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1.
Braz J Microbiol ; 51(4): 1719-1727, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32856241

RESUMO

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.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cryptococcus gattii/efeitos dos fármacos , Ergosterol/metabolismo , Taninos Hidrolisáveis/farmacologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida albicans/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/metabolismo , Cryptococcus gattii/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Transmissão
2.
Biopreserv Biobank ; 18(3): 196-203, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32213084

RESUMO

The choice of a suitable preservation method is critical for long-term microorganisms' viability. The strains should be preserved for long periods using reliable and reproducible methods that minimize genotypic and phenotypic variations and viability losses. The methodologies are usually designed for a better performance in isolated microorganisms. However, atypical primary isolates of Cryptococcus neoformans or Cryptococcus gattii, such as mixed species or even different species of a species complex, are a challenge for long-term preservation and taxonomic review studies. The aim of this study was to evaluate which of the four preservation methods tested presented better performance in the preservation of simulated coexistence strains of C. neoformans and C. gattii. Two environmental strains, one C. gattii and one C. neoformans, were mixed in vitro to test four different preservation methods (freezing at -20°C, -80°C, -196°C, and freeze-drying). The colony-forming units from each preservation method were evaluated, and colonies were randomly selected and cultivated in canavanine glycine bromothymol blue (CGB) agar to evaluate the amounts of CGB-positive (C. gattii) and CGB-negative (C. neoformans) colonies resulting from each preservation method after 1 week, 15 days, 1 month, 6 months, and 1 year. According to our results, cryopreservation at -20°C demonstrated was preferable for C. neoformans species, and further studies after long-term storage are necessary. Recovery of yeast cells after freeze-dried preservation in skim milk is better for both species. Ultrafreezing methods evaluated (-80°C and -196°C) also showed better results in the maintenance of C. gattii. Freeze-drying should be preferred for the maintenance of multilineage isolates from the C. neoformans and C. gattii species complexes.


Assuntos
Criopreservação/métodos , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus neoformans/crescimento & desenvolvimento , DNA Fúngico/genética , Árvores/microbiologia , Cryptococcus gattii/genética , Cryptococcus gattii/isolamento & purificação , Cryptococcus neoformans/genética , Cryptococcus neoformans/isolamento & purificação , Meios de Cultura , Liofilização , Congelamento , Instabilidade Genômica , Viabilidade Microbiana , Fenótipo , Temperatura
3.
PLoS One ; 14(8): e0220989, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31398236

RESUMO

Cryptococcus gattii is a capsular fungal pathogen, which causes life-threatening cryptococcosis in immunocompetent individuals. This emerging pathogen is less likely to be recognized by innate immunity compared to traditional Cryptococcus neoformans strains. Previous studies indicate that C-type lectin receptors (CLRs), including dectin-1 and dectin-2, play a role in recognizing cryptococcal cells; however, it remains to be elucidated whether the receptors physically associate with C. gattii yeast cell surfaces. Based on the previous findings, we hypothesized that culture conditions influence the expression or exposure of CLR ligands on C. gattii. Therefore, in the present study, we first investigated the culture conditions that induce exposure of CLR ligands on C. gattii yeast cells via the binding assay using recombinant fusion proteins of mouse CLR and IgG Fc, Fc dectin-1 and Fc dectin-2. Common fungal culture media, such as yeast extract-peptone-dextrose (YPD) broth, Sabouraud broth, and potato dextrose agar, did not induce the exposure of dectin-1 ligands, including ß-1,3-glucan, on both capsular and acapsular C. gattii strains, in contrast to Fc dectin-1 and Fc dectin-2 bound to C. gattii cells growing in the conventional synthetic dextrose (SD) medium [may also be referred to as a yeast nitrogen base with glucose medium]. The medium also induced the exposure of dectin-1 ligands on C. neoformans, whereas all tested media induced dectin-1 and dectin-2 ligands in a control fungus Candida albicans. Notably, C. gattii did not expose dectin-1 ligands in SD medium supplemented with yeast extract or neutral buffer. In addition, compared to YPD medium-induced C. gattii, SD medium-induced C. gattii more efficiently induced the phosphorylation of Syk, Akt, and Erk1/2 in murine dendritic cells (DCs). Afterwards, the cells were considerably engulfed by DCs and remarkably induced DCs to secrete the inflammatory cytokines. Overall, the findings suggest that C. gattii alters its immunostimulatory potential in response to the environment.


