Cryptococcus neoformans: History
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Zena Birkenfeld

Cryptococcus neoformans var. neoformans is the second most prevalent agent of cryptococcosis in central Europe. Infections mostly present with localized skin and disseminated infections. Clinical isolates of C. neoformans var. neoformans present a substantial phenotypic variability. Median survival of G. mellonella varied between 6 and 14 days. C. neoformans var. neoformans isolates from disseminated infections showed stronger melanization and larger capsules.

  • cryptococcosis
  • Cryptococcus neoformans

1. Introduction

Cryptococcosis is the most prevalent fungal infection of the central nervous system. Currently, the nomenclature of the agents of cryptococcosis is being reevaluated [1][2]. Here, we adhere to the nomenclature using Cryptococcus neoformans species complex. C. neoformans var. grubii is the most prevalent agent isolated from AIDS patients with meningoencephalitis worldwide [3]. Several virulence factors including the polysaccharide capsule and production of melanin were characterized. Although clinical isolates differ in these phenotypes, their contribution to clinical illness is poorly understood [4].
In Europe, up to 20% of human cryptococcosis cases are caused by C. neoformans var. neoformans [5][6]. Infection models generally suggest a lower virulence of C. neoformans var. neoformans strains as compared to C. neoformans var. grubii [7]. These infections may present with diverse clinical syndromes including primary cutaneous cryptococcosis, often in non-immunocompromised hosts [8].
Molecular typing of clinical and environmental C. neoformans isolates from Europe using multilocus sequence typing (MLST) covering parts of six housekeeping genes and an intergenic spacer suggests different trajectories of clinical isolates from both varieties cultivated in Europe, i.e., mostly clonal expansion of C. neoformans var. grubii versus recombination of C. neoformans var. neoformans [6][9][10]. Previous studies did not find a correlation between genotypes, as assessed by MLST and clinical presentation [10]. Correlation between genotype and fungal phenotype is increasingly being recognized and may be responsible for clinical presentation and outcome of patients with cryptococcosis. However, isolates of the same genotype, as determined by MLST may dramatically differ in virulence traits, suggesting that typing methods with higher resolution might be needed to understand genetic determinants of virulence and clinical presentation [4][11]. In addition, isolates of different genotypes may show comparable virulence determinants as identified by infection models that are associated with the clinical presentation of cryptococcosis.

2. Interaction of Cryptococcus with Acanthamoeba

A. castellanii 1BU (ATCC PRA-105, Manassas, VA, USA) was obtained from a stock culture maintained at the Robert Koch Institute. Trophozoites were incubated at 29 °C with 4.5% CO2 in 20 mL peptone yeast glucose medium (PYG; ATCC 712, Manassas, VA, USA) containing 1% (v/v) of Penicillin/Streptomycin. Amoebas were passaged weekly and one day before each experimental unit by transferring the culture into a new cell culture flask with filter cap and diluting the culture 1:10 with fresh medium.
Phagocytosis by and cytotoxicity for A. castellanii was determined following the procedure described before by Steenbergen et al. [12]. In brief, phagocytosis indices were determined by coincubation of fungi (2 × 105) and amoeba (1 × 105) in 100 µL PYG medium at 29 °C and 4.5% CO2 for two hours in the dark. Cells were stained, viewed by microscope and the ratio of amoebas with engulfed yeast cells was calculated. For determination of cytotoxicity, fungi (1 × 104) and amoeba (1 × 104) were coincubated in 100 µL PBS at 29 °C and 4.5% CO2 for 48 h in the dark. The trypan blue exclusion assay (Sigma-Aldrich, Burlington, MA, USA) was performed to determine the percentage of dead amoebas [13]. As negative control, amoebas were incubated alone in PBS. The reference strain B3501A and the acapsular strain cap67 were used to determine the detectable range of uptake and cytotoxicity of the coincubation experiments. For each experimental setup, three individual experiments were performed with eight technical replicates each.

