RESUMO
Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.
Assuntos
Cryptococcus neoformans , Lacase , Melaninas , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/enzimologia , Lacase/metabolismo , Melaninas/biossíntese , Melaninas/metabolismo , Ensaios Enzimáticos/métodosRESUMO
Cryptococcus is a globally distributed fungal pathogen that primarily afflicts immunocompromised individuals. The therapeutic options are limited and include mostly amphotericin B or fluconazole, alone or in combination. The extensive usage of antifungals allowed the selection of resistant pathogens posing threats to global public health. Histone deacetylase genes are involved in Cryptococcus virulence, and in pathogenicity and resistance to azoles in Candida albicans. Aiming to assess whether histone deacetylase genes are involved in antifungal response and in synergistic drug interactions, we evaluated the activity of amphotericin B, fluconazole, sulfamethoxazole, sodium butyrate or trichostatin A (histone deacetylase inhibitors), and hydralazine or 5- aza-2'-deoxycytidine (DNA methyl-transferase inhibitors) against different Cryptococcus neoformans strains, C. neoformans histone deacetylase null mutants and Cryptococcus gattii NIH198. The drugs were employed alone or in different combinations. Fungal growth after photodynamic therapy mediated by an aluminium phthalocyanine chloride nanoemulsion, alone or in combination with the aforementioned drugs, was assessed for the C. neoformans HDAC null mutant strains. Our results showed that fluconazole was synergistic with sodium butyrate or with trichostatin A for the hda1Δ/hos2Δ double mutant strain. Sulfamethoxazole was synergistic with sodium butyrate or with hydralazine also for hda1Δ/hos2Δ. These results clearly indicate a link between HDAC impairment and drug sensitivity. Photodynamic therapy efficacy on controlling the growth of the HDAC mutant strains was increased by amphotericin B, fluconazole, sodium butyrate or hydralazine. This is the first study in Cryptococcus highlighting the combined effects of antifungal drugs, histone deacetylase or DNA methyltransferase inhibitors and photodynamic therapy in vitro.
Assuntos
Antifúngicos/metabolismo , Proteínas de Bactérias/genética , Criptococose/tratamento farmacológico , Cryptococcus neoformans/enzimologia , Epigênese Genética/efeitos dos fármacos , Histona Desacetilases/genética , Indóis/metabolismo , Compostos Organometálicos/metabolismo , Fotoquimioterapia/métodos , Anfotericina B/química , Ácido Butírico/química , Sinergismo Farmacológico , Emulsões/química , Fluconazol/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/química , Indóis/farmacologia , Nanopartículas/química , Compostos Organometálicos/farmacologia , Sulfametoxazol/químicaRESUMO
Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans, we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis.IMPORTANCECryptococcus neoformans is a yeast that causes severe disease, primarily in immunosuppressed people. It has many attributes that allow it to survive and cause disease, such as a polysaccharide capsule and the dark pigment melanin produced by the laccase enzyme. Upon infection, the yeast is ingested by cells called macrophages, whose function is to kill them. Instead, these fungal cells can exit from macrophages in a process called nonlytic exocytosis. We know that this process is controlled by both host and fungal factors, only some of which are known. As part of an ongoing study, we observed that C. neoformans isolates that produce melanin faster are more-frequent targets of nonlytic exocytosis. Further experiments showed that this is probably due to higher production of laccase, because fungi lacking this enzyme are nonlytically exocytosed less often. This shows that laccase is an important signal/regulator of nonlytic exocytosis of C. neoformans from macrophages.
Assuntos
Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Exocitose , Lacase/metabolismo , Macrófagos/microbiologia , Animais , Brasil , Células Cultivadas , Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus neoformans/genética , Humanos , Hospedeiro Imunocomprometido , Lacase/análise , Lacase/biossíntese , Lacase/genética , Melaninas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , VirulênciaRESUMO
Gti1/Pac2 transcription factors occur exclusively in fungi and their roles vary according to species, including regulating morphological transition and virulence, mating and secondary metabolism. Many of these functions are important for fungal pathogenesis. We therefore hypothesized that one of the two proteins of this family in Cryptococcus neoformans, a major pathogen of humans, would also control virulence-associated cellular processes. Elimination of this protein in C. neoformans results in reduced polysaccharide capsule expression and defective cytokinesis and growth at 37°C. The mutant loses virulence in a mouse model of cryptococcal infection and retains only partial virulence in the Galleria mellonella alternative model at 30°C. We performed RNA-Seq experiments on the mutant and found abolished transcription of genes that, in combination, are known to account for all the observed phenotypes. The protein has been named Required for cytokinesis and virulence 1 (Rcv1).