Assuntos
Cryptococcus gattii/imunologia , Meio Ambiente , Imunomodulação , Animais , Células da Medula Óssea/metabolismo , Membrana Celular/metabolismo , Cryptococcus gattii/crescimento & desenvolvimento , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Ligantes , Camundongos Endogâmicos C57BL , Ligação Proteica , Solubilidade
4.
mSphere ; 3(6)2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30404928

RESUMO

Cryptococcus neoformans is an environmental pathogenic fungus with a worldwide geographical distribution that is responsible for hundreds of thousands of human cryptococcosis cases each year. During infection, the yeast undergoes a morphological transformation involving capsular enlargement that increases microbial volume. To understand the factors that play a role in environmental dispersal of C. neoformans and C. gattii, we evaluated the cell density of Cryptococcus using Percoll isopycnic gradients. We found differences in the cell densities of strains belonging to C. neoformans and C. gattii species complexes. The buoyancy of C. neoformans strains varied depending on growth medium. In minimal medium, the cryptococcal capsule made a major contribution to the cell density such that cells with larger capsules had lower density than those with smaller capsules. Removing the capsule, by chemical or mechanical methods, increased the C. neoformans cell density and reduced buoyancy. Melanization of the C. neoformans cell wall, which also contributes to virulence, produced a small but consistent increase in cell density. Encapsulated C. neoformans sedimented much more slowly in seawater as its density approached the density of water. Our results suggest a new function for the capsule whereby it can function as a flotation device to facilitate transport and dispersion in aqueous fluids.IMPORTANCE The buoyancy of a microbial cell is an important physical characteristic that may affect its transportability in fluids and interactions with tissues during infection. The polysaccharide capsule surrounding C. neoformans is required for infection and dissemination in the host. Our results indicate that the capsule has a significant effect on reducing cryptococcal cell density, altering its sedimentation in seawater. Modulation of microbial cell density via encapsulation may facilitate dispersal for other important encapsulated pathogens.


Assuntos
Cápsulas/metabolismo , Fenômenos Químicos , Cryptococcus neoformans/química , Cryptococcus neoformans/fisiologia , Centrifugação Isopícnica , Cryptococcus gattii/química , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/fisiologia , Cryptococcus neoformans/crescimento & desenvolvimento , Meios de Cultura/química , Povidona , Dióxido de Silício
5.
Sci Rep ; 8(1): 15260, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30323314

RESUMO

Natural and artificial hybridization has been frequently reported among divergent lineages within and between the two closely related human pathogenic fungi Cryptococcus gattii species complex and Cryptococcus neoformans species complex. However, the biological effects of such hybridization are not well known. Here we used five strains of the C. neoformans species complex and twelve strains of the C. gattii species complex to investigate the potential effects of selected environmental and genetic factors on the germination of their basidiospores from 29 crosses. We found that the germination rates varied widely among crosses and environmental conditions, ranging from 0% to 98%. Overall, the two examined media showed relatively little difference on spore germination while temperature effects were notable, with the high temperature (37 °C) having an overall deleterious effect on spore germination. Within the C. gattii species complex, one intra-lineage VGIII × VGIII cross had the highest germination rates among all crosses at all six tested environmental conditions. Our analyses indicate significant genetic, environmental, and genotype-environment interaction effects on the germination of basidiospores within the C. gattii species complex.


Assuntos
Cryptococcus gattii/classificação , Cryptococcus gattii/genética , Cryptococcus gattii/fisiologia , Meio Ambiente , Esporos Fúngicos , Basidiomycota/classificação , Basidiomycota/genética , Basidiomycota/crescimento & desenvolvimento , Cryptococcus gattii/crescimento & desenvolvimento , Ecossistema , Evolução Molecular , Interação Gene-Ambiente , Genes Fúngicos Tipo Acasalamento/genética , Especiação Genética , Variação Genética/fisiologia , Genótipo , Filogenia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento
6.
Mycoses ; 61(12): 959-962, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30047168

RESUMO

Isolation of representatives of the Cryptococcus neoformans/Cryptococcus gattii species complex can be made using dopamine containing media, such as Niger seed agar and l-DOPA agar. Here, we describe an alternative medium that uses banana flowers. Banana is a dopamine containing fruit and is widely available in tropical and subtropical countries that have high numbers of cryptococcosis patients. This banana blossom-based agar is useful for the enrichment of isolates of the C. neoformans/C. gattii species complex from environmental and clinical materials. The banana blossom agar (BABA) with and without creatinine can differentiate between the melanin forming isolates of the C. neoformans/C. gattii species complex from other yeasts that do not form melanin.


