RESUMO
Cryptococcus gattii, an environmental fungus, is one of the agents of cryptococcosis. The influence of agrochemicals on fungal resistance to antifungals is widely discussed. However, the effects of benomyl (BEN) on fungal interaction with different hosts is still to be understood. Here we studied the influence of adaptation to BEN in the interaction with a plant model, phagocytes and with Tenebrio molitor. First, the strain C. gattii L24/01 non-adapted (NA), adapted (A) to BEN, and adapted with further culture on drug-free media (10p) interact with Nicotiana benthamiana, with a peak in the yeast burden on the 7th day post-inoculation. C. gattii L24/01 A and 10p provided lower fungal burden, but these strains increased cell diameter and capsular thickness after the interaction, together with decreased fungal growth. The strains NA and A showed reduced ergosterol levels, while 10p exhibited increased activity of laccase and urease. L24/01 A recovered from N. benthamiana was less engulfed by murine macrophages, with lower production of reactive oxygen species. This phenotype was accompanied by increased ability of this strain to grow inside macrophages. Otherwise, L24/01 A showed reduced virulence in the T. molitor larvae model. Here, we demonstrate that the exposure to BEN, and interaction with plants interfere in the morphophysiology and virulence of the C. gattii.
Assuntos
Cryptococcus gattii , Nicotiana , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/metabolismo , Cryptococcus gattii/fisiologia , Animais , Camundongos , Nicotiana/microbiologia , Macrófagos/microbiologia , Criptococose/microbiologia , Tenebrio/microbiologia , Agroquímicos/farmacologia , Antifúngicos/farmacologia , Doenças das Plantas/microbiologiaRESUMO
This is the first study that describes the antifungal and anti-biofilm potential of O-alkylamidoximes against strains of Cryptococcus neoformans and Cryptococcus gattii. In vitro tests have shown that O-alkylamidoximes are capable of inhibiting fungal growth and biofilm formation of the C. neoformans and C. gattii strains, suggesting, from molecular docking, the potential for interaction with the Hsp90. The associations between O-alkylamidoximes and amphotericin B were beneficial. Therefore, O-alkylamidoximes can be a useful alternative to contribute to the limited arsenal of drugs, since they showed a powerful action against the primary agents of Cryptococcosis.
Assuntos
Antifúngicos , Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Oximas , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Oximas/química , Oximas/farmacologiaRESUMO
Paraquat (1,10-dimethyl-4,4-bipyridinium dichloride; PQ) is a free-radical producing herbicide that affects cell membranes and can upset the environmental balance of microorganisms present in soil, such as Cryptococcus spp. This study aimed to evaluate the in vitro activity of PQ against Cryptococcus spp. in planktonic and biofilm forms, as well as the protective effect of antioxidant agents against the antifungal effect of PQ and the kinetics of melanin production in response to PQ. Susceptibility to PQ was evaluated by microdilution. Cryptococcus sp. strains exposed to PQ were grown in media with ascorbic acid (AA) and glutathione (GSH). Melanin production was assessed in the presence of l-3,4-dihydroxyphenylalanine (l-DOPA) + PQ. The minimum inhibitory concentration of PQ against Cryptococcus spp. ranged from 8 to 256 µg/mL. Furthermore, PQ reduced biofilm formation. AA and GSH restored the fungal growth of Cryptococcus spp. exposed to PQ. In addition, l-DOPA + PQ delayed melanin production by 24 and 48 h for C. deuterogattii and C. neoformans sensu lato, respectively, suggesting that PQ induces a fitness trade-off in melanin production. Taken together, our data suggest that the antifungal effect of PQ against Cryptococcus spp. possibly exerts selective pressures interfering with biofilm formation and melanin production by these yeasts.
