RESUMO

Stress management is an adaptive advantage for survival in adverse environments. Pathogens face this challenge during host colonization, requiring an appropriate stress response to establish infection. The fungal pathogen Cryptococcus neoformans undergoes thermal, oxidative, and osmotic stresses in the environment and animal host. Signaling systems controlled by Ras1, Hog1, and calcineurin respond to high temperatures and osmotic stress. Cationic stress caused by Na+, K+, and Li+ can be overcome with glycerol, the preferred osmolyte. Deleting the glycerol phosphate phosphatase gene (GPP2) prevents cells from accumulating glycerol due to a block in the last step of its biosynthetic pathway. Gpp2 accumulates in a phosphorylated form in a cna1Δ strain, and a physical interaction between Gpp2 and Cna1 was found; moreover, the gpp2Δ strain undergoes slow growth and has attenuated virulence in animal models of infection. We provide biochemical evidence that growth in 1 M NaCl increases glycerol content in the wild type, whereas gpp2Δ, cna1Δ, and cnb1Δ mutants fail to accumulate it. The deletion of cnb1Δ or cna1Δ renders yeast cells sensitive to cationic stress, and the Gfp-Gpp2 protein assumes an abnormal localization. We suggest a mechanism in which calcineurin controls Gpp2 at the post-translational level, affecting its localization and activity, leading to glycerol biosynthesis. Also, we showed the transcriptional profile of glycerol-deficient mutants and established the cationic stress response mediated by calcineurin; among the biological processes differentially expressed are carbon utilization, translation, transmembrane transport, glutathione metabolism, oxidative stress response, and transcription regulation. To our knowledge, this is the first time that this transcriptional profile has been described. These results have implications for pathogen stress adaptability.

RESUMO

BACKGROUND: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera collected from Colombian patients with non-HIV-associated cryptococcosis in a retrospective national cohort from 1997 to 2016. METHODS: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs against GM-CSF in 30 HIV negative adults with cryptococcosis (13 caused by C. gattii and 17 caused by C. neoformans). RESULTS: We detected neutralizing auto-Abs against GM-CSF in the sera of 10 out of 13 (77%) patients infected with C. gattii and one out of 17 (6%) patients infected with C. neoformans. CONCLUSIONS: We report eleven Colombian patients diagnosed with cryptococcosis who had auto-Abs that neutralize GM-CSF. Among these patients, ten were infected with C. gattii and only one with C. neoformans.

Assuntos

Anticorpos Neutralizantes , Autoanticorpos , Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Masculino , Colômbia , Feminino , Adulto , Cryptococcus gattii/imunologia , Pessoa de Meia-Idade , Cryptococcus neoformans/imunologia , Criptococose/imunologia , Criptococose/diagnóstico , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Estudos Retrospectivos , Soronegatividade para HIV/imunologia , Adulto Jovem , Idoso

RESUMO

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

Assuntos

Antifúngicos , Criptococose , Cryptococcus neoformans , Saccharomyces cerevisiae , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Animais , Camundongos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Modelos Animais de Doenças , Macrófagos/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Testes de Sensibilidade Microbiana , Caspofungina/farmacologia , Feminino , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Anfotericina B/farmacologia

RESUMO

Resumen Introducción : La criptococosis meníngea (CM) es una causa frecuente de meningoencefalitis en personas que viven con HIV (PVHIV) y produce una importante morbi-mortalidad (20-55%). Se describen las características clínicas, la letalidad y las variables de mal pronóstico en PVHIV con CM, en unidades de cuidados intensivos (UCI). Métodos : Estudio observacional y retrospectivo. Pe ríodo 21/11/2006 a 24/05/2023. Población evaluada: 154 PVHIV adultos, admitidos en UCI con diagnóstico de CM. Los porcentajes y valores absolutos, fueron comparados mediante Chi-Cuadrado o test de Fisher y las medianas mediante test de Mann-Whitney. La asociación con mortalidad se evaluó por regresión logística. Se utilizó el programa SPSS 23.0. Un valor p<0.05 fue considerado significativo. Resultados : Los pacientes que fallecieron y los que so brevivieron fueron comparables en edad y sexo (p>0.05). El análisis univariado, observó que un estado funcional y nutricional alterado, falta de tratamiento antirretroviral previo (TARV), CD4 <100 células/μl, APACHE II ≥13 y un score pronóstico de PVHIV ≥8 puntos, requerir ventilación mecánica (VM), sufrir insuficiencia respiratoria, renal, disfunción neurológica o sepsis, podrían estar asociados (p<0.05) con mortalidad. La regresión logística estableció que un estado funcional y nutricional alterado, un score pronóstico PVHIV ≥8, necesitar VM y sufrir sepsis serían variables independientes asociadas a mortalidad. Conclusión : Los resultados indican que el estado funcional y nutricional alterado, un score pronóstico PVHIV ≥8 puntos, requerir VM y sufrir sepsis al ingreso a UCI podrían servir como variables independientes para predecir un mayor riesgo de mortalidad.

