Titan cell formation is unique to Cryptococcus species complex.

Members of the Cryptococcus species complex stand out by unique virulence factors that allowed evolutionary transition to pathogenesis. Among the factors contributing to cryptococcosis is a morphological transformation into giant (Titan) cells. It remains unclear whether species outside of the C. neoformans/C. gattii species complex are capable of titanization. We utilized two recently developed protocols that allow obtaining Titan cells in vitro to test if titanization occurs in non-C. neoformans/C. gattii species. We find that none of the tested strains, representing 10 species of basidiomycetous yeasts and the ascomycetous yeast Saccharomyces cerevisiae, undergo significant titanization under conditions that promote robust Titan cell formation in C. neoformans/C. gattii species complex. C. terreus formed occasional enlarged cells through a mechanism potentially similar to that of titanization. Our findings suggest that titanization is a rare phenomenon among basidiomycetous yeasts that occurs mostly in members of the C. neoformans/C. gattii species complex.

Anticryptococcal activity of hexane extract from Spondias tuberosa Arruda and associated cellular events.

Cryptococcosis is an opportunistic systemic mycosis whose treatment is limited to three drugs. In this work, we evaluated the antifungal activity of a hexane extract (HE) from Spondias tuberosa leaves against Cryptococcus neoformans and Cryptococcus gattii. Minimal inhibitory concentrations (MIC) were determined, and putative mechanisms were evaluated by flow cytometry. In addition, an in vivo infection assay was performed using Tenebrio molitor larvae. Treatment with HE inhibited the growth of standard and clinical isolates of C. neoformans and C. gattii (MICs ranging from 0.78 to 3.12mg/mL), significantly (P<0.05) increased mitochondrial superoxide anion levels, and induced mitochondrial membrane depolarization, loss of lysosomal membrane integrity, and phosphatidylserine externalization. The mean survival time of C. gattii-infected T. molitor larvae significantly (P<0.05) increased from 1.225 days in control to 3.067 and 3.882 days in HE-treated groups (78 and 156mg/kg, respectively). In conclusion, HE showed anticryptococcal activity, induced mitochondrial and lysosomal damage in yeast cells, and exhibited anti-infective action against C. gattii in T. molitor larvae.

Activity of 3'-hydroxychalcone against Cryptococcus gattii and toxicity, and efficacy in alternative animal models.

AIM: This work aimed to evaluate the activity of 3'-hydroxychalcone against Cryptococcus gattii in planktonic and biofilm forms and their toxicity using alternative animal models. MATERIALS & METHODS: Minimum inhibitory concentration and minimum fungicide concentration were determined. Biofilm formation and the susceptibility tests were performed by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H-tetrazolium hydroxide assay. Toxicity and efficacy were checked in Danio rerio and Galleria mellonella models. RESULTS: The compound 3'-hydroxychalcone showed fungicidal activity against C. gattii in both planktonic and biofilm forms. The toxicity in zebrafish embryos revealed a low lethal concentration. In G. mellonella, the compound did not show antifungal activity and larvae toxicity. CONCLUSION: Because of the activity of 3'-hydroxychalcone against C. gattii in vitro, molecular modifications should be made to improve efficacy and to reduce toxicity in vivo. [Formula: see text].

Antifungal activity of eicosanoic acids isolated from the endophytic fungus Mycosphaerella sp. against Cryptococcus neoformans and C. gattii.

The antifungal effects of two eicosanoic acids, 2-amino-3,4-dihydroxy-2-25-(hydroxymethyl)-14-oxo-6,12-eicosenoic acid (compound 1) and myriocin (compound 2), isolated from Mycosphaerella sp. were evaluated against Cryptococcus neoformans and C. gattii. The compounds displayed antifungal activities against several isolates of C. neoformans and C. gattii, with minimal inhibitory concentration (MIC) values ranging from 0.49 to 7.82 µM for compound 1 and 0.48-1.95 µM for compound 2. In the checkerboard microtiter test, both compounds exhibited synergistic activity with amphotericin B against C. gattii. Ultrastructural analysis revealed several signs of damage in C. gattii and C. neoformans cells treated with compounds 1 and 2, including deformities in cell shape, depressions on the surface, and withered cells. The cells of C. gattii treated with compounds 1 and 2 showed less loss of cellular material in comparison to those treated with amphotericin B. The difference in cellular material loss increased in a test compound concentration-dependent manner. Consistent with this observation, compounds 1 and 2 were able to internalize propidium iodide (PI) in C. gattii cells. In addition, compound 2 induced the formation of several pseudohyphae, suggesting that it could reduce virulence in C. gattii cells. The study results show that these natural products led to membrane damage; however, this may not be the main target of action. These compounds have potential antifungal activity and could be useful in further studies for developing more effective combination therapies with amphotericin B and reducing side effects in patients.

Etiological factors of cryptococcosis - what makes them pathogens ?