Assuntos
Cryptococcus gattii , Cryptococcus neoformans , Herbicidas , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Cryptococcus gattii/metabolismo , Cryptococcus neoformans/metabolismo , Herbicidas/metabolismo , Herbicidas/farmacologia , Levodopa/metabolismo , Levodopa/farmacologia , Melaninas/metabolismo , Melaninas/farmacologia , Testes de Sensibilidade Microbiana , Paraquat/metabolismo , Paraquat/farmacologiaRESUMO
Cryptococcus gattii is one of the causes of cryptococcosis, a life-threatening disease generally characterized by pneumonia and/or meningitis. Zinc is an essential element for life, being required for the activity of many proteins with catalytic and structural roles. Here, we characterize ZRG1 (zinc-related gene 1), which codes a product involved in zinc metabolism. Transcriptional profiling revealed that zinc availability regulated the expression of ZRG1, and its null mutants demonstrated impaired growth in zinc- and nitrogen-limiting conditions. Moreover, zrg1 strains displayed alterations in the expression of the zinc homeostasis-related genes ZAP1 and ZIP1. Notably, cryptococcal cells lacking Zrg1 displayed upregulation of autophagy-like phenotypes. Despite no differences were detected in the classical virulence-associated traits; cryptococcal cells lacking ZRG1 displayed decreased capacity for survival inside macrophages and attenuated virulence in an invertebrate model. Together, these results indicate that ZRG1 plays an important role in proper zinc metabolism, and is necessary for cryptococcal fitness and virulence.
Assuntos
Proteínas de Transporte de Cátions/genética , Cryptococcus gattii/genética , Proteínas Fúngicas/genética , Animais , Autofagia , Proteínas de Transporte de Cátions/metabolismo , Cryptococcus gattii/metabolismo , Cryptococcus gattii/patogenicidade , Proteínas Fúngicas/metabolismo , Camundongos , Mutação , Células RAW 264.7 , Zinco/metabolismoRESUMO
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 TransmissãoRESUMO
Cryptococcus gattii is an etiologic agent of cryptococcosis, a potentially fatal disease that affects humans and animals. The successful infection of mammalian hosts by cryptococcal cells relies on their ability to infect and survive in macrophages. Such phagocytic cells present a hostile environment to intracellular pathogens via the production of reactive nitrogen and oxygen species, as well as low pH and reduced nutrient bioavailability. To overcome the low-metal environment found during infection, fungal pathogens express high-affinity transporters, including members of the ZIP family. Previously, we determined that functional zinc uptake driven by Zip1 and Zip2 is necessary for full C.gattiivirulence. Here, we characterized the ZIP3 gene of C. gattii, an ortholog of the Saccharomyces cerevisiae ATX2, which codes a manganese transporter localized to the membrane of the Golgi apparatus. Cryptococcal cells lacking Zip3 were tolerant to toxic concentrations of manganese and had imbalanced expression of intracellular metal transporters, such as the vacuolar Pmc1 and Vcx1, as well as the Golgi Pmr1. Moreover, null mutants of the ZIP3 gene displayed higher sensitivity to reactive oxygen species (ROS) and substantial alteration in the expression of ROS-detoxifying enzyme-coding genes. In line with these phenotypes, cryptococcal cells displayed decreased virulence in a non-vertebrate model of cryptococcosis. Furthermore, we found that the ZIP3 null mutant strain displayed decreased melanization and secretion of the major capsular component glucuronoxylomannan, as well as an altered extracellular vesicle dimensions profile. Collectively, our data suggest that Zip3 activity impacts the physiology, and consequently, several virulence traits of C. gattii.