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Abstract Introduction : Meningeal cryptococcosis (MC) is a frequent cause of meningoencephalitis in people living with HIV (PLHIV), leading to substantial morbidity (20- 55%). Clinical characteristics, lethality and adverse prog nostic factors in PLHIV with MC admitted to intensive care units (ICUs) are described. Methods : A retrospective observational study. Period from 11/21/2006 to 05/24/2023. It involved 154 adult PLHIV diagnosed with MC and admitted to ICUs. Percen tages and absolute values were compared by Chi-Square or Fisher's test and medians by Mann-Whitney test. The association with mortality was assessed by logistic re gression. SPSS 23.0 software was used. A p-value <0.05 was considered significant. Results : Patients who died and those who survived were comparable in age and sex (p>0.05). Univariate analysis showed that impaired functional and nutritio nal status, lack of previous highly active antiretroviral therapy, CD4 <100 cells, APACHE II ≥13 and a PLHIV prognostic score ≥8 points, requiring mechanical venti lation (MV), respiratory failure, renal failure, neurological dysfunction or sepsis could be associated (p<0.05) with mortality. Logistic regression established that impaired functional and nutritional status, a PLHIV prognostic score ≥8, need for MV and presence of sepsis would be independent variables associated with mortality. Conclusion : The results indicate that altered functio nal and nutritional status, a PLHIV prognostic score ≥ 8 points, requiring MV and suffering sepsis on admission to the ICU are more frequent in deceased patients, and they could therefore serve as independent variables to predict a higher risk of mortality.

RESUMO

This chapter describes methodological details for preparing specimens of Cryptococcus neoformans (although it can be applied to any species of the genus) and their subsequent analysis by scanning and transmission electron microscopy. Adaptations to conventional protocols for better preservation of the sample, as well as to avoid artifacts, are presented. The protocols may be used to examine both the surface ultrastructure and the interior of this pathogenic fungus in detail.

Assuntos

Artefatos , Cryptococcus neoformans , Cryptococcus neoformans/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Microscopia Eletrônica de Varredura/métodos , Manejo de Espécimes/métodos

RESUMO

The interaction between macrophages and Cryptococcus neoformans is crucial in the pathogenesis of cryptococcosis. These phagocytes are important immune effectors, but also a niche in which facultative intracellular parasites, such as C. neoformans, thrive. Consequently, phagocytosis of cryptococcal cells and its outcomes are very frequently studied. One major issue with several of the tests used for this, however, is that macrophage-C. neoformans interaction does not always result in phagocytosis, as fungi may be attached to the external surface of the phagocyte. The most used methodologies to study phagocytosis of cryptococcal cells have varying degrees of precision in separating fungi that are truly internalized from those that are outside macrophages. Here we describe two assays to measure phagocytosis that can differentiate internal from external C. neoformans cells.

