Dissertations / Theses on the topic 'Cryptococcus neoformans'

This thesis examines C. neoformans and the meningitis it causes, based largely in Papua New Guinea (PNG). Investigation of possible environmental associations in PNG revealed that few E. camaldulensis survive experimental planting, while E. tereticornis is endemic. In Port Moresby E. confertiflora, E. papuana and E. alba are common. Examination of 1130 specimens from plant, bird and animal sources, failed to identify the ecological niche of C. neoformans in PNG. Epidemiological study of 96 patients presenting to Port Moresby General Hospital with cryptococcal meningitis from 1972-1993 showed an annual incidence of 33 cases per million population of Central Province and the National Capital District. 21 of these were infected with var. gattii and 12 with var. neoformans. On average 11 cases of cryptococcal meningitis present here annually. In this study geographical clustering, male predominance and possible seasonal variation were found. Eleven patients with cryptococcal meningitis were prospectively diagnosed and isolates biotyped. Seven isolates were var. gattii (one patient with diabetes mellitus) and four were var. neoformans. The latter came from adult patient with HVI infection, tuberculosis or Plasmodium vivax malaria. Five patients (45.5%) died; the 2 var. neoformans HIVI infected men and 3 adult var. gattii patients. In PNG, where var. gattii has been predominant in the immunocompetent, var. neoformans is emerging amongst immunosuppressed patients, notably those with HIVI infection. These studies confirm the high prevalence in PNG of meningitis caused by C. neoformans var. gattii in immunocompetent individuals. Potential mammal and plant sources are similar to those found in Australia.

The opportunistic fungal pathogen Cryptococcus neoformans has been previously shown to use heme as a sole iron source, but the mechanisms for heme utilization are unknown. The goal of this study was to begin a genetic analysis of heme utilization in C. neoformans by deletion of candidate genes and phenotypic characterization. The first hypothesis was that a putative heme oxygenase protein, Hmx1, was responsible for degrading heme to release iron. However, an hmx1 deletion strain was capable of growth on heme, indicating that the gene is not required for heme utilization. The expression pattern of HMX1 showed down-regulation in the presence of heme and hemoglobin indicating that HMX1 likely plays a regulatory role within the cell. Because loss of the heme-related gene HMX1 did not reveal any phenotypes related to heme as an iron source, the role of the vacuolar protein Vps41 in iron and heme utilization was also examined. The work on Vps41 was designed to test a second more general hypothesis that the vacuole is involved in heme or iron storage and utilization. It was found that vps41 mutants had heme and iron growth defects, as well as increased sensitivity to excess levels of both heme and inorganic iron. Analysis of the wild-type strain grown with heme led to the surprising discovery of dark intracellular aggregates that were visible with light microscopy. These aggregates were reminiscent of the crystallized heme (hemozoin) found in malaria parasites. In contrast, the cells of vps41 mutants became filled with diffuse heme throughout the cell, indicating that an intact vacuole was required for aggregate formation. The inability of the mutant to sequester heme in the aggregates may contribute to the observed sensitivity of the strain to heme toxicity. Overall, these results provide new insights into heme utilization and storage in C. neoformans.

La cryptococcose est une infection opportuniste causée par la levure ubiquitaire Cryptococcus neoformans. L'interaction avec les macrophages est importante pour le développement de la maladie chez l'homme. La physiopathologie évolue en trois étapes: (i) primo-infection, (ii) dormance, (iii) réactivation en cas d'immunodépression cellulaire. La présentation clinique et l'évolution de la cryptococcose sont variables en fonction des individus. L'objectif de mon travail était de comprendre l'impact de la diversité de la levure sur l'évolution de l'infection. Nous avons étudiés (i) les souches cliniques et leur diversité d'interaction avec les macrophages; (ii) la corrélation entre le phénotype in vitro et l'évolution clinique de l'infection chez l'homme; (iii) la diversité d'adaptation à l'hôte. Ainsi, nous avons développés de nouveaux outils (cytométrie de flux quantitative, microscopie en temps réel, single-cell quantitative PCR) pour permettre l'analyse de populations de levures rares. Nous avons trouvés une grande variabilité d'interaction de 54 isolats cliniques avec le macrophage. L'échec mycologique et la mortalité chez l'homme était associés à des phénotypes d'interaction particuliers. A partir de l'analyse de 9 isolats cliniques, nous avons pu mettre en évidence que la virulence des isolats et l'expression de certains gènes de virulence connus étaient variables également. En nous focalisant sur la réponse au stress et la prolifération, nous avons observé l'apparition de différentes populations au cours de l'infection. Après tri des différentes populations, nous avons identifié une population ayant une faible réponse au stress et montré qu'elle était potentiellement dormante. Ces données suggèrent que la cryptococcose et les infections fongiques en générale sont des infections complexes résultants d'une grande diversité, plasticité et adaptation des champignons à l'hôte
Cryptococcosis is an opportunistic infection due to the ubiquitous yeast Cryptococcus neoformans. This pathogen is a facultative intracellular organism. Interaction with immune cells including monocytes/macrophages lineage and dendritic cells are of major importance in the natural history of the infection. In humans, the pathophysiology of the infection evolves in three steps (i) primoinfection in childhood, (ii) dormancy, demonstrated from epidemiological and genotypic data in the lab few years ago (Garcia-Hermoso et al. 1999), (iii) reactivation upon immunosuppression. In terms of disease, clinical presentation and outcome of cryptococcosis are known to be diverse among individuals even those sharing the same underlying diseases. To address the question of the impact of fungal diversity on the natural course of the infection at the macrophage-level (standardized model of yeasts/ j774 macrophages in vitro interaction), murine model-level (murine model of cryptococcosis in OF1 outbred mice) and human-level (CryptoA/D database), we studied (i) the diversity of C. Neoformans/macrophages interactions using well characterized clinical isolates (ii) the correlation between in vitro phenotype of the isolate and clinical outcome in humans (iii) the diversity of adaptation to the host. We developed new assays and new tools using flow cytometry I (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), gene expression analysis (single-cell quantitative real time PCR) to overcome technical issues. We found high variation in phagocytic, 2 hours-, 48hours-intracellular proliferation indexes among the 54 ClinCn compared to H99. No correlation with the genotype was observed. The lack of sterilization at week 2 despite active antifungal therapy was significantly associated with a lower phagocytic index, whereas treatment failure at month 3 and death from cryptococcosis were significantly related to a higher 2 hours-intracellular proliferation. Among 9 selected clinical isolates compared to H99, (i) the virulence in mice was significantly different, intracellular expression of some virulence factors correlated with (ii) intracellular proliferation and (iii) phagocytic indexes. With a focus on multiplication and stress response and considering the H99 reference strain, we observed the appearance of various populations of yeasts during mice and macrophage infections. After sorting yeasts populations, we observed that a specific one was less prone and dependent of serum to grow compared to the other p'opulations. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. We found a high diversity of C. Neoformans upon interaction with macrophages considering 54 clinical isolates in correlation with clinical outcome in humans, but also a considerable adaptation to host in our two models considering the reference strains H99. We observed also even more diversity of fungal adaptation to host when clinical isolates were considered. Ail together, these data suggest that cryptococcosis and fungal disease in general could be more complex diseases since diversity, plasticity and adaptation of the fungal organism to hosts is high and heterogeneous