Assuntos
Proteínas de Transporte de Cátions/genética , Cryptococcus gattii/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Animais , Criptococose/genética , Criptococose/microbiologia , Criptococose/patologia , Cryptococcus gattii/metabolismo , Cryptococcus gattii/patogenicidade , Humanos , Macrófagos/metabolismo , Manganês/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Virulência/genéticaRESUMO
Cryptococcus neoformans/Cryptococcus gattii complex species are etiological agents of cryptococcosis, a systemic mycosis that cause respiratory infection and meningoencephalitis. To establish the infection, these yeasts produce virulence factors, such as melanin, which contribute to pathogenicity and antifungal tolerance. The aim of this study was to investigate melanin production by the C. neoformans/C. gattii complex in the presence of different precursors of melanogenesis and evaluate the effect of melanization on the antifungal susceptibility of these species to fluconazole, flucytosine and amphotericin B. Epinephrine, norepinephrine, dopamine and caffeic acid were used as substrates for melanin production, and l-dopa was used as positive control. The susceptibility of melanized strains (n = 6), after exposure to epinephrine or l-dopa, was evaluated by broth microdilution assay, and non-melanized strains were used as control. The antifungal activity of amphotericin B against melanized strains was also investigated by time kill assay. All Cryptococcus spp. strains produced melanin after exposure to the tested substrates. After exposure to epinephrine, minimum inhibitory concentration (MIC) ranges were 1-8 µg/mL for fluconazole, 2-8 µg/mL for flucytosine and 0.125-1 µg/mL for amphotericin B, while, after exposure to l-dopa, MIC ranges were 2-8 µg/mL for fluconazole, 4-8 µg/mL for flucytosine, and 0.125-0.5 µg/mL for amphotericin B. Similar results were observed for non-melanized strains. The production of melanin after exposure to epinephrine was higher than that induced by l-dopa. Melanized cells of both species were more tolerant to amphotericin B than the non-melanized control, emphasizing the importance of melanin production for fungal virulence.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Cryptococcus gattii/metabolismo , Cryptococcus neoformans/metabolismo , Epinefrina/farmacologia , Melaninas/metabolismo , Animais , Antibacterianos , Ácidos Cafeicos/metabolismo , Ácidos Cafeicos/farmacologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Dopamina/metabolismo , Dopamina/farmacologia , Epinefrina/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Norepinefrina/metabolismo , Norepinefrina/farmacologiaRESUMO
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
OBJECTIVES: Cryptococcus gattii from the North American Northwest (NW) have higher azole MICs than do non-NW C. gattii or Cryptococcus neoformans. Since mechanisms of azole resistance in C. gattii are not known, we identified C. gattii and C. neoformans plasma membrane azole efflux pumps and characterized their properties. METHODS: The C. gattii R265 genome was searched for orthologues of known fungal azole efflux genes, expression of candidate genes was assessed by RT-PCR and the expressed genes' cDNAs were cloned and expressed in Saccharomyces cerevisiae. Azole MICs and intracellular [(3)H]fluconazole were measured in C. gattii and C. neoformans and in S. cerevisiae expressing each cDNA of interest, as was [(3)H]fluconazole uptake by post-Golgi vesicles (PGVs) isolated from S. cerevisiae sec6-4 mutants expressing each cDNA of interest. RESULTS: Intracellular [(3)H]fluconazole concentrations were inversely correlated with fluconazole MICs only in 25 NW C. gattii strains. S. cerevisiae expressing three C. gattii cDNAs (encoded by orthologues of C. neoformans AFR1 and MDR1 and the previously unstudied gene AFR2) and their C. neoformans counterparts had higher azole MICs and lower intracellular [(3)H]fluconazole concentrations than did empty-vector controls. PGVs from S. cerevisiae expressing all six Cryptococcus cDNAs also accumulated more [(3)H]fluconazole than did controls, and [(3)H]fluconazole transport by all six transporters of interest was ATP dependent and was inhibited by excess unlabelled fluconazole, voriconazole, itraconazole and posaconazole. CONCLUSIONS: We conclude that C. gattii and C. neoformans AFR1, MDR1 and AFR2 encode ABC transporters that pump multiple azoles out of S. cerevisiae cells, thereby causing azole resistance.
Assuntos
Antifúngicos/metabolismo , Azóis/metabolismo , Cryptococcus gattii/enzimologia , Cryptococcus neoformans/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico Ativo , Clonagem Molecular , Criptococose/microbiologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/isolamento & purificação , Cryptococcus gattii/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Marcação por Isótopo , Testes de Sensibilidade Microbiana , Noroeste dos Estados Unidos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Análise de Sequência de DNARESUMO
Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In the literature, the competition for iron between microbes and mammalian hosts during infection is well documented. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation. A total of 15 and 29 genes were identified as having altered expression levels due to iron depletion after 3 h and 12 h, respectively. Of these, eight genes were identified in both libraries. The transcripts were related to many biological processes, such as cell cycle, ergosterol metabolism, cell wall organization, transportation, translation, cell respiration and the stress response. These data suggest a remodeling of C. gattii metabolism during conditions of iron deprivation.