Assuntos

Criptococose , Cryptococcus neoformans , Macrófagos , Fagocitose , Cryptococcus neoformans/imunologia , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Criptococose/microbiologia , Criptococose/imunologia , Animais , Camundongos , Humanos , Interações Hospedeiro-Patógeno/imunologia

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/métodos

RESUMO

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

Assuntos

Anticorpos Antifúngicos , Anticorpos Monoclonais , Cryptococcus neoformans , Hibridomas , Cryptococcus neoformans/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Animais , Humanos , Hibridomas/imunologia , Anticorpos Antifúngicos/imunologia , Anticorpos Antifúngicos/isolamento & purificação , Camundongos , Linfócitos B/imunologia , Criptococose/imunologia , Criptococose/diagnóstico , Antígenos de Fungos/imunologia , Imunização

RESUMO

Cryptococcosis is one of the major life-threatening opportunistic/systemic fungal diseases of worldwide occurrence, which can be asymptomatic or establish pneumonia and meningoencephalitis mainly in immunosuppressed patients, caused by the Cryptococcus neoformans and C. gattii species complexes. Acquisition is by inhaling fungal propagules from avian droppings, tree hollows and decaying wood, and the association of the molecular types with geographic origin, virulence and antifungal resistance have epidemiological importance. Since data on cryptococcosis in Alagoas are limited, we sought to determine the molecular types of etiological agents collected from clinical and environmental sources. We evaluated 21 isolates previously collected from cerebrospinal fluid and from environment sources (pigeon droppings and tree hollows) in Maceió-Alagoas (Brazil). Restriction fragment length polymorphism of URA5 gene was performed to characterize among the eight standard molecular types (VNI-VNIV and VGI-VGIV). Among isolates, 66.67% (14) were assigned to C. neoformans VNI - 12 of them (12/14) recovered from liquor and 2 from a tree hollow (2/14). One isolate from pigeon droppings (4.76%) corresponded to C. neoformans VNIV, while five strains from tree hollows and one from pigeon droppings (6, 28.57%) to C. gattii VGII. VNI-type was present in clinical and environmental samples and most C. neoformans infections were observed in HIV-positive patients, while types VNIV and VGII were prevalent in environmental sources in Alagoas. This is the first molecular characterization of Cryptococcus spp. in Alagoas, our study provides additional information on the ecoepidemiology of Cryptococcus spp. in Brazil, contributing to a closer view of the endemic species.

Assuntos

Columbidae , Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Microbiologia Ambiental , Cryptococcus neoformans/genética , Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/classificação , Brasil/epidemiologia , Criptococose/microbiologia , Criptococose/epidemiologia , Cryptococcus gattii/genética , Cryptococcus gattii/isolamento & purificação , Cryptococcus gattii/classificação , Humanos , Animais , Columbidae/microbiologia , Técnicas de Tipagem Micológica , Polimorfismo de Fragmento de Restrição , Fezes/microbiologia , Genótipo

RESUMO

INTRODUCTION: Meningeal cryptococcosis (MC) is a frequent cause of meningoencephalitis in people living with HIV (PLHIV), leading to substantial morbidity (20-55%). Clinical characteristics, lethality and adverse prognostic factors in PLHIV with MC admitted to intensive care units (ICUs) are described. METHODS: A retrospective observational study. Period from 11/21/2006 to 05/24/2023. It involved 154 adult PLHIV diagnosed with MC and admitted to ICUs. Percentages and absolute values were compared by Chi-Square or Fisher's test and medians by Mann-Whitney test. The association with mortality was assessed by logistic regression. SPSS 23.0 software was used. A p-value <0.05 was considered significant. RESULTS: Patients who died and those who survived were comparable in age and sex (p>0.05). Univariate analysis showed that impaired functional and nutritional status, lack of previous highly active antiretroviral therapy, CD4 <100 cells, APACHE II ≥ 13 and a PLHIV prognostic score ≥ 8 points, requiring mechanical ventilation (MV), respiratory failure, renal failure, neurological dysfunction or sepsis could be associated (p<0.05) with mortality. Logistic regression established that impaired functional and nutritional status, a PLHIV prognostic score ≥ 8, need for MV and presence of sepsis would be independent variables associated with mortality. CONCLUSION: The results indicate that altered functional and nutritional status, a PLHIV prognostic score ≥ 8 points, requiring MV and suffering sepsis on admission to the ICU are more frequent in deceased patients, and they could therefore serve as independent variables to predict a higher risk of mortality.