Assuntos
Cryptococcus gattii/isolamento & purificação , Cryptococcus neoformans/isolamento & purificação , Meios de Cultura/química , Técnicas Microbiológicas/métodos , Criptococose/diagnóstico , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus neoformans/crescimento & desenvolvimento , Dopamina/metabolismo , Microbiologia Ambiental , Flores/metabolismo , Humanos , Melaninas/metabolismo , Musa/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-29891599

RESUMO

Cryptococcal meningitis (CM), caused primarily by Cryptococcus neoformans, is uniformly fatal if not treated. Treatment options are limited, especially in resource-poor geographical regions, and mortality rates remain high despite current therapies. Here we evaluated the in vitro and in vivo activity of several compounds, including APX001A and its prodrug, APX001, currently in clinical development for the treatment of invasive fungal infections. These compounds target the conserved Gwt1 enzyme that is required for the localization of glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins in fungi. The Gwt1 inhibitors had low MIC values, ranging from 0.004 µg/ml to 0.5 µg/ml, against both C. neoformans and C. gattii APX001A and APX2020 demonstrated in vitro synergy with fluconazole (fractional inhibitory concentration index, 0.37 for both). In a CM model, APX001 and fluconazole each alone reduced the fungal burden in brain tissue (0.78 and 1.04 log10 CFU/g, respectively), whereas the combination resulted in a reduction of 3.52 log10 CFU/g brain tissue. Efficacy, as measured by a reduction in the brain and lung tissue fungal burden, was also observed for another Gwt1 inhibitor prodrug, APX2096, where dose-dependent reductions in the fungal burden ranged from 5.91 to 1.79 log10 CFU/g lung tissue and from 7.00 and 0.92 log10 CFU/g brain tissue, representing the nearly complete or complete sterilization of lung and brain tissue at the higher doses. These data support the further clinical evaluation of this new class of antifungal agents for the treatment of CM.


Assuntos
Amidoidrolases/antagonistas & inibidores , Aminopiridinas/farmacologia , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , Isoxazóis/farmacologia , Meningite Criptocócica/tratamento farmacológico , Organofosfatos/farmacologia , Pró-Fármacos/farmacologia , Administração Oral , Amidoidrolases/genética , Amidoidrolases/metabolismo , Aminopiridinas/síntese química , Aminopiridinas/farmacocinética , Animais , Antifúngicos/síntese química , Antifúngicos/farmacocinética , Encéfalo/efeitos dos fármacos , Encéfalo/microbiologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/enzimologia , Cryptococcus gattii/genética , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Sinergismo Farmacológico , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Injeções Intraperitoneais , Isoxazóis/síntese química , Isoxazóis/farmacocinética , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Masculino , Meningite Criptocócica/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Organofosfatos/síntese química , Organofosfatos/farmacocinética , Pró-Fármacos/síntese química , Pró-Fármacos/farmacocinética
8.
Med Mycol J ; 59(2): E25-E30, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29848908

RESUMO

The use of embryonated egg as an alternative in the study of the pathogenesis of fungi is evolving. Although murine models are the "gold standard," embryonated egg models are also used to screen determinants of virulence among fungi species. This study was aimed at determining the virulence potential of Cryptococcus gattii strains R265, R272, and EJB18, and Malassezia sympodialis using chorioallantoic membrane (CAM) of embryonated egg. At a concentration of 107 cfu/ml, C. gattii R272 was more virulent than R265 in the egg model, while EJB18 had low virulence. The CAM model supported the growth of Malassezia sympodialis strain and induced the formation of hyphae. The formation of lesions by the organism and its re-isolation from CAM suggest that the model can be used for evaluating the virulence of C. gattii. Histopathology of CAM from both strains also revealed massive disruption of CAM. This study suggests that embryonated egg is a useful alternative tool to pre-screen Cryptococcus gattii strains to select strains for subsequent testing in murine models and could also be a potential medium for studying the hyphal growth in Malassezia species.