3. Infection of Galleria mellonella Larvae

Larvae of G. mellonella were obtained in a shop for fishing supplies (Angelbedarfsladen Malchow, Berlin, Germany) and experiments were started immediately after obtaining the larvae. One day before inoculation, animals were weighed and collected in experimental groups of 20. The larvae were divided into equal weight groups (250–600 mg) and weights were visualized via boxplots to obtain a cross-section of the total population but equally distributed between the groups to ensure comparability and thereby exclude body volume as a determining factor. Larvae with dark spots were excluded because melanization might be evidence for infection [14]. The animals were incubated overnight at 37 °C in Petri dishes (Ø = 10 cm) containing wood chips before inoculation.
Cryptococcus cells were harvested from overnight cultures, washed and adjusted to a concentration of 0.5 × 108 cells/mL in 0.85% NaCl. The cell suspensions were carefully vortexed and taken up by an insulin syringe. Larvae were disinfected using a cotton swab containing 70% ethanol and inoculated through injection of 20 µL into the last left proleg. Each animal was infected with 1 × 106 cells. The inoculum was controlled by plating (5 µL) as a serial dilution on YPD and brain heart infusion (BHI) agar. In each experiment, control groups were used to estimate a traumatic effect of inoculation (injection of 20 µL sterile 0.85% NaCl), to estimate the virulence potential of unviable C. neoformans var. neoformans cells (H99 inactivated at 60 °C for one hour), and to monitor for larval viability (no intervention). Larvae were incubated at 37 °C in Petri dishes containing wood chips and controlled for survival (movement upon stimulus) every day. Petri dishes with fresh wood chips were changed daily.

4. Researches and Findings

C. neoformans var. neoformans isolates cultivated in Germany show a remarkable phenotypic diversity including capsule size, production of melanin, urease and phospholipase activity. This may impact the differences in uptake into phagocytic amoebae, cytotoxicity for amoebae and virulence in the G. mellonella infection model. Isolates from disseminated infections showed larger capsules, cell bodies and higher melanin production as compared to isolates from localized skin infections. Although isolates from disseminated infections showed variable degrees of urease and phospholipase production, they demonstrated higher uptake into amoeba and cytotoxicity and shorter survival of G. mellonella.
Epidemiologic studies performed in the context of disseminated infections by C. neoformans var. grubii in HIV-infected patients demonstrate that fungal genotype may affect clinical outcomes [4]. It was suggested that cell surface changes, including capsule size, melanization and changes in cell size, i.e., large titan cells and small micro cells are phenotypic changes that correlate with virulence potential when environmental fungi enter hosts [4]. It was demonstrated that isolates from human cryptococcosis patients, mainly C. neoformans var. grubii and C. gattii, show substantial variation in virulence in infection models including the G. mellonella infection model [15][16].
So far, few studies have been performed to correlate the virulence potential of C. neoformans var. neoformans and the clinical presentation of these infections. C. neoformans var. neoformans are the second most prevalent agents of cryptococcosis in Europe [5][6]. In addition, they can be cultivated from environmental sources including trees, dust, pigeon droppings and animals including cats, cows and pigeons [9]. Based on environmental sampling from non-random sites, it was suggested that this fungus may be adapted to colder, wetter climates currently found in central Europe [10]. However, a recent report on the cultivation from Saudi Arabian desert soil suggests that this fungus is more widespread and potentially more adaptable to various environmental conditions than previously thought [17]. Interestingly, the population structure of C. neoformans var. neoformans isolates from Europe shows high genotypic diversity potentially facilitated by sexual reproduction [9]. As this process may generate phenotypic diverse offspring, laboratory tests might be needed address to virulence potential and therefore the public health potential of C. neoformans var. neoformans isolates.
Different models were used to study fungal virulence. Amoebas are a relatively easy and cheap model system. They have gained interest as some aspects of the interaction between amoeba and fungi resemble the interaction between fungi and phagocytic cells. The capsule, melanin production and phospholipase activity were shown to be important for the survival of Cryptococcus in coincubation with amoeba, while urease activity and mating-type were not. This model does not reflect other disease determinants such as immune-mediated host damage, an important part of animal cryptococcosis [18]. However, the significant differences in uptake by amoeba and cytotoxicity suggest that this model may be used to screen for the potential of strains to cause disseminated infections.
G. mellonella has emerged as a model system to explore fungal virulence and pathogenesis. G. mellonella and the mammalian immune system share some similarities including pathogen recognition, inflammatory responses, phagocytosis of pathogens and phagocyte responses to fungal uptake [19].
Primary cutaneous cryptococcosis typically presents as a solitary skin infection site after transcutaneous injury with a contaminated source. These infections are thought to be mostly caused by C. neoformans var. neoformans (genotype VNIV) isolates and generally have a favorable outcome without systemic signs of infection and mostly negative serum antigen. They are often diagnosed in patients without underlying immunosuppression [8]. The absence of dissemination in these cases might be a consequence of the host’s immune system and fungal attributes. Genotype VNIV strains were shown to be less virulent in animal models than VNI strains. In addition, cryptococci with the mating-type are less virulent in infection models and this was confirmed for C. neoformans var. neoformans [7][20].
Nevertheless, even Da strains may cause disseminated infections despite low virulence potential in infection models as demonstrated here by the isolate 19-0346 from a disseminated infection and low virulence in G. mellonella. This has previously been documented for another Da isolate from a disseminated infection in an AIDS patient and low virulence in a mouse model [7].

This entry is adapted from the peer-reviewed paper 10.3390/microorganisms10020321

References

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