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Introducción: La criptococosis meníngea (CM) es una causa frecuente de meningoencefalitis en personas que viven con HIV (PVHIV) y produce una importante morbimortalidad (20-55%). Se describen las características clínicas, la letalidad y las variables de mal pronóstico en PVHIV con CM, en unidades de cuidados intensivos (UCI). Métodos: Estudio observacional y retrospectivo. Período 21/11/2006 a 24/05/2023. Población evaluada: 154 PVHIV adultos, admitidos en UCI con diagnóstico de CM. Los porcentajes y valores absolutos, fueron comparados mediante Chi-Cuadrado o test de Fisher y las medianas mediante test de Mann-Whitney. La asociación con mortalidad se evaluó por regresión logística. Se utilizó el programa SPSS 23.0. Un valor p<0.05 fue considerado significativo. Resultados: Los pacientes que fallecieron y los que sobrevivieron fueron comparables en edad y sexo (p>0.05). El análisis univariado, observó que un estado funcional y nutricional alterado, falta de tratamiento antirretroviral previo (TARV), CD4 <100 células/µl, APACHE II ≥ 13 y un score pronóstico de PVHIV ≥ 8 puntos, requerir ventilación mecánica (VM), sufrir insuficiencia respiratoria, renal, disfunción neurológica o sepsis, podrían estar asociados (p<0.05) con mortalidad. La regresión logística estableció que un estado funcional y nutricional alterado, un score pronóstico PVHIV ≥ 8, necesitar VM y sufrir sepsis serían variables independientes asociadas a mortalidad. Conclusión: Los resultados indican que el estado funcional y nutricional alterado, un score pronóstico PVHIV ≥ 8 puntos, requerir VM y sufrir sepsis al ingreso a UCI podrían servir como variables independientes para predecir un mayor riesgo de mortalidad.

Assuntos

Infecções Oportunistas Relacionadas com a AIDS , Unidades de Terapia Intensiva , Meningite Criptocócica , Humanos , Masculino , Feminino , Estudos Retrospectivos , Adulto , Meningite Criptocócica/mortalidade , Meningite Criptocócica/complicações , Pessoa de Meia-Idade , Infecções Oportunistas Relacionadas com a AIDS/mortalidade , Infecções Oportunistas Relacionadas com a AIDS/complicações , Unidades de Terapia Intensiva/estatística & dados numéricos , Prognóstico , Fatores de Risco , Infecções por HIV/complicações , Infecções por HIV/mortalidade

RESUMO

Antifungal infections are becoming a major concern to human health due to antimicrobial resistance. Echinocandins have been promising agents against resistant fungal infections, primarily caspofungin, which has a more effective mechanism of action than azoles and polyenes. However, fungi such as Cryptococcus neoformans appear to be inheritably resistant to these drugs, which is concerning due to the high clinical importance of C. neoformans. In this review, we review the history of C. neoformans and the treatments used to treat antifungals over the years, focusing on caspofungin, while highlighting the C. neoformans problem and possible explanations for its inherent resistance.

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Caspofungin is a drug used to treat several types of fungal infections. Resistance to caspofungin is a huge problem, especially in those that are immunocompromised. It is important to understand the history of caspofungin discovery, its clinical applications and its mechanism of action, as well as if a new drug target could be used overcome resistance. This review may perform guide new studies combining caspofungin with other drugs and indicate new potential targets for caspofungin.

Assuntos

Antifúngicos , Caspofungina , Criptococose , Cryptococcus neoformans , Farmacorresistência Fúngica , Caspofungina/uso terapêutico , Caspofungina/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Antifúngicos/uso terapêutico , Antifúngicos/farmacologia , Humanos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Equinocandinas/uso terapêutico , Equinocandinas/farmacologia , Animais , Testes de Sensibilidade Microbiana , Lipopeptídeos/uso terapêutico , Lipopeptídeos/farmacologia