Assuntos
Membrana Corioalantoide/microbiologia , Cryptococcus gattii/patogenicidade , Animais , Embrião de Galinha , Galinhas , Cryptococcus gattii/crescimento & desenvolvimento , Malassezia/crescimento & desenvolvimento , Malassezia/patogenicidade , Camundongos , Virulência
9.
Artigo em Inglês | MEDLINE | ID: mdl-29158283

RESUMO

Resistance to antimicrobials is a growing problem in both developed and developing countries. In nations where AIDS is most prevalent, the human fungal pathogen Cryptococcus neoformans is a significant contributor to mortality, and its growing resistance to current antifungals is an ever-expanding threat. We investigated octapeptin C4, from the cationic cyclic lipopeptide class of antimicrobials, as a potential new antifungal. Octapeptin C4 was a potent, selective inhibitor of this fungal pathogen with an MIC of 1.56 µg/ml. Further testing of octapeptin C4 against 40 clinical isolates of C. neoformans var. grubii or neoformans showed an MIC of 1.56 to 3.13 µg/ml, while 20 clinical isolates of C. neoformans var. gattii had an MIC of 0.78 to 12.5 µg/ml. In each case, the MIC values for octapeptin C4 were equivalent to, or better than, current antifungal drugs fluconazole and amphotericin B. The negatively charged polysaccharide capsule of C. neoformans influences the pathogen's sensitivity to octapeptin C4, whereas the degree of melanization had little effect. Testing synthetic octapeptin C4 derivatives provided insight into the structure activity relationships, revealing that the lipophilic amino acid moieties are more important to the activity than the cationic diaminobutyric acid groups. Octapeptins have promising potential for development as anticryptococcal therapeutic agents.


Assuntos
Antifúngicos/farmacologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Anfotericina B/farmacologia , Antifúngicos/síntese química , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Candida glabrata/efeitos dos fármacos , Candida glabrata/crescimento & desenvolvimento , Candida parapsilosis/efeitos dos fármacos , Candida parapsilosis/crescimento & desenvolvimento , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus neoformans/crescimento & desenvolvimento , Fluconazol/farmacologia , Humanos , Lipopeptídeos/síntese química , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/síntese química , Relação Estrutura-Atividade
10.
Environ Microbiol ; 19(10): 4318-4325, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28892309

RESUMO

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe and Mediterranean area.


Assuntos
Microambiente Celular/fisiologia , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus neoformans/crescimento & desenvolvimento , Microbiologia Ambiental , Microbiologia do Solo , Criptococose/microbiologia , Cryptococcus gattii/metabolismo , Cryptococcus neoformans/metabolismo , Europa (Continente) , Região do Mediterrâneo , Estações do Ano , Solo/química , Tempo (Meteorologia)
11.
Mycoses ; 60(10): 697-702, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28699287

RESUMO

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/biossíntese
12.
Mycoses ; 60(7): 447-453, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28338245

RESUMO

Pathogenesis of cryptococcosis in the central nervous system (CNS) is a topic of ongoing research, including the mechanisms by which this fungus invades and infects the brain. Astrocytes, the most common CNS cells, play a fundamental role in the local immune response. Astrocytes might participate in cryptococcosis either as a host or by responding to fungal antigens. To determine the infectivity of Cryptococcus neoformans var. grubii and Cryptococcus gattii in a human astrocytoma cell line and the induction of major histocompatibility complex (MHC) molecules. A glioblastoma cell line was infected with C. neoformans var. grubii and C. gattii blastoconidia labelled with FUN-1 fluorescent stain. The percentage of infection and expression of HLA class I and II molecules were determined by flow cytometry. The interactions between the fungi and cells were observed by fluorescence microscopy. There was no difference between C. neoformans var. grubii and C. gattii in the percentage infection, but C. neoformans var. grubii induced higher expression of HLA class II than C. gattii. More blastoconidia were recovered from C. neoformans-infected cells than from C. gattii infected cells. Cryptococcus neoformans var. grubii may have different virulence mechanisms that allow its survival in human glia-derived cells.


