBS, resuspended in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, USA), and further cultured for 48 h at 30°C.…”

“…Finally, 0.1 ml of the fungal culture was mixed with an equal volume of India ink (Phygene, China), and the capsule was visualized under the microscope. Similarly, to evaluate the effect of paeonol on melanin production in C. neoformans H99, the assay was conducted as previously described ( Nichols, 2021 ). A total of 5 µl of C. neoformans H99 cells (approximately 1 × 10 6 CFUs ml −1 ) were spotted on melanin-inducing minimal medium (15 mM glucose, 10 mM MgSO 4 , 21.4 mM KH 2 PO 4 , 13 mM glycine, 3 µM thiamine, 1.5% (w v −1 ) Bacto agar, pH 5.5) supplemented with 1 mM l -DOPA and different concentrations of paeonol (0, 1/4 MIC, 1/2 MIC, and MIC) and further cultured at 30°C for 48 h at 150 rpm and protected from light.…”

Distinct pathways of adaptive evolution inCryptococcus neoformansreveal a point mutation in adenylate cyclase with drastic tradeoffs for pathogenicity

“…Melanization of different strains was analyzed following previously established protocols 65 . Briefly, overnight cultures of C. neoformans strains were pelleted by centrifugation (3000xg, 5 minutes), washed twice in sterile water and adjusted to an OD of 0.25.…”