Assuntos
Criptococose/patologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Fatores de Transcrição/metabolismo , Animais , Criptococose/microbiologia , Cryptococcus neoformans/crescimento & desenvolvimento , Citocinese , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Lepidópteros , Camundongos , Polissacarídeos/metabolismo , Análise de Sequência de RNA , Temperatura , Fatores de Transcrição/genética , VirulênciaRESUMO
The human fungal pathogen Cryptococcus neoformans undergoes many phenotypic changes to promote its survival in specific ecological niches and inside the host. To explore the role of chromatin remodeling on the expression of virulence-related traits, we identified and deleted seven genes encoding predicted class I/II histone deacetylases (HDACs) in the C. neoformans genome. These studies demonstrated that individual HDACs control non-identical but overlapping cellular processes associated with virulence, including thermotolerance, capsule formation, melanin synthesis, protease activity and cell wall integrity. We also determined the HDAC genes necessary for C. neoformans survival during in vitro macrophage infection and in animal models of cryptococcosis. Our results identified the HDA1 HDAC gene as a central mediator controlling several cellular processes, including mating and virulence. Finally, a global gene expression profile comparing the hda1Δ mutant versus wild-type revealed altered transcription of specific genes associated with the most prominent virulence attributes in this fungal pathogen. This study directly correlates the effects of Class I/II HDAC-mediated chromatin remodeling on the marked phenotypic plasticity and virulence potential of this microorganism. Furthermore, our results provide insights into regulatory mechanisms involved in virulence gene expression that are likely shared with other microbial pathogens.
Assuntos
Criptococose/genética , Cryptococcus neoformans/enzimologia , Histona Desacetilases/genética , Virulência/genética , Animais , Parede Celular , Criptococose/enzimologia , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/genética , Genoma Fúngico/genética , Histona Desacetilases/classificação , Humanos , Macrófagos/microbiologia , Macrófagos/patologiaRESUMO
Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i) interesting physiological property of C. neoformans regarding its amino acid uptake system; (ii) an important aspect of virulence, which is the need for amino acid permeases during thermal and oxidative stress resistance and, hence, host invasion and colonization; and (iii) provides a convenient prototype for antifungal development, which are the amino acid permeases Aap4/Aap5 and their inhibitor.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Criptococose/microbiologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Sistemas de Transporte de Aminoácidos/genética , Animais , Antifúngicos/farmacologia , Carbono/metabolismo , Criptococose/mortalidade , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/metabolismo , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Glucose/metabolismo , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Nitrogênio/metabolismo , Estresse Oxidativo , Fenótipo , Especificidade por Substrato , Temperatura , Virulência/genéticaRESUMO
Introducción: la criptocococis es una micosis sistémica causada por C. neoformans y C. gattii, es frecuente y oportunista en inmunocomprometidos y patógeno primario en personas inmunocompetentes. C. neoformans tiene una distribución mundial y se ha aislado desde las excretas de palomas. C. gattii se considera restringida a regiones con clima tropical, subtropical, y templadas, se encuentra asociada frecuentemente a detritos de especies de Eucalyptus sp. La virulencia de estas levaduras le permite desarrollar patogénesis en mamíferos y supervivencia en el ambiente. Objetivo: Identificar y determinar la actividad de proteinasas y fosfolipasas, de C. neoformans y C. gattii aisladas desde las oquedades de árboles en lugares con alta afluencia de público. Materiales y Métodos: Se tomaron 200 muestras de hisopado desde distintas especies de árboles desde sectores de la región de OHiggins y el Maule. Se siembran en ASG, se aíslan y mantienen en ASD. Identificación con tinta china, Urea de Christensen, crecimiento a 37°C, asimilación y fermentación de azucares, y siembra en medio CGB. Se mide índice de actividad enzimática Prz de proteinasas y fofolipasas. Resultados y Conclusiones: Se obtuvieron 109 cepas de C. neoformans aisladas desde las oquedades de diferentes especies arbóreas y 3 cepas presuntivas de C. gattii desde Eucalyptus sp. y Prunus cerasifera artropurpurea. El 88,1 por ciento de las cepas C. neoformans y 100 por ciento de C. gattii, presentaron alta actividad proteolítica, El 49,5 por ciento de las cepas de C. neoformans y 33,3 por ciento de C. gattii mostraron alta actividad de fosfolipasas.