Assuntos
Astrócitos/microbiologia , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus neoformans/crescimento & desenvolvimento , Linhagem Celular , Citometria de Fluxo , Antígenos HLA/análise , Humanos , Viabilidade Microbiana , Microscopia de Fluorescência
13.
Eur J Pharm Sci ; 92: 235-43, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27235581

RESUMO

The emergence of fluconazole-resistant Cryptococcus gattii is a global concern, since this azole is the main antifungal used worldwide to treat patients with cryptococcosis. Although pharmacokinetic (PK) and pharmacodynamic (PD) indices are useful predictive factors for therapeutic outcomes, there is a scarcity of data regarding PK/PD analysis of antifungals in cryptococcosis caused by resistant strains. In this study, PK/PD parameters were determined in a murine model of cryptococcosis caused by resistant C. gattii. We developed and validated a suitable liquid chromatography-electrospray ionization tandem mass spectrometry method for PK studies of fluconazole in the serum, lungs, and brain of uninfected mice. Mice were infected with susceptible or resistant C. gattii, and the effects of different doses of fluconazole on the pulmonary and central nervous system fungal burden were determined. The peak levels in the serum, lungs, and brain were achieved within 0.5h. The AUC/MIC index (area under the curve/minimum inhibitory concentration) was associated with the outcome of anti-cryptococcal therapy. Interestingly, the maximum concentration of fluconazole in the brain was lower than the MIC for both strains. In addition, the treatment of mice infected with the resistant strain was ineffective even when high doses of fluconazole were used or when amphotericin B was tested, confirming the cross-resistance between these drugs. Altogether, our novel data provide the correlation of PK/PD parameters with antifungal therapy during cryptococcosis caused by resistant C. gattii.


Assuntos
Antifúngicos , Criptococose , Cryptococcus gattii , Farmacorresistência Fúngica , Fluconazol , Anfotericina B/uso terapêutico , Animais , Antifúngicos/sangue , Antifúngicos/farmacocinética , Antifúngicos/uso terapêutico , Encéfalo/metabolismo , Encéfalo/microbiologia , Cromatografia Líquida , Contagem de Colônia Microbiana , Criptococose/tratamento farmacológico , Criptococose/metabolismo , Criptococose/microbiologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/isolamento & purificação , Modelos Animais de Doenças , Fluconazol/sangue , Fluconazol/farmacocinética , Fluconazol/uso terapêutico , Pulmão/metabolismo , Pulmão/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Modelos Biológicos , Espectrometria de Massas por Ionização por Electrospray
14.
J Appl Microbiol ; 121(2): 373-9, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27107205

RESUMO

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 Microbiana
15.
PLoS One ; 11(4): e0153219, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27049762

RESUMO

Cryptococcus gattii is a resurgent fungal pathogen that primarily infects immunocompetent hosts. Thus, it poses an increasingly significant impact on global public health; however, the mechanisms underlying its pathogenesis remain largely unknown. We conducted a detailed characterization of the deubiquitinase Ubp5 in the biology and virulence of C. gattii using the hypervirulent strain R265, and defined its properties as either distinctive or shared with C. neoformans. Deletion of the C. gattii Ubp5 protein by site-directed disruption resulted in a severe growth defect under both normal and stressful conditions (such as high temperature, high salt, cell wall damaging agents, and antifungal agents), similar to the effects observed in C. neoformans. However, unlike C. neoformans, the C. gattii ubp5Δ mutant displayed a slight enhancement of capsule and melanin production, indicating the evolutionary convergence and divergence of Ubp5 between these two sibling species. Attenuated virulence of the Cg-ubp5Δ mutant was not solely due to its reduced thermotolerance at 37°C, as shown in both worm and mouse survival assays. In addition, the assessment of fungal burden in mammalian organs further indicated that Ubp5 was required for C. gattii pulmonary survival and, consequently, extrapulmonary dissemination. Taken together, our work highlights the importance of deubiquitinase Ubp5 in the virulence composite of both pathogenic cryptococcal species, and it facilitates a better understanding of C. gattii virulence mechanisms.


