RESUMO
A criptococose é uma doença infecciosa sistêmica de origem fúngica, causada pelas espécies: Cryptococcus neoformans e Cryptococcus gattii. É considerada uma micose oportunista capaz de infectar mamíferos domésticos, animais silvestres e seres humanos, sendo classificada como uma doença zoonótica. Esse patógeno é encontrado principalmente em ambientes contaminados por fezes de pombos (Columba livia), que atuam como importantes fontes de infecção do fungo. De acordo com sua disseminação para os tecidos do organismo, a doença pode causar diferentes síndromes tanto em seres humanos como em animais síndrome respiratória, síndrome neurológica, síndrome ocular e síndrome cutânea. O diagnóstico pode ser realizado através da pesquisa de antígeno polissacarídeo circulante no soro ou líquor, por meio da prova de látex. Testes imunoenzimáticos (ELISA) também podem ser realizados para detecção de antígenos dessa levedura. Exame citológico, histopatológico e cultura fúngica para a identificação do agente tornam o diagnóstico da criptococose mais fácil. O tratamento é baseado no uso de antifúngicos e sua escolha é realizada através da avaliação dos sinais clínicos observados. Diante do exposto, o objetivo deste trabalho é relatar um caso de criptococose respiratória e cutânea em felino doméstico de vida livre da cidade de Sobral/CE.
Cryptococcosis is a systemic infectious disease of fungal origin caused by the species: Cryptococcus neoformans and Cryptococcus gattii. It is considered an opportunistic mycosis capable of infecting domestic mammals, wild animals, and humans, being classified as a zoonotic disease. This pathogen is mainly found mainly in environments contaminated by pigeon (Columba livia) feces, which act as important sources of fungal infection. According to its spread to the body's tissues, the disease can cause different syndromes in both humans and animals: respiratory syndrome, neurological syndrome, ocular syndrome, and cutaneous syndrome. The diagnosis can be made through the investigation of circulating polysaccharide antigen in serum or cerebrospinal fluid using the latex test. Immunoenzymatic tests (ELISA) can also be performed to detect yeast antigens. Cytological examination, histopathological examination, and fungal culture to identify the agent make the diagnosis of cryptococcosis easier. The treatment is based on the use of antifungals, and its choice is made through the evaluation of the observed clinical signs. In this context, this work aims to report a case of respiratory and cutaneous cryptococcosis in a free-range domestic cat in the city of Sobral/CE.
Assuntos
Animais , Gatos , Doenças do Gato , Zoonoses , Criptococose/veterinária , Cryptococcus neoformans/patogenicidade , Cryptococcus gattii/patogenicidadeRESUMO
In the airways, the adhesion of Cryptococcus neoformans with airway epithelial cells is crucial for the establishment of cryptococcosis. Tobacco smoke is considered a risk factor for cryptococcosis. Here, we evaluated the effects of cigarette smoke extract (CSE) on human bronchial epithelial cells (BEAS-2B) stimulated with C. neoformans. Multiplicities of infection (MOIs) of 1-100 of C. neoformans per cell led to increased IL-8 production and no cytotoxic effects when compared to those of controls. C. neoformans (MOI 100) also significantly increased the concentration of IL-6. In cells stimulated with CSE doses (1.0, 2.5 and 5.0%) from one or five cigarettes, increased IL-1ß production was observed only in doses from one (1.0%) and five (2.5%) cigarettes when compared to that of controls. However, only 1.0% CSE failed to show cytotoxic effects. In addition, CSE significantly increased the concentration of IL-8. Cells stimulated with both CSE and C. neoformans demonstrated a reduction in IL-6/STAT3 signalling compared to that in cells stimulated by C. neoformans. In addition, a significant increase in IL-10 production was also observed. No alterations in NF-kB or ICAM-1 expression were observed among the groups. The combination of CSE and C. neoformans favoured the increase of fungal numbers and extracellular adhering of C. neoformans on BEAS-2B cells. In addition, the internalization of C. neoformans on BEAS-2B cells was reduced after CSE stimulation. In conclusion, the association of CSE and C. neoformans induced an anti-inflammatory effect in bronchial epithelial cells, which might favour the development of C. neoformans infection in the airways.