Introduction: criptocococis is a systemic mycosis caused by C. neoformans and C. gattii, frequent and opportunistic in immunocompromised and primary pathogen in immunocompetent persons. C. neoformans has a worldwide distribution and has been isolated from the excreta of pigeons. C. gattii is considered restricted to regions with tropical, subtropical, and temperate, is often associated with species of Eucalyptus sp. The virulence of these yeasts develop pathogenesis allows survival in mammals and the environment. Objective: To identify and determine the activity of proteinases and phospholipases of C. neoformans and C. gattii isolated from the hollows of trees in places with high turnout. Materials and Methods: 200 swab samples were taken from different species of trees from areas of the region of OHiggins and Maule. Planted in ASG, they are isolated and kept in ASD. Identification with ink, Urea Christensen, growth at 37 ° C, assimilation and fermentation of sugars, and planting medium CGB. Prz index proteinase enzyme activity is measured and phospholipases. Results and Conclusions: We manage to get 109 strains of C. neoformans isolated from the hollows of different tree species and 3 presumptive strains of C. gattii from Eucalyptus sp. and Prunus cerasifera artropurpurea. 88.1 percent of the strains C. neoformans and C. gattii 100 percent , they showed high proteolytic activity, 49.5 percent of the strains of C. neoformans and C. gattii 33.3 percent showed high activity phospholipases.
Assuntos
Humanos , Cryptococcus gattii/isolamento & purificação , Cryptococcus gattii/enzimologia , Cryptococcus gattii/patogenicidade , Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Peptídeo Hidrolases , Fosfolipases , Árvores/microbiologia , Chile , Criptococose/etiologia , Eucalyptus/microbiologia , Pneumopatias Fúngicas , Prunus/microbiologiaRESUMO
Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology.
Assuntos
Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Animais , Desenho de Fármacos , Farmacorresistência Fúngica , Humanos , Melaninas/biossíntese , Polissacarídeos/metabolismo , VirulênciaRESUMO
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
A small series of C-glycosides containing the methoxyaryl moieties was tested for the inhibition of the ß-class carbonic anhydrases (CAs, EC 4.2.1.1) from Cryptococcus neoformans and Brucella suis. Many compounds showed activities in the micromolar or submicromolar range and excellent selectivity for pathogen CAs over human isozymes. The deprotected glycosides incorporating the 6-methoxy-2-naphthyl moiety showed the best inhibition profile and therefore represent leads for the development of novel anti-infectives with a new mechanism of action.
Assuntos
Brucella suis/enzimologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Cryptococcus neoformans/enzimologia , Glicosídeos/farmacologia , Naftalenos/farmacologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Glicosídeos/síntese química , Glicosídeos/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Naftalenos/químicaRESUMO
Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1Δ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1Δ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection.