RESUMO

Cryptococcus neoformans is responsible for over 100 000 deaths annually, and the treatment of this fungal disease is expensive and not consistently effective. Unveiling new therapeutic avenues is crucial. Previous studies have suggested that the anthelmintic drug fenbendazole is an affordable and nontoxic candidate to combat cryptococcosis. However, its mechanism of anticryptococcal activity has been only superficially investigated. In this study, we examined the global cellular response of C. neoformans to fenbendazole using a proteomic approach (data are available via ProteomeXchange with identifier PXD047041). Fenbendazole treatment mostly impacted the abundance of proteins related to metabolic pathways, RNA processing, and intracellular traffic. Protein kinases, in particular, were significantly affected by fenbendazole treatment. Experimental validation of the proteomics data using a collection of C. neoformans mutants led to the identification of critical roles of five protein kinases in fenbendazole's antifungal activity. In fact, mutants lacking the expression of genes encoding Chk1, Tco2, Tco3, Bub1, and Sch9 kinases demonstrated greater resistance to fenbendazole compared to wild-type cells. In combination with the standard antifungal drug amphotericin B, fenbendazole reduced the cryptococcal burden in mice. These findings not only contribute to the elucidation of fenbendazole's mode of action but also support its use in combination therapy with amphotericin B. In conclusion, our data suggest that fenbendazole holds promise for further development as an anticryptococcal agent.

Assuntos

Antifúngicos , Criptococose , Cryptococcus neoformans , Fenbendazol , Proteínas Quinases , Proteômica , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Antifúngicos/farmacologia , Animais , Fenbendazol/farmacologia , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Camundongos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Anfotericina B/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Testes de Sensibilidade Microbiana , Modelos Animais de Doenças , Farmacorresistência Fúngica/genética

RESUMO

Background: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Autoantibodies (auto-Abs) neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera from Colombian patients with non-HIV related cryptococcosis in a retrospective national cohort collected from 1997 to 2016. Methods: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs in 30 HIV (-) adults presenting cryptococcosis (13 by C. gattii, and 17 by C. neoformans). Results: We detected auto-Abs neutralizing GM-CSF in the plasma of 9 out of 13 (69%) patients infected with C. gattii and 1 out of 17 (6%) patients with C. neoformans. Conclusions: We report ten Colombian patients with cryptococcosis due to auto-Abs neutralizing GM-CSF. Nine of the ten patients were infected with C. gattii, and only one with C. neoformans.

RESUMO

Fungal infections are a global public health challenge, especially among immunocompromised patients. Basidiomycetous yeasts, such as Rhodotorula mucilaginosa, have emerged as opportunistic pathogens, but have received less attention than Cryptococcus neoformans. This study aimed to characterize the polysaccharides of R. mucilaginosa and compare them with those of C. neoformans, analyzing their clinical implications. Comprehensive physicochemical, mechanical, and ultrastructural analyses of polysaccharides from both species were performed, revealing correlations with virulence and pathogenicity. R. mucilaginosa cells are surrounded by a capsule smaller than that produced by C. neoformans, but with similar polysaccharides. Those polysaccharides are also secreted by R. mucilaginosa. Cross-reactivity with R. mucilaginosa was observed in a diagnostic C. neoformans antigen test, using both in vitro and in vivo samples, highlighting the need for more reliable tests. Some R. mucilaginosa strains exhibited virulence comparable to that of C. neoformans in an invertebrate experimental model (Tenebrio molitor). This study contributes to a deeper understanding of yeast pathogenicity and virulence, highlighting the need for more accurate diagnostic tests to improve the differential diagnosis of infections caused by basidiomycetous yeasts.

RESUMO

Cryptococcus neoformans is an encapsulated yeast that can cause cryptococcosis and cryptococcal meningitis, which conventional treatment involves antifungal drugs such as polyenes, flucytosine, azoles, and their combinations. However, the high cost, toxicity, and increase in fungi resistance to antifungal agents stimulate the search for therapeutic strategies such as drug repurposing and combination therapy. This study evaluated the activity of the antihypertensive verapamil (VEH) alone and combined with amphotericin B (AmB) against C. neoformans. VEH exhibited antifungal activity against C. neoformans with minimum inhibitory concentration and minimum fungicidal concentration of 118 µg per mL. The combination of VEH and AmB exhibited synergism, reducing at least eightfold both drugs' concentrations. Moreover, the combination decreased the size and glucuronoxylomannnan content of C. neoformans capsule. However, no difference was observed in ergosterol levels of C. neoformans after treatment with VEH and AmB in combination. Altogether, VEH in combination with AmB exhibits potential as a candidate as for the development of anti-cryptococcal drug.