Assuntos
Cryptococcus gattii/genética , Cryptococcus gattii/metabolismo , Perfilação da Expressão Gênica , Ferro/metabolismo , Estresse Fisiológico , Cryptococcus gattii/fisiologia , Genes FúngicosRESUMO
Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In the literature, the competition for iron between microbes and mammalian hosts during infection is well documented. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation. A total of 15 and 29 genes were identified as having altered expression levels due to iron depletion after 3 h and 12 h, respectively. Of these, eight genes were identified in both libraries. The transcripts were related to many biological processes, such as cell cycle, ergosterol metabolism, cell wall organization, transportation, translation, cell respiration and the stress response. These data suggest a remodeling of C. gattii metabolism during conditions of iron deprivation.
Assuntos
Cryptococcus gattii/genética , Cryptococcus gattii/metabolismo , Perfilação da Expressão Gênica , Ferro/metabolismo , Estresse Fisiológico , Cryptococcus gattii/fisiologia , Genes FúngicosRESUMO
Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In the literature, the competition for iron between microbes and mammalian hosts during infection is well documented. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation. A total of 15 and 29 genes were identified as having altered expression levels due to iron depletion after 3 h and 12 h, respectively. Of these, eight genes were identified in both libraries. The transcripts were related to many biological processes, such as cell cycle, ergosterol metabolism, cell wall organization, transportation, translation, cell respiration and the stress response. These data suggest a remodeling of C. gattii metabolism during conditions of iron deprivation.
Assuntos
Cryptococcus gattii/genética , Cryptococcus gattii/metabolismo , Perfilação da Expressão Gênica , Ferro/metabolismo , Estresse Fisiológico , Cryptococcus gattii/fisiologia , Genes FúngicosRESUMO
Mass-spectrometry-based shotgun proteomics has become a widespread technology for analyzing complex protein mixtures. Here we describe a new module integrated into PatternLab for Proteomics that allows the pinpointing of differentially expressed domains. This is accomplished by inferring functional domains through our cloud service, using HMMER3 and Pfam remotely, and then mapping the quantitation values into domains for downstream analysis. In all, spotting which functional domains are changing when comparing biological states serves as a complementary approach to facilitate the understanding of a system's biology. We exemplify the new module's use by reanalyzing a previously published MudPIT dataset of Cryptococcus gattii cultivated under iron-depleted and replete conditions. We show how the differential analysis of functional domains can facilitate the interpretation of proteomic data by providing further valuable insight.
Assuntos
Cryptococcus gattii/química , Cryptococcus gattii/metabolismo , Bases de Dados de Proteínas , Proteínas Fúngicas , Proteoma , Proteômica/métodos , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Estrutura Terciária de Proteína , Proteoma/biossíntese , Proteoma/químicaRESUMO
OBJECTIVES: Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. In the present study, the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus Cryptococcus gattii were examined. METHODS: We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by these drugs in C. gattii cells: lipid peroxidation, ergosterol content, ROS and peroxynitrite production, enzymatic activity of the antioxidant system and the in vitro interaction of antifungal drugs with a peroxidase inhibitor, a superoxide dismutase inhibitor and a peroxynitrite scavenger. RESULTS: The data demonstrated that itraconazole led to ROS formation and lipid peroxidation in C. gattii cells in the early stages of the treatment; this did not occur with fluconazole. This phenomenon strongly increased the activities of enzymes of the antioxidant system. These results were confirmed by synergism observed between the catalase inhibitor and itraconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased peroxidase activity. These data were confirmed by the synergism between the catalase/superoxide dismutase inhibitors and amphotericin B. In addition, the effect of this antifungal was antagonized by the peroxynitrite scavenger. CONCLUSIONS: Oxidative and nitrosative bursts play an important role in the antifungal activity of itraconazole and amphotericin B against C. gattii.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Cryptococcus gattii/efeitos dos fármacos , Itraconazol/farmacologia , Nitrosação , Explosão Respiratória , Cryptococcus gattii/metabolismo , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidadeRESUMO
Zinc homeostasis is essential for fungal growth, as this metal is a critical structural component of several proteins, including transcription factors. The fungal pathogen Cryptococcus gattii obtains zinc from the stringent zinc-limiting milieu of the host during the infection process. To characterize the zinc metabolism in C. gattii and its relationship to fungal virulence, the zinc finger protein Zap1 was functionally characterized. The C. gattii ZAP1 gene is an ortholog of the master regulatory genes zafA and ZAP1 that are found in Aspergillus fumigatus and Saccharomyces cerevisiae, respectively. There is some evidence to support an association between Zap1 and zinc metabolism in C. gattii: (i) ZAP1 expression is highly induced during zinc deprivation, (ii) ZAP1 knockouts demonstrate impaired growth in zinc-limiting conditions, (iii) Zap1 regulates the expression of ZIP zinc transporters and distinct zinc-binding proteins and (iv) Zap1 regulates the labile pool of intracellular zinc. In addition, the deletion of ZAP1 reduces C. gattii virulence in a murine model of cryptococcosis infection. Based on these observations, we postulate that proper zinc metabolism plays a crucial role in cryptococcal virulence.