Assuntos
Cryptococcus neoformans/enzimologia , Proteínas Fúngicas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Polissacarídeos/biossíntese , Vesículas Secretórias/metabolismo , Animais , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Feminino , Proteínas Fúngicas/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Polissacarídeos/metabolismo , Via Secretória , Virulência/genéticaRESUMO
The Ca(2+)-calcineurin signaling pathway in the human fungal pathogen Cryptococcus neoformans is essential for adaptation to the host environment during infection. Calcium transporters regulate cytosolic calcium concentrations, providing Ca(2+) loading into storage organelles. The three calcium transporters that have been characterized in C. neoformans, Cch1, Eca1 and Vcx1, are required for fungal virulence, supporting a role for calcium-mediated signaling in cryptococcal pathogenesis. In the present study, we report the functional characterization of the putative vacuolar calcium ATPase Pmc1 in C. neoformans. Our results demonstrate that Pmc1 provides tolerance to high Ca(2+) concentrations. The double knockout of C. neoformans PMC1 and VCX1 genes impaired the intracellular calcium transport, resulting in a significant increase in cytosolic calcium levels. Furthermore, Pmc1 was essential for both the progression of pulmonary infection and brain colonization in mice, emphasizing the crucial role of calcium signaling and transport for cryptococcal pathogenesis.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Cryptococcus neoformans/enzimologia , Proteínas Fúngicas/metabolismo , Animais , ATPases Transportadoras de Cálcio/classificação , ATPases Transportadoras de Cálcio/genética , Criptococose/metabolismo , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Camundongos , Filogenia , Vacúolos/enzimologia , VirulênciaRESUMO
The effects of the protease inhibitors saquinavir, darunavir, ritonavir, and indinavir on growth inhibition, protease and phospholipase activities, as well as capsule thickness of Cryptococcus neoformans were investigated. Viral protease inhibitors did not reduce fungal growth when tested in concentrations ranging from 0.001 to 1.000 mg/L. A tendency toward increasing phospholipase activity was observed with the highest tested drug concentration in a strain-specific pattern. However, these drugs reduced protease activity as well as capsule production. Our results confirm a previous finding that antiretroviral drugs affect the production of important virulence factors of C. neoformans.
Assuntos
Antirretrovirais/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Indinavir/farmacologia , Ritonavir/farmacologia , Saquinavir/farmacologia , Fatores de Virulência/genéticaRESUMO
In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [(3)H]dihydrosphingosine or [(3)H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B(1), a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasite's CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B(1) did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasite's CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1Δ lac1Δ double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B(1) than the parental strain.
Assuntos
Genoma de Protozoário , Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Acil Coenzima A/metabolismo , Acil Coenzima A/farmacologia , Clonagem Molecular , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Meios de Cultura , Ativação Enzimática , Ensaios Enzimáticos , Fumonisinas/farmacologia , Genes de Protozoários , Teste de Complementação Genética , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Filogenia , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Deleção de Sequência , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Tetra-Hidronaftalenos/farmacologia , Fatores de Tempo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genéticaRESUMO
The increased incidence of infections caused by the opportunistic pathogen Cryptococcus neoformans, which mainly affects immunocompromised patients but can also infect immunocompetent individuals, has needed additional studies on this micro-organism's pathogenicity and factors related to virulence, such as enzyme production, for a better understanding of the aetiology of cryptococcosis. The aim of this study was to verify the applicability of non-denaturing PAGE for analysis of laccases by quantification of the amount of melanin pigment produced by clinical and environmental strains of C. neoformans. After incubation of the gel with the substrate L-dopa, strains produced melanin spots of a bright brown to black colour. Quantification of these spots was performed by densitometry analysis and the amount of melanin produced was calculated and compared among the strains. All strains showed laccase activity. Serotype B strains showed a higher melanin intensity than serotype A strains. Over half of the clinical strains (56.2%) showed the lowest melanin intensities, suggesting that melanin production may not be the main virulence factor against host defence. The clinical strain ICB 88 revealed two melanin spots on the gel, indicating the presence of two laccase isoforms. The environmental strains showed the highest values of melanin intensity, which may be related to previous exposure to environmental stress conditions.
