Miltefosine Has a Postantifungal Effect and Induces Apoptosis in Cryptococcus Yeasts.

Cryptococcus spp. are common opportunistic fungal pathogens, particularly in HIV patients. The approved drug miltefosine (MFS) has potential as an alternative antifungal against cryptococcosis; however, the mechanism of action of MFS in Cryptococcus is poorly understood. Here, we examined the effects of MFS on C. neoformans and C. gattii yeasts (planktonic and biofilm lifestyles) to clarify its mechanism of action. MFS presented inhibitory and fungicidal effects against planktonic Cryptococcus cells, with similar activities against dispersion biofilm cells, while sessile biofilm cells were less sensitive to MFS. Interestingly, MFS had postantifungal effect on Cryptococcus, with a proliferation delay of up to 8.15 h after a short exposure to fungicidal doses. MFS at fungicidal concentrations increased the plasma membrane permeability, likely due to a direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. Moreover, MFS reduced the mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and induced DNA fragmentation and condensation, all of which are hallmarks of apoptosis. Transmission electron microscopy analysis showed that MFS-treated yeasts had a reduced mucopolysaccharide capsule (confirmed by morphometry with light microscopy), plasma membrane irregularities, mitochondrial swelling, and a less conspicuous cell wall. Our results suggest that MFS increases the plasma membrane permeability in Cryptococcus via an interaction with ergosterol and also affects the mitochondrial membrane, eventually leading to apoptosis, in line with its fungicidal activity. These findings confirm the potential of MFS as an antifungal against C. neoformans and C. gattii and warrant further studies to establish clinical protocols for MFS use against cryptococcosis.

An alternative method for the analysis of melanin production in Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato.

Melanin is an important virulence factor for several microorganisms, including Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato, thus, the assessment of melanin production and its quantification may contribute to the understanding of microbial pathogenesis. The objective of this study was to standardise an alternative method for the production and indirect quantification of melanin in C. neoformans 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 grayscale 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 pigmentation. 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.

Fisetin as a promising antifungal agent against Cryptocococcus neoformans species complex.

AIMS: The aim of this study was to investigate the mechanisms of action of fisetin, a flavonol with antifungal activity previously evaluated against the Cryptococcus neoformans species complex. METHODS AND RESULTS: Ergosterol content and flow cytometry analysis were determined for the C. neoformans species complex in the presence of fisetin and ultrastructural analysis of morphology was performed on Cryptococcus gattii and C. neoformans. Decrease in the total cellular ergosterol content after exposure to fisetin ranged from 25·4% after exposure to 128 µg ml(-1) to 21·6% after exposure to 64 µg ml(-1) of fisetin compared with the control (without fisetin). The fisetin effects obtained with flow cytometry showed metabolic impairment, and alterations in its normal morphology caused by fisetin in C. neoformans cells were verified using scanning electron microscopy. CONCLUSIONS: Fisetin is a compound that acts in the biosynthesis of ergosterol. Flow cytometry showed that fisetin reduced viability of the metabolically active cells of C. gattii, while morphological changes explain the action of fisetin in inhibiting growth of these fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the idea that fisetin may represent a good starting point for the development of future therapeutic substances for cryptococcosis.

[Fungal infections of the central nervous system in the immunocompetent host]. / Pilzinfektionen des Zentralnervensystems bei immunkompetentem Wirt.

The majority of mycoses which lead to mycotic tumors in patients without any predisposing underlying disease are either caused by Cryptococcus gattii and C. neoformans or by dematiaceous fungi which include Cladophialophora bantiana, Ramichloridium mackenziei, Exophiala and Fonsecaea species. The detection of hyphae in granuloma in the brain should lead to screening for pigmented fungi, which are recognized best in hematoxylin eosin (HE) or sometimes also in periodic acid-Schiff (PAS) stained sections. In patients who survive a near drowning accident and those who develop brain abscesses, scedosporiosis should always be considered as a possible infection.

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Punicalagin triggers ergosterol biosynthesis disruption and cell cycle arrest in Cryptococcus gattii and Candida albicans : Action mechanisms of punicalagin against yeasts.

Punicalagin is a phenolic compound extracted from Lafoensia pacari A. St.-Hil (Lythraceae) leaves. It has demonstrated interesting activity against pathogenic fungi, e.g., Cryptococcus gattii and Candida albicans, by inhibiting fungi growth in a minimum inhibitory concentration (MIC) at 4 µg/mL. However, the mechanisms behind its antifungal action are not well understood. In this study, certain parameters were investigated, by transmission electron microscopy, ergosterol synthesis inhibition, and flow cytometry analyses, to gain insight into the possible biological targets of punicalagin (4 or 16 µg/mL) against yeast cells. Data showed that, in contrast to untreated cells, punicalagin triggered severe ultrastructural changes in C. gattii and C. albicans, such as disorganization of cytoplasmic content and/or thickened cell walls. In addition, it caused a decrease in yeast plasma membrane ergosterol content in a concentration-dependent manner. However, it was unable to bring about significant fungal cell membrane rupture. On the other hand, punicalagin (16 µg/mL) significantly arrested C. albicans and C. gattii cells at the G0/G1 phase, with a consequent reduction in cells at the G2/M phase in both fungi isolates, and thereby prevented progression of the normal yeast cell cycle. However, these alterations showed no involvement of reactive oxygen species overproduction in C. albicans and C. gattii cells, although punicalagin triggered a significant loss of mitochondrial membrane potential in C. albicans. These findings suggest that punicalagin is a promising plant-derived compound for use in developing new antifungal therapies.

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.

Phenotypic characteristics and transcriptome profile of Cryptococcus gattii biofilm.

In this study, we characterized Cryptococcus gattii biofilm formation in vitro. There was an increase in the density of metabolically active sessile cells up to 72 h of biofilm formation on polystyrene and glass surfaces. Scanning electron microscopy and confocal laser scanning microscopy analysis revealed that in the early stage of biofilm formation, yeast cells adhered to the abiotic surface as a monolayer. After 12 h, extracellular fibrils were observed projecting from C. gattii cells, connecting the yeast cells to each other and to the abiotic surface; mature biofilm consisted of a dense network of cells deeply encased in an extracellular polymeric matrix. These features were also observed in biofilms formed on polyvinyl chloride and silicone catheter surfaces. We used RNA-Seq-based transcriptome analysis to identify changes in gene expression associated with C. gattii biofilm at 48 h compared to the free-floating planktonic cells. Differential expression analysis showed that 97 and 224 transcripts were up-regulated and down-regulated in biofilm, respectively. Among the biological processes, the highest enriched term showed that the transcripts were associated with cellular metabolic processes, macromolecule biosynthetic processes and translation.

Extracellular fibrils of pathogenic yeast Cryptococcus gattii are important for ecological niche, murine virulence and human neutrophil interactions.

Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40-100 nm diameter x500-3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12alpha mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN- mediated killing.