Assuntos
Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/patogenicidade , Proteases Específicas de Ubiquitina/metabolismo , Animais , Antifúngicos/farmacologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/genética , Genes Fúngicos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Proteases Específicas de Ubiquitina/genética , Virulência
16.
J Appl Microbiol ; 120(1): 41-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26442997

RESUMO

AIM: The aim of this study was to investigate the in vitro and in vivo activities of pure curcumin, as well as its combination with fluconazole, against Cryptococcus gattii. METHODS AND RESULTS: The minimal inhibitory concentrations (MIC) of curcumin and its interactions with fluconazole against C. gattii were assessed in vitro using standard methods. This same combination was used to treat C. gattii-induced cryptococcosis in mice. The behavioural and functional assessment of the mice during treatment was also performed. The average MIC for curcumin was 19·8 µg ml(-1) . Its combination with fluconazole resulted in FICΣ (fractional inhibitory concentration index) values between 0·79 and 2·29. Curcumin (alone or combined with fluconazole) significantly reduced pulmonary damage and fungal burden in the brain. No colonies were found in the brain following combination treatment, which was also confirmed by the improved behaviour of mice. CONCLUSIONS: The combination therapy with curcumin and fluconazole was the most effective among the treatments tested, as in addition to reducing the fungal burden and damage on lung tissues, it was able to eliminate the fungal burden in the brain, enhancing the survival of mice. SIGNIFICANCE AND IMPACT OF THE STUDY: This study points to the possibility of using curcumin in combination with fluconazole as a clinical treatment of cryptococcosis.


Assuntos
Antifúngicos/administração & dosagem , Criptococose/tratamento farmacológico , Cryptococcus gattii/efeitos dos fármacos , Curcumina/administração & dosagem , Fluconazol/administração & dosagem , Animais , Criptococose/microbiologia , Cryptococcus gattii/crescimento & desenvolvimento , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL
17.
Microbiology (Reading) ; 162(2): 309-317, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26645478

RESUMO

Heat-shock proteins (Hsps) are chaperones required for the maintenance of cellular homeostasis in different fungal pathogens, playing an important role in the infectious process. This study investigated the effect of pharmacological inhibition of Hsp90 by radicicol on the Cryptococcus neoformans/Cryptococcus gattii species complex--agents of the most common life-threatening fungal infection amongst immunocompromised patients. The influence of Hsp90 inhibition was investigated regarding in vitro susceptibility to antifungal agents of planktonic and sessile cells, ergosterol concentration, cell membrane integrity, growth at 37 °C, production of virulence factors in vitro, and experimental infection in Caenorhabditis elegans. Hsp90 inhibition inhibited the in vitro growth of planktonic cells of Cryptococcus spp. at concentrations ranging from 0.5 to 2 µg ml(-1) and increased the in vitro inhibitory effect of azoles, especially fluconazole (FLC) (P < 0.05). Inhibition of Hsp90 also increased the antifungal activity of azoles against biofilm formation and mature biofilms of Cryptococcus spp., notably for Cryptococcus gattii. Furthermore, Hsp90 inhibition compromised the permeability of the cell membrane, and reduced planktonic growth at 37 °C and the capsular size of Cryptococcus spp. In addition, Hsp90 inhibition enhanced the antifungal activity of FLC during experimental infection using Caenorhabditis elegans. Therefore, our results indicate that Hsp90 inhibition can be an important strategy in the development of new antifungal drugs.


Assuntos
Antifúngicos/farmacologia , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans/microbiologia , Cryptococcus gattii/patogenicidade , Cryptococcus neoformans/patogenicidade , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Plâncton/efeitos dos fármacos , Anfotericina B/farmacologia , Animais , Biofilmes/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Criptococose/patologia , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus neoformans/crescimento & desenvolvimento , Ergosterol/metabolismo , Fluconazol/farmacologia , Humanos , Itraconazol/farmacologia , Melaninas/biossíntese , Testes de Sensibilidade Microbiana , Voriconazol/farmacologia
18.
Emerg Infect Dis ; 21(11): 1989-96, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26484590

RESUMO

Vancouver Island, Canada, reports the world's highest incidence of Cryptococcus gattii infection among humans and animals. To identify key biophysical factors modulating environmental concentrations, we evaluated monthly concentrations of C. gatti in air, soil, and trees over a 3-year period. The 2 study datasets were repeatedly measured plots and newly sampled plots. We used hierarchical generalized linear and mixed effect models to determine associations. Climate systematically influenced C. gattii concentrations in all environmental media tested; in soil and on trees, concentrations decreased when temperatures were warmer. Wind may be a key process that transferred C. gattii from soil into air and onto trees. C. gattii results for tree and air samples were more likely to be positive during periods of higher solar radiation. These results improve the understanding of the places and periods with the greatest C. gattii colonization. Refined risk projections may help susceptible persons avoid activities that disturb the topsoil during relatively cool summer days.