A significant participation in the growing prevalence of fungal infections have basidiomycete yeasts of the genus of Cryptococcus. Among about seventy actually known species belonging to Cryptococcus genus there are only two: Cryptococcus neoformans and Cryptococcus gattii, which as anamorphs in parasitic stage of its life fully evolved the mechanisms predisposing them to be a human and animal pathogens. Specific attributes important in the pathogenesis of cryptococcosis called virulence factors allow them to colonize the mammalian tissues. Undoubtedly, the most important role in the pathogenesis play: the presence of polysaccharide capsule, synthesis of melanin, and the ability to grow in the range of temperatures 35-40°C.

Unisexual reproduction of Cryptococcus gattii.

Cryptococcus gattii is a basidiomycetous human fungal pathogen that typically causes infection in tropical and subtropical regions and is responsible for an ongoing outbreak in immunocompetent individuals on Vancouver Island and in the Pacific Northwest of the US. Pathogenesis of this species may be linked to its sexual cycle that generates infectious propagules called basidiospores. A marked predominance of only one mating type (α) in clinical and environmental isolates suggests that a-α opposite-sex reproduction may be infrequent or geographically restricted, raising the possibility of an alternative unisexual cycle involving cells of only α mating type, as discovered previously in the related pathogenic species Cryptococcus neoformans. Here we report observation of hallmark features of unisexual reproduction in a clinical isolate of C. gattii (isolate 97/433) and describe genetic and environmental factors conducive to this sexual cycle. Our results are consistent with population genetic evidence of recombination in the largely unisexual populations of C. gattii and provide a useful genetic model for understanding how novel modes of sexual reproduction may contribute to evolution and virulence in this species.

Phenotypic differences of Cryptococcus molecular types and their implications for virulence in a Drosophila model of infection.

Compared to Cryptococcus neoformans, little is known about the virulence of the molecular types in Cryptococcus gattii. We compared in vitro virulence factor production and survival data using a Drosophila model of infection to further characterize the phenotypic features of different cryptococcal molecular types. Forty-nine different isolates were inoculated into wild-type flies and followed for survival. In vitro, isolates were assessed for growth at 30 and 37°C, melanin production, capsule size, resistance to H(2)O(2), and antifungal susceptibility. A mediator model was used to assess molecular type and virulence characteristics as predictors of survival in the fly model. VGIII was the most virulent molecular type in flies (P < 0.001). At 30°C, VGIII isolates grew most rapidly; at 37°C, VNI isolates grew best. C. gattii capsules were larger than those of C. neoformans (P < 0.001). Mediator model analysis found a strong correlation of Drosophila survival with molecular type and with growth at 30°C. We found molecular-type-specific differences in C. gattii in growth at different temperatures, melanin production, capsule size, ability to resist hydrogen peroxide, and antifungal susceptibility, while growth at 30°C and the VGIII molecular type were strongly associated with virulence in a Drosophila model of infection.

Time-course proteome analysis reveals the dynamic response of Cryptococcus gattii cells to fluconazole.

Cryptococcus gattii is an encapsulated fungus capable of causing fatal disease in immunocompetent humans and animals. As current antifungal therapies are few and limited in efficacy, and resistance is an emerging issue, the development of new treatment strategies is urgently required. The current study undertook a time-course analysis of the proteome of C. gattii during treatment with fluconazole (FLC), which is used widely in prophylactic and maintenance therapies. The aims were to analyze the overall cellular response to FLC, and to find fungal proteins involved in this response that might be useful targets in therapies that augment the antifungal activity of FLC. During FLC treatment, an increase in stress response, ATP synthesis and mitochondrial respiratory chain proteins, and a decrease in most ribosomal proteins was observed, suggesting that ATP-dependent efflux pumps had been initiated for survival and that the maintenance of ribosome synthesis was differentially expressed. Two proteins involved in fungal specific pathways were responsive to FLC. An integrative network analysis revealed co-ordinated processes involved in drug response, and highlighted hubs in the network representing essential proteins that are required for cell viability. This work demonstrates the dynamic cellular response of a typical susceptible isolate of C. gattii to FLC, and identified a number of proteins and pathways that could be targeted to augment the activity of FLC.

Variability of phenotypic traits in Cryptococcus varieties and species and the resulting implications for pathogenesis.

Variability of phenotypic characteristics in Cryptococcus neoformans var. grubii and var. neoformans as well as Cryptococcus gattii can have diverse effects on the virulence of these fungi and are thus important for pathogenesis. This article summarizes the diverse phenotypic changes that these fungi can manifest. We divide changes into those that affect the entire fungal population and are predominantly induced by environmental signals, and those that involve subpopulations of the fungal population and have to be selected. Last, the article summarizes the experimental evidence that epitopes on the polysaccharide capsule also vary, which may have implications for the pathogenesis as these findings would further diversify the fungal population.