Assuntos
Cryptococcus gattii/metabolismo , Cryptococcus gattii/patogenicidade , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/metabolismo , Virulência/fisiologia , Zinco/metabolismo , Animais , Criptococose/microbiologia , Feminino , Proteínas Fúngicas/genética , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genética , Virulência/genéticaRESUMO
Iron is essential and ubiquitous in living organisms. The competition for this micronutrient between the host and its pathogens has been related to disease establishment. Cryptococcus gattii is an encapsulated yeast that causes cryptococcosis mainly in immunocompetent individuals. In this study, we analyzed the proteomic profile of the C. gattii R265 Vancouver Island isolate under iron-depleted and -repleted conditions by multidimensional protein identification technology (MudPIT) and by 2D-GE. Proteins and key mechanisms affected by alteration of iron levels such as capsule production, cAMP-signaling pathway, response to stress, and metabolic pathways related to mitochondrial function were identified. Our results also show both proteomic methodologies employed to be complementary.
Assuntos
Cryptococcus gattii/metabolismo , Proteínas Fúngicas/metabolismo , Ferro/fisiologia , Proteoma/metabolismo , Vias Biossintéticas , Cryptococcus gattii/genética , Cryptococcus gattii/crescimento & desenvolvimento , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , Anotação de Sequência Molecular , Proteoma/classificação , Proteoma/genética , ProteômicaRESUMO
INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.
Assuntos
Cryptococcus/metabolismo , Meios de Cultura/farmacologia , Melaninas/biossíntese , Metildopa/farmacologia , Ágar , Cryptococcus/classificação , Cryptococcus/crescimento & desenvolvimento , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/metabolismo , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/metabolismo , Meios de Cultura/química , Especificidade da EspécieRESUMO
INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.
INTRODUÇÃO: A produção de melanina por espécies de Cryptococcus é uma característica amplamente utilizada em laboratórios de micologia para caracterização do complexoC. neoformans. O objetivo deste estudo foi verificar a eficácia da metildopa na forma farmacêutica de comprimido, como substrato para a produção de melanina por Cryptococcus, comparar diferentes bases de meios de cultura acrescidas de metildopa para produção de melanina e comparar o pigmento produzido nestes meios com o produzido em ágar Níger e ágar girassol por C. neoformans, C. laurentii e C. albidus. MÉTODOS: Foram testados dois isolados de cada uma das espécies, C. neoformans, C.laurentii e C.albidus, e um de C. albicans para avaliar a produção de melanina nos meios de cultura ágar Müeller-Hinton (MH), ágar brain heart infusion (BHI), ágar base sangue (BS), meio mínimo (MM), todos acrescidos de metildopa, e ainda ágar girassol e ágar Níger. RESULTADOS: Todos os isolados cresceram na maioria dos meios após 24h. O crescimento nos meios BS e BHI somente ocorreu após 48h. Todos os isolados produziram melanina nos meios MM, MH, girassol e Niger. CONCLUSÕES: A metildopa de origem farmacêutica pode ser utilizada como substrato para a produção de melanina por espécies de Cryptococcus; o MM acrescido de metildopa mostrou-se mais eficiente na produção de melanina do que os meios BS, MH e BHI; ágar girassol e ágar Níger seguidos de MM acrescido de metildopa foram os mais eficientes na produção de melanina pelos isolados estudados.