Assuntos
Cryptococcus gattii/classificação , Microbiologia do Ar , Colúmbia Britânica , Criptococose , Cryptococcus gattii/genética , Cryptococcus gattii/crescimento & desenvolvimento , Humanos , Ilhas , Microbiologia do Solo , Árvores/microbiologia
19.
Rev. iberoam. micol ; 32(3): 197-199, jul.-sept. 2015. tab
Artigo em Inglês | IBECS | ID: ibc-142081

RESUMO

Background. Both Cryptococcus neoformans and Cryptococcus gattii have been isolated from a variety of environmental sources in Colombia. Aim. To determine the viability of C. neoformans/C. gattii isolates in stored soil samples, filtrates and bird droppings from which these yeasts were previously recovered. Methods. A total of 964 samples collected between 2003 and 2009, and kept at room temperature were processed. From them, 653 samples were from trees decaying wood, 274 from soil filtrates and 37 from bird droppings. When C. neoformans or C. gattii were recovered, the molecular type of each isolate was established by PCR fingerprinting using the single primer (GTG)5. Results. Among the processed samples, 161 isolates were recovered. From those, 81 (50.3%) corresponded to C. gattii recovered from decaying wood of Eucalyptus spp., Corymbia ficifolia, Terminalia catappa and Ficus spp. trees, and 80 (49.7%) corresponded to C. neoformans recovered from Ficus spp. and eucalyptus trees, as well as from bird droppings. The most prevalent molecular type among the C. gattii and C. neoformans isolates was VGII and VNI, respectively. Conclusions . The re-isolation of C. neoformans/C. gattii from 10-year stored samples suggests that these yeasts are able to keep viable in naturally colonized samples (AU)


Antecedentes. Cryptococcus neoformans y Cryptococcus gattii han sido aislados de diversas fuentes ambientales en Colombia. Objetivos. Determinar la viabilidad de C. neoformans/C. gattii en muestras de suelo, filtrados y excrementos de aves en las que se habían aislado las levaduras previamente. Métodos. Se procesaron 964 muestras recogidas entre los años 2003 y 2009, y almacenadas a temperatura ambiente. De estas, 653 muestras provenían de detritos, 274 de filtrados de suelo y 37 de excrementos de aves. Una vez recuperados C. neoformans y C. gattii, se determinó el patrón molecular mediante la técnica de PCR huella digital con el iniciador (GTG)5. Resultados. Entre las muestras procesadas, se recuperaron 161 aislamientos. De estos, 81 (50,3%) fueron C. gattii recuperados de detritos de Eucalyptus spp., Corymbia ficifolia, Terminalia catappa y Ficus spp., y 80 (49,7%) eran C. neoformans recuperados de Ficus spp. y eucaliptos, así como de excrementos de aves. El patrón molecular más prevalente entre C. gattii y C. neoformans fue VGII y VNI, respectivamente. Conclusiones. El reaislamiento de C. neoformans/C. gattii de muestras almacenadas durante 10 años evidencia que estas levaduras son capaces de mantenerse viables en muestras colonizadas naturalmente (AU)


Assuntos
Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus gattii/crescimento & desenvolvimento , Criptococose/microbiologia , Sobrevivência Celular , Viabilidade Microbiana , Leveduras/crescimento & desenvolvimento
20.
PLoS One ; 9(12): e113147, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25478697

RESUMO

Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO2, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of ß-carbonic anhydrase (ß-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. High CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by ß-CA independent mechanism(s).


Assuntos
Anidrases Carbônicas/biossíntese , Cryptococcus gattii/crescimento & desenvolvimento , Hifas/crescimento & desenvolvimento , Esporos/crescimento & desenvolvimento , Dióxido de Carbono/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Hifas/efeitos dos fármacos , Hifas/genética , Mutação , Reprodução/efeitos dos fármacos
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