Assuntos
Criptococose/microbiologia , Cryptococcus neoformans/fisiologia , Lacase/metabolismo , Melaninas/biossíntese , Brasil , Criptococose/imunologia , Cryptococcus neoformans/classificação , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/isolamento & purificação , Humanos , Hospedeiro Imunocomprometido , SorotipagemRESUMO
Se detectó la actividad de fosfolipasa en 19 cepas clínicas y 17 aviarias de C. neoformans var. neoformans, usando Agar Sabouraud con yema de huevo, incubándose a 37ºC por 5 días. Se determinó el índice Pz estableciéndose los siguientes rangos: Pz muy alto (0.9-1), alto (0.89-0.80), bajo (0.79-0.70) y muy bajo (<0.69). El 84 por ciento de las cepas clínicas presentaron índice Pz muy bajo, el 5 por ciento bajo y 11 por ciento muy alto. Mientras en las cepas aviarias el 82 por ciento presentaron índice muy bajo y un 18 por ciento muy alto. Los valores de Pz promedio fueron muy bajos en todos los aislamientos, sin existir diferencias significativas (p > 0,05) entre las cepas clínicas y aviarias, lo que implica una alta actividad enzimática. La susceptibilidad in vitro a Fluconazol se realizó por el método de difusión con discos y el 89,5 por ciento de las cepas clínicas fueron sensibles y el 10,5 por ciento resistentes, mientras en las cepas aviarias, el 59 por ciento fueron sensibles, 29 por ciento sensible dosis dependiente y un12 por ciento resistentes.
Phospholipase activity was determined to 19 clinical and 17 aviars trains of C. neoformans var. neoformans, incubating the yeast for 5 days at 37º C on Sabouraud Agar supplemented with egg yolk. Pz values were determined and the following ranges were established: very high (0.9-1), high (0.89-0.80), low (0.79-0.70) and very low (<0.69). The 84 percent of the clinical isolates showed Pz values very low (5 percent) and 11 percent very high. On the other hand, the 82 percent of the aviars strains presented Pz values very low and 18 percent very high. Average Pz values were very low in all isolates , there were no statically significant differences (p>0.05) implying a high enzymatic activity. Susceptibility in vitro testing to Fluconazole was performed by a disk diffusion method (M44-A).The 89.5 percent of the clinical isolates were susceptible and 10.5 percent resistant, while in avian strains, 59 por ciento were susceptible, 29 percent susceptible dose dependent and 12 percent resistant.
Assuntos
Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/enzimologia , Fluconazol , Testes de Sensibilidade Microbiana , FosfolipasesRESUMO
Different phenotypic characteristics associated with virulence of the Cryptococcus neoformans species complex have shown an important role in their pathogenicity. In this study we have determined the role of phenotypically and genotypically factors of some virulence factors from clinical isolates in the two species of the complex; 35 C. neoformans and 19 Cryptococcus gattii. Growth at 37 degrees C, macroscopic and microscopic morphology, switching phenomenon, activity of 23 extracellular enzymes, variability of the colonies in agar with phloxin B; phospholipase B gene, and the mating type were determined by PCR; the molecular pattern was determined by URA5 RFLP. All isolates grew at 37 degrees C, the capsular size was greater in C. gattii (1.87 microm -/+1.47 microm) than in C. neoformans (0.83 microm -/+0.15 microm). Switching was observed mainly in isolates of C. gattii. All isolates expressed the enzyme urease, a lower activity of the proteases (Pz= 0.54), but a higher activity of the phospholipase (Pz=0.43) and phenoloxidase (Pz=0.003) was determined for C. gattii.
Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Fatores de Virulência/genética , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/isolamento & purificação , HumanosRESUMO
The capacity of Cryptococcus spp to produce melanin in media containing phenol compounds is widely used for identifying these species in the laboratory. The aim of the present study was to compare the production of this pigment by Cryptococcus spp. in four culture media. Sixteen strains of Cryptococcus neoformans, 17 of Cryptococcus albidus, 13 of Cryptococcus laurentii and two of Cryptococcus uniguttulatus were tested in the following media: potato-carrot agar, Niger seed agar, sunflower seed agar and L-dopa agar. The melanin production was evaluated on the basis of colony pigmentation. Its production after five days of incubation was demonstrated by 93.8% of the strains of Cryptococcus neoformans in the media of potato-carrot agar, sunflower seed agar and L-dopa agar. From the isolates of Cryptococcus albidus, 29.4% produced the pigment in potato-carrot agar and L-dopa agar, 11.8% in Niger seed agar and 36% in sunflower seed agar. From Cryptococcus laurentii, 53.8% produced the pigment in potato-carrot agar and sunflower seed agar, 61.5% in L-dopa agar and 84.6% in Niger seed agar. Only one strain of Cryptococcus uniguttulatus presented slight production of the pigment, in potato-carrot agar.