Assuntos

Criptococose , Cryptococcus neoformans , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Flucitosina/farmacologia , Flucitosina/uso terapêutico , Testes de Sensibilidade Microbiana

RESUMO

Background: Cryptococcus neoformans is an opportunistic fungal pathogen that causes infections mainly in immunosuppressed individuals, such as transplant recipients. Aims: This study investigated the effects of rapamycin, an immunosuppressant drug, on the cellular organization, biophysical characteristics, and main virulence factors of C. neoformans. Methods: Morphological, structural, physicochemical and biophysical analyses of cells and secreted polysaccharides of the reference H99 C. neoformans strain were investigated under the effect of subinhibitory concentrations of rapamycin. Results: Rapamycin at a minimum inhibitory concentration of 2.5 µM reduced C. neoformans cell viability by 53%, decreased capsule, increased cell size, chitin and lipid body formation, and changed peptidase and urease activity. Conclusion: Further studies are needed to assess how rapamycin affects the virulence factors and pathogenicity of C. neoformans.

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Cryptococcosis is a fungal infection caused by a type of fungus called Cryptococcus. Among the Cryptococcus group, Cryptococcus neoformans is often linked to fungal infections in people who have a weak immune system (known as being immunosuppressed). The main aim of this work was to look at the effect of an immunosuppressant called rapamycin, which is commonly used to prevent organ transplant rejection, on the ability of C. neoformans to cause infection. The results showed that this drug stopped the growth of the fungus, dampening its ability to cause disease.

Assuntos

Criptococose , Cryptococcus neoformans , Humanos , Fatores de Virulência , Sirolimo/farmacologia , Criptococose/microbiologia , Virulência

RESUMO

Cryptococcosis is a systemic mycosis affecting immunosuppressed individuals, caused by various Cryptococcus species. The current treatment utilizes a combination of antifungal drugs, but issues such as nephrotoxicity, restricted or limited availability in certain countries, and resistance limit their effectiveness. Repurposing approved drugs presents a viable strategy for developing new antifungal options. This study investigates the potential of glatiramer acetate (Copaxone®) as a chemotherapy candidate for Cryptococcus neoformans infection. Various techniques are employed to evaluate the effects of glatiramer acetate on the fungus, including microdilution, XTT analysis, electron and light microscopy, and physicochemical measurements. The results demonstrate that glatiramer acetate exhibits antifungal properties, with an IC50 of 0.470 mg/mL and a minimum inhibitory concentration (MIC) of 2.5 mg/mL. Furthermore, it promotes enhanced cell aggregation, facilitates biofilm formation, and increases the secretion of fungal polysaccharides. These findings indicate that glatiramer acetate not only shows an antifungal effect but also modulates the key virulence factor-the polysaccharide capsule. In summary, repurposing glatiramer acetate as a potential chemotherapy option offers new prospects for combating C. neoformans infection. It addresses the limitations associated with current antifungal therapies by providing an alternative treatment approach.

RESUMO

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a life-threatening fungal infection affecting mostly immunocompromised patients. In fact, cryptococcal meningitis accounts for about 19% of AIDS-related deaths in the world. Because of long-term azole therapies to treat this mycosis, resistance to fluconazole leading to treatment failure and poor prognosis has long been reported for both fungal species. Among the mechanisms implicated in resistance to azoles, mutations in the ERG11 gene, encoding the azole target enzyme lanosterol 14-α-demethylase, have been described. This study aimed to establish the amino acid composition of ERG11 of Colombian clinical isolates of C. neoformans and C. gattii and to correlate any possible substitution with the in vitro susceptibility profile of the isolates to fluconazole, voriconazole, and itraconazole. Antifungal susceptibility testing results showed that C. gattii isolates are less susceptible to azoles than C. neoformans isolates, which could correlate with differences in the amino acid composition and structure of ERG11 of each species. In addition, in a C. gattii isolate with high MICs for fluconazole (64 µg/mL) and voriconazole (1 µg/mL), a G973T mutation resulting in the substitution R258L, located in substrate recognition site 3 of ERG11, was identified. This finding suggests the association of the newly reported substitution with the azole resistance phenotype in C. gattii. Further investigations are needed to determine the exact role that R258L plays in the decreased susceptibility to fluconazole and voriconazole, as well as to determine the participation of additional mechanisms of resistance to azole drugs. IMPORTANCE The fungal species Cryptococcus neoformans and C. gattii are human pathogens for which drug resistance or other treatment and management challenges exist. Here, we report differential susceptibility to azoles among both species, with some isolates displaying resistant phenotypes. Azoles are among the most commonly used drugs to treat cryptococcal infections. Our findings underscore the necessity of testing antifungal susceptibility in the clinical setting in order to assist patient management and beneficial outcomes. In addition, we report an amino acid change in the sequence of the target protein of azoles, which suggests that this change might be implicated in resistance to these drugs. Identifying and understanding possible mechanisms that affect drug affinity will eventually aid the design of new drugs that overcome the global growing concern of antifungal resistance.