Assuntos
Criptococose/patologia , Cryptococcus neoformans/patogenicidade , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/microbiologia , Linhagem Celular , Sobrevivência Celular , Criptococose/microbiologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Fagocitose/efeitos dos fármacos , Fatores de Risco , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills almost 200,000 people worldwide each year. It is acquired when mammalian hosts inhale the infectious propagules; these are deposited in the lung and, in the context of immunocompromise, may disseminate to the brain and cause lethal meningoencephalitis. Once inside the host, C. neoformans undergoes a variety of adaptive processes, including secretion of virulence factors, expansion of a polysaccharide capsule that impedes phagocytosis, and the production of giant (Titan) cells. The transcription factor Pdr802 is one regulator of these responses to the host environment. Expression of the corresponding gene is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. Direct targets of Pdr802 include the quorum sensing proteins Pqp1, Opt1, and Liv3; the transcription factors Stb4, Zfc3, and Bzp4, which regulate cryptococcal brain infectivity and capsule thickness; the calcineurin targets Had1 and Crz1, important for cell wall remodeling and C. neoformans virulence; and additional genes related to resistance to host temperature and oxidative stress, and to urease activity. Notably, cryptococci engineered to lack Pdr802 showed a dramatic increase in Titan cells, which are not phagocytosed and have diminished ability to directly cross biological barriers. This explains the limited dissemination of pdr802 mutant cells to the central nervous system and the consequently reduced virulence of this strain. The role of Pdr802 as a negative regulator of Titan cell formation is thus critical for cryptococcal pathogenicity.IMPORTANCE The pathogenic yeast Cryptococcus neoformans presents a worldwide threat to human health, especially in the context of immunocompromise, and current antifungal therapy is hindered by cost, limited availability, and inadequate efficacy. After the infectious particle is inhaled, C. neoformans initiates a complex transcriptional program that integrates cellular responses and enables adaptation to the host lung environment. Here, we describe the role of the transcription factor Pdr802 in the response to host conditions and its impact on C. neoformans virulence. We identified direct targets of Pdr802 and also discovered that it regulates cellular features that influence movement of this pathogen from the lung to the brain, where it causes fatal disease. These findings significantly advance our understanding of a serious disease.
Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Células Gigantes/fisiologia , Interações Hospedeiro-Patógeno , Fatores de Transcrição/genética , Animais , Feminino , Proteínas Fúngicas/metabolismo , Deleção de Genes , Células Gigantes/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismoRESUMO
Cryptococcosis is a life-threatening fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii species complex. Most cases are recorded in patients suffering from HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome). However, this infection also occurs in non-HIV patients with a proportion of 10-30% of all cases. The study aimed at the clinical and molecular characterization of non-HIV patients diagnosed with cryptococcosis at the Tropical Medicine Foundation (FMT-HVD) from July 2016 to June 2019. Medical records of respective patients were analyzed to describe the course of cryptococcosis in non-HIV patients. In addition, multi-locus sequence typing (MLST) was applied to identify the sequence types of the isolated Cryptococcus strains, to perform phylogenetic analysis, and to evaluate the isolates' genetic relationship to global reference strains. Antifungal susceptibility profiles to amphotericin B, fluconazole, and itraconazole were assessed by broth microdilution. From a total of 7 patients, 4 were female, the age range varied between 10 and 53 years (median of 36.3 years). Cryptococcal meningitis was the common clinical manifestation (100%). The period between onset of symptoms and confirmed diagnosis ranged from 15 to 730 days (mean value of 172.9 days), and the observed mortality was 57.1%. Of note, comorbidities of the assessed cryptococcosis patients comprised hypertension, diabetes mellitus, and intestinal tuberculosis. Genotyping applying PCR-RFLP of the URA5 gene identified all clinical isolates as C. gattii genotype VGII. Using MLST, it was possible to discriminate the sequence types ST20 (n = 4), ST5 (n = 3), and the newly identified sequence type ST560 (n = 1). The antifungals amphotericin B, fluconazole, and itraconazole showed satisfactory inhibitory activity (microdilution test) against all C. gattii VGII strains.
Assuntos
Cryptococcus gattii/genética , Cryptococcus neoformans/genética , Meningite Criptocócica/epidemiologia , Adolescente , Adulto , Antifúngicos/farmacologia , Brasil/epidemiologia , Criança , Cryptococcus gattii/classificação , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/patogenicidade , Cryptococcus neoformans/classificação , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/patogenicidade , Feminino , Geografia , Infecções por HIV , Humanos , Masculino , Meningite Criptocócica/microbiologia , Meningite Criptocócica/mortalidade , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Técnicas de Tipagem Micológica , Filogenia , Estudos Prospectivos , Adulto JovemRESUMO
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
Intracellular calcium (Ca2+) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signaling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+ binding mediated by the EF-hand domains of the Ca2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+ homeostasis under stress and nonstress conditions, as the ncs1Δ mutant is sensitive to a high Ca2+ concentration and has an elevated basal Ca2+ level. Furthermore, NCS1 expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to the case with Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca2+ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ and cam1Δ), and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1 deficiency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data show that Ncs1 has a significant role as a Ca2+ sensor in C. neoformans, working with calcineurin to regulate Ca2+ homeostasis and, consequently, promote fungal growth and virulence.IMPORTANCECryptococcus neoformans is the major cause of fungal meningitis in HIV-infected patients. Several studies have highlighted the important contributions of Ca2+ signaling and homeostasis to the virulence of C. neoformans Here, we identify the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression, and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signaling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.
