RESUMO
This chapter describes methodological details for preparing specimens of Cryptococcus neoformans (although it can be applied to any species of the genus) and their subsequent analysis by scanning and transmission electron microscopy. Adaptations to conventional protocols for better preservation of the sample, as well as to avoid artifacts, are presented. The protocols may be used to examine both the surface ultrastructure and the interior of this pathogenic fungus in detail.
Assuntos
Artefatos , Cryptococcus neoformans , Cryptococcus neoformans/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Microscopia Eletrônica de Varredura/métodos , Manejo de Espécimes/métodosRESUMO
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.
Assuntos
Anacardiaceae/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Criptococose/tratamento farmacológico , Hexanos/química , Animais , Antifúngicos/efeitos adversos , Antifúngicos/uso terapêutico , Criptococose/patologia , Cryptococcus gattii/citologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/ultraestrutura , Cryptococcus neoformans/citologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/ultraestrutura , Hexanos/farmacologia , Humanos , Larva/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/fisiologia , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Fitoterapia , Extratos Vegetais/química , Tenebrio/efeitos dos fármacos , Tenebrio/crescimento & desenvolvimento , Testes de ToxicidadeRESUMO
Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.
Assuntos
Acanthamoeba castellanii/metabolismo , Fungos/patogenicidade , Lectina de Ligação a Manose/metabolismo , Acanthamoeba castellanii/química , Acanthamoeba castellanii/microbiologia , Acanthamoeba castellanii/ultraestrutura , Animais , Candida albicans/patogenicidade , Candida albicans/ultraestrutura , Concanavalina A/metabolismo , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/ultraestrutura , Histoplasma/patogenicidade , Histoplasma/ultraestrutura , Interações Hospedeiro-Patógeno , Larva/microbiologia , Lepidópteros/microbiologia , Manose/química , Manose/metabolismo , Lectina de Ligação a Manose/química , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestrutura , Saccharomyces cerevisiae/patogenicidade , Saccharomyces cerevisiae/ultraestrutura , Fatores de Tempo , Imagem com Lapso de Tempo , Virulência , Fatores de Virulência/metabolismoRESUMO
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.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Anfotericina B/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus gattii/metabolismo , Cryptococcus gattii/ultraestrutura , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/ultraestrutura , Fragmentação do DNA/efeitos dos fármacos , Ergosterol/metabolismo , Cápsulas Fúngicas/efeitos dos fármacos , Cápsulas Fúngicas/metabolismo , Cápsulas Fúngicas/ultraestrutura , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/microbiologia , Fosforilcolina/farmacologia , Plâncton/efeitos dos fármacos , Plâncton/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Criptococose/microbiologia , Cryptococcus gattii/metabolismo , Cryptococcus neoformans/metabolismo , Melaninas/biossíntese , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/patogenicidade , Cryptococcus gattii/ultraestrutura , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/ultraestrutura , Meios de Cultura , Humanos , Melaninas/análise , Fatores de Virulência/análise , Fatores de Virulência/biossínteseRESUMO
Invasive fungal infections, including cryptococcosis, are a growing threat to immunocompromised patients. Although Cryptococcus neoformans and Cryptococcus gattii are the main agents of human cryptococcosis, opportunistic infections by environmental species, such as C. liquefaciens, have been observed recently. The main Cryptococcus virulence factor is the production and secretion of polysaccharides (PS). Previously, we showed that both species produce PS of similar composition. Here, we examined the ultrastructure and biological activity of capsular and secreted PS from C. liquefaciens, and yeast pathogenicity to an invertebrate host, in comparison with C. neoformans. Ultrastructural analysis by high-resolution microscopy showed that both species produce large and complex capsules. PS from both species had indistinguishable effects on phagocytosis levels, NO production and the secretion of a variety of immune mediators. Challenge with C. liquefaciens or C. neoformans led to complete lethality of G. mellonella larvae. Treatment with C. liquefaciens PS could not protect mice against infection with C. neoformans. We conclude that polysaccharides of the environmental yeast C. liquefaciens have strikingly similar ultrastructural and biological properties to those of C. neoformans, highlighting the importance of monitoring the emergence of new fungal pathogens for which thermotolerance may be an important transitional step towards pathogenesis in humans.
Assuntos
Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Polissacarídeos Fúngicos/efeitos adversos , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Mariposas/crescimento & desenvolvimento , Fagocitose , Animais , Criptococose/metabolismo , Cryptococcus neoformans/classificação , Cryptococcus neoformans/ultraestrutura , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Óxido Nítrico/metabolismo , Células THP-1RESUMO
AIM: In this study, we aimed to analyze the relationship of phosphorus-rich structures with surface architecture in Cryptococcus neoformans. METHODS: Phosphorus-rich structures in C. neoformans were analyzed by combining fluorescence microscopy, biochemical extraction, scanning electron microscopy, electron probe x-ray microanalysis and 3D reconstruction of high pressure frozen and freeze substituted cells by focused ion beam-scanning electron microscopy (FIB-SEM). RESULTS & CONCLUSION: Intracellular and surface phosphorus-enriched structures were identified. These molecules were required for capsule assembly, as demonstrated in experiments using polysaccharide incorporation by capsule-deficient cells and mutants with defects in polyphosphate synthesis. The demonstration of intracellular and cell wall-associated polyphosphates in C. neoformans may lead to future studies involving their participation in both physiologic and pathogenic events.
Assuntos
Cápsulas Bacterianas/química , Cryptococcus neoformans/metabolismo , Fósforo/análise , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/ultraestrutura , Cryptococcus neoformans/genética , Cryptococcus neoformans/ultraestrutura , Microscopia Eletrônica de Varredura , Fósforo/metabolismoRESUMO
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.
Assuntos
Antifúngicos/farmacologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Flavonoides/farmacologia , Criptococose/tratamento farmacológico , Criptococose/parasitologia , Cryptococcus gattii/química , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/ultraestrutura , Cryptococcus neoformans/química , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/ultraestrutura , Ergosterol/análise , Flavonóis , Testes de Sensibilidade MicrobianaRESUMO
Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. Its main virulence factor is an extracellular polysaccharide capsule whose structure, assembly and dynamics remain poorly understood. In this study, we apply improved protocols for sample preparation and recently-developed scanning microscopy techniques to visualize the ultrastructure of the C. neoformans capsule at high-resolution (up to 1 nm) and improved structural preservation. Although most capsule structures in nature consist of linear polymers, we show here that the C. neoformans capsule is a 'microgel-like' structure composed of branched polysaccharides. Moreover, we imaged the capsule-to-cell wall link, which is formed by thin fibers that branch out of thicker capsule filaments, and have one end firmly embedded in the cell wall structure. Together, our findings provide compelling ultrastructural evidence for a branched and complex capsule conformation, which may have important implications for the biological activity of the capsule as a virulence factor.