Assuntos

Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Humanos , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Cryptococcus gattii/genética , Fluconazol/farmacologia , Azóis/farmacologia , Voriconazol/farmacologia , Lanosterol/farmacologia , Lanosterol/uso terapêutico , Esterol 14-Desmetilase/genética , Esterol 14-Desmetilase/metabolismo , Esterol 14-Desmetilase/farmacologia , Cryptococcus neoformans/genética , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Testes de Sensibilidade Microbiana , Farmacorresistência Fúngica/genética , Aminoácidos

RESUMO

Cryptococcus neoformans and C. gattii species complexes (phylum: Basidiomycota) are environmental yeasts and are the main cause of human cryptococcosis worldwide. The most recent molecular typing studies in Latin America have focused on the intertropical region. Thus, this study aimed to update the knowledge of human cryptococcosis in the South American temperate region. We obtained and analyzed 116 C. neoformans/C. gattii species complexes isolates from the Public Health Surveillance Laboratory between 2008-2013 and 2017-2021 (C. gattii species complex = 1 and C. neoformans species complex = 115). The average patient age was 45 years, with an overall male:female ratio of 3.1:1. The proportion of HIV-negative patients was significantly higher in the second study period. Restriction fragment length polymorphism typing of URA5 gene revealed that the C. neoformans species complex comprised 75.7% VNI, 2.6% VNII, 0.9% VNIII, 1.7% VNIV, 17.4% VNII/VNIV hybrids, and one C. neoformans isolate (0.9%) misidentified as VGI; the C. gattii species complex isolates comprised one VGII. The overall case fatality rate was 49.5%, with no differences in lethality between VNI and hybrid isolates. Of the four isolates responsible for episodes of reoccurrence, only one had a genotype different from the first episode. Antifungal susceptibility testing revealed that most isolates fell below the local epidemiological cut-off value. This study provides additional information for the analysis of C. neoformans/C. gattii species complexes dynamics in the South American temperate region.

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This study describes the epidemiological and molecular trends of human cryptococcosis according to the public health Uruguayan surveillance network. The findings provide additional information for analyzing the Cryptococcusneoformans/C. gattii species complexes in the South American temperate region.

Assuntos

Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Humanos , Masculino , Feminino , Animais , Antifúngicos/farmacologia , Uruguai/epidemiologia , Criptococose/epidemiologia , Criptococose/microbiologia , Criptococose/veterinária , Tipagem Molecular/veterinária , Genótipo , Técnicas de Tipagem Micológica/veterinária

RESUMO

This study aimed to investigate the effects of cyclosporine on the morphology, cell wall structure, and secretion characteristics of Cryptococcus neoformans. The minimum inhibitory concentration (MIC) of cyclosporine was found to be 2 µM (2.4 µg/mL) for the H99 strain. Yeast cells treated with cyclosporine at half the MIC showed altered morphology, including irregular shapes and elongated projections, without an effect on cell metabolism. Cyclosporine treatment resulted in an 18-fold increase in chitin and an 8-fold increase in lipid bodies, demonstrating changes in the fungal cell wall structure. Cyclosporine also reduced cell body and polysaccharide capsule diameters, with a significant reduction in urease secretion in C. neoformans cultures. Additionally, the study showed that cyclosporine increased the viscosity of secreted polysaccharides and reduced the electronegativity and conductance of cells. The findings suggest that cyclosporine has significant effects on C. neoformans morphology, cell wall structure, and secretion, which could have implications for the development of new antifungal agents.