Assuntos
Calcineurina/genética , Proteínas de Ligação ao Cálcio/genética , Divisão Celular/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Animais , Cryptococcus neoformans/química , Feminino , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Virulência/genéticaRESUMO
BACKGROUND: Cryptococcus neoformans/ Cryptococcus gattii species complex is composed of encapsulated yeast species that are causative agents of cryptococcosis. The characterisation of pathogenic Cryptococcus species provides useful data for epidemiological studies as well as the clinical diagnosis and treatment of patients. OBJECTIVES: This study aimed to characterise the epidemiology, antifungal susceptibility and virulence of 72 clinical strains isolated from cryptococcosis cases between 2012 and 2017 in a tertiary reference hospital in south-eastern Brazil. METHODS: Species and molecular types were molecularly assessed by PCR and PCR-restriction fragment length polymorphism (RFLP) of the URA5 gene. Antifungal susceptibility testing was performed according to the CLSI protocols. The virulence was studied in a Galleria mellonella infection model. RESULTS: The most frequently isolated strain was C. neoformans molecular type VNI (61/72; 84.7%), although C. neoformans molecular type VNII (3/72; 4.2%) was also isolated. Additionally, C. deuterogattii molecular type VGII (8/72; 11.1%) was present, but most frequently from non-HIV-infected patients. Non-wild-type phenotype to the antifungals was observed in 26.4% (19/72) of the C. neoformans and C. deuterogattii clinical isolates, and the latter demonstrated higher MIC to fluconazole and itraconazole than C. neoformans clinical isolates. Finally, the virulence of C. neoformans and C. deuterogattii clinical isolates was diverse in G mellonella larvae and uncorrelated with the virulence factors of melanin and capsule. CONCLUSIONS: The assessment of the spread of cryptococcal species and molecular types as well as the pattern of corresponding antifungal susceptibility and virulence aids in surveil the emergence of resistant strains, ensuring more accurate management of the cryptococcal infection.
Assuntos
Antifúngicos/farmacologia , Criptococose/microbiologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/genética , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Brasil , Criança , Cryptococcus gattii/patogenicidade , Cryptococcus neoformans/patogenicidade , Farmacorresistência Fúngica , Feminino , Humanos , Larva , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem Molecular , Mariposas , Técnicas de Tipagem Micológica , Polimorfismo de Fragmento de Restrição , Estudos Retrospectivos , Virulência , Adulto JovemRESUMO
In nature, microorganisms often exhibit competitive behavior for nutrients and limited space, allowing them to alter the virulence determinants of pathogens. The human pathogenic yeast Cryptococcus neoformans can be found organized in biofilms, a complex community composed of an extracellular matrix which confers protection against predation. The aim of this study was to evaluate and characterize antagonistic interactions between two cohabiting microorganisms: C. neoformans and the bacteria Serratia marcescens. The interaction of S. marcescens with C. neoformans expressed a negative effect on biofilm formation, polysaccharide capsule, production of urease, and melanization of the yeast. These findings evidence that competition in mixed communities can result in dominance by one species, with direct impact on the physiological modulation of virulence determinants. Such an approach is key for understating the response of communities to the presence of competitors and, ultimately, rationally designing communities to prevent and treat certain diseases.
Assuntos
Biofilmes , Cryptococcus neoformans , Interações Microbianas , Serratia marcescens , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/fisiologia , Interações Microbianas/fisiologia , Serratia marcescens/patogenicidade , Serratia marcescens/fisiologia , Fatores de Virulência/metabolismoRESUMO
Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.
Assuntos
Criptococose/genética , Cryptococcus neoformans/genética , Ergosterol/biossíntese , Metiltransferases/genética , Antifúngicos/farmacologia , Azóis/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Retículo Endoplasmático/efeitos dos fármacos , Ergosterol/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação/efeitos dos fármacos , Virulência/genéticaRESUMO
Cryptococcus is an important fungal pathogen worldwide, causing serious clinical manifestations that can affect immunocompetent patients and can be particularly severe for immunocompromised patients. The Cryptococcus gattii s.s. (AFLP4/VGI), Cryptococcus tetragattii (AFLP/VGIV), Cryptococcus neoformans, and Cryptococcus deneoformans have been isolated from both clinical and environmental sources in Europe. We aim to quantify the people in Europe and the entire Mediterranean area who are under risk associated with each of the three fungal pathogens in a spatially explicit way, generating a series of maps and population statistics per country. Niche modeling was applied to estimate the potential distribution of each fungal pathogen, then these models were overlapped with a map of population density to estimate risk levels. The potential number of people per risk level and per country was quantified using a map of population count per pixel. Prevalence of HIV per country was also included in the analysis to quantify the HIV-infected population under potential risk. People under risk associated with exposure to C. gattii species (C. gattii s.s. and C. tetragattii) reached 137.65 million, whereas those exposed to C. neoformans and C. deneoformans were 268.58 and 360.78 million people, respectively. More than a half million HIV-infected patients are exposed to each of the two species of the C. neoformans species complex, and more than 200,000 to the C. gattii species complex. The present results can be useful for public health planning by European governments, focusing on the provision of inputs for a "screen-and-treat" approach, availability of medical resources, and continuous monitoring programs in risk zones.