Assuntos
Ágar , Cryptococcus neoformans/enzimologia , Cryptococcus/enzimologia , Meios de Cultura , Melaninas/biossíntese , Cryptococcus/classificaçãoRESUMO
A capacidade de Cryptococcus spp produzir melanina em meios contendo compostos fenólicos é amplamente utilizada na identificação destas espécies no laboratório. O objetivo do presente trabalho foi comparar a produção desse pigmento em quatro meios de cultura por Cryptococcus sp. Foram testadas 16 cepas de Cryptococcus neoformans, 17 de Cryptococcus albidus, 13 de Cryptococcus laurentii, e 2 de Cryptococcus uniguttulatus nos meios: ágar batata e cenoura, ágar alpiste, ágar semente de girassol e ágar L-dopa. A produção de melanina foi avaliada com base na pigmentação das colônias, e demonstrada em 5 dias de incubação por 93,8 por cento das cepas de Cryptococcus neoformans nos meios ágar batata e cenoura, ágar semente de girassol e ágar L-dopa. Dos isolados de Cryptococcus albidus, 29,4 por cento produziram o pigmento em ágar batata e cenoura e L-dopa, 11,8 por cento em ágar alpiste, e 36 por cento em ágar girassol. De Cryptococcus laurentii, 53,8 por cento produziram em batata e cenoura e em semente de girassol, 61,5 por cento em L-dopa, 84,6 por cento em ágar alpiste. Somente uma cepa de Cryptococcus uniguttulatus produziu fracamente o pigmento em ágar batata e cenoura.
The capacity of Cryptococcus spp to produce melanin in media containing phenol compounds is widely used for identifying these species in the laboratory. The aim of the present study was to compare the production of this pigment by Cryptococcus spp. in four culture media. Sixteen strains of Cryptococcus neoformans, 17 of Cryptococcus albidus, 13 of Cryptococcus laurentii and two of Cryptococcus uniguttulatus were tested in the following media: potato-carrot agar, Niger seed agar, sunflower seed agar and L-dopa agar. The melanin production was evaluated on the basis of colony pigmentation. Its production after five days of incubation was demonstrated by 93.8 percent of the strains of Cryptococcus neoformans in the media of potato-carrot agar, sunflower seed agar and L-dopa agar. From the isolates of Cryptococcus albidus, 29.4 percent produced the pigment in potato-carrot agar and L-dopa agar, 11.8 percent in Niger seed agar and 36 percent in sunflower seed agar. From Cryptococcus laurentii, 53.8 percent produced the pigment in potato-carrot agar and sunflower seed agar, 61.5 percent in L-dopa agar and 84.6 percent in Niger seed agar. Only one strain of Cryptococcus uniguttulatus presented slight production of the pigment, in potato-carrot agar.
Assuntos
Ágar , Meios de Cultura , Cryptococcus neoformans/enzimologia , Cryptococcus/enzimologia , Melaninas/biossíntese , Cryptococcus/classificaçãoRESUMO
There is increasing evidence in the literature showing that fungal pathogens express biologically active ectoenzymes. The expression of surface phosphatases at the cell surface of Cryptococcus neoformans, the etiologic agent of cryptococcosis, was evaluated in the present study. Different isolates of C. neoformans express ectophosphatase activity, which is not influenced by capsule size or serotype. The cryptococcal enzyme is an acid phosphatase, inhibited by classic inhibitors of ectophosphatases, including ammonium molybdate and sodium salts of fluoride and orthovanadate. Only the inhibition of enzyme activity caused by sodium orthovanadate has been shown to be irreversible. The cryptococcal ectoenzyme is also inhibited by Zn2+ and inorganic phosphate, the final product of reactions catalyzed by phosphatases. The ectophosphatase from C. neoformans efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate removal when phosphothreonine is used as a substrate. Yeast cells with irreversibly inhibited ectophosphatases are less capable of adhering to animal epithelial cells than fungi fully expressing enzyme activity, suggesting that ectoenzyme expression can contribute to the pathogenesis of C. neoformans.