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2013.
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O advento de pacientes portadores do HIV e de pacientes imunocomprometidos provocou um aumento na incidência de micoses fúngicas, como a criptococcose. Existem várias classes de antifúngicos disponíveis comercialmente para o tratamento dessas infecções que são utilizadas de acordo com a patologia e o patógeno. No entanto, são relatados casos de resistência a essas drogas e existe uma grande preocupação em relação aos efeitos adversos que elas podem causar nos pacientes. Nesse contexto, o desenvolvimento de novas drogas antifúngicas poderia ser uma solução para a problemática atual das drogas disponíveis e isso requer o estudo de novos alvos moleculares. O gene ERG6 codifica a enzima esterol C-24 metiltransferase que atua na conversão de zimosterol em fecosterol. Constitui uma etapa que ocorre em fungos para a biossíntese do ergosterol, mas não ocorre nos hospedeiros animais, que possuem enzimas específicas para a conversão do zimosterol até colesterol. No presente trabalho, a função do gene ERG6 foi investigada e caracterizada no fungo Cryptococcus neoformans demonstrando que a ausência desse gene gera diversas alterações fenotípicas, tais como sensibilidade ao estresse osmótico, ao estresse oxidativo e maior susceptibilidade a diferentes drogas antifúngicas. Além disso, foi ainda observado que a capacidade de crescer em meios contento diferentes estressores de parede se mostra alterada. Em relação aos fatores de virulência conhecidos para C. neoformans, o fungo foi incapaz de crescer a temperatura de 37°C, porém não teve sua produção de cápsula ou melanina afetadas. No entanto, em testes para observação da sua capacidade de virulência in vitro utilizando macrófagos e in vivo com lagartas da espécie Galleria mellonella mostraram uma redução na virulência em mutantes de ERG6. Por fim a análise dos esteróis de membrana mostrou que ocorre uma alteração em toda a composição de esteróis da membrana celular. Dessa forma, por ser uma enzima encontrada principalmente em fungos, a Erg6 pode ser um alvo molecular potencial para drogas antifúngicas. ______________________________________________________________________________ ABSTRACT
The advent of HIV patients and immunocompromised patients caused an increase in the incidence of fungal mycoses, such as cryptococcosis. There are several antifungal agents commercially available for the treatment of such infections that are chosen based on the disease and the pathogen. However, cases of resistance are reported to these drugs and there is great concern about the adverse effects that they can cause on patients. In this context, the development of new antifungal drugs could be a solution to the current problem of the available drugs and it requires the study of potential molecular targets. The gene ERG6 encodes the sterol C-24 methyltransferase, an enzyme that acts on the conversion zimosterol in fecosterol. This is a step that occurs in fungi in the ergosterol biosynthesis, but does not occur in the animal hosts which have specific enzymes for the conversion of zymosterol to cholesterol. In this study, gene function of ERG6 was investigated and characterized in the Cryptococcus neoformans demonstrating the absence of this gene generates several phenotypic changes, such as sensitivity to osmotic stress, oxidative stress and increased susceptibility to different antifungal drugs. Furthermore, it was also observed that the ability to grow on media with different cell wall stressors was also altered. It was observed that the lack of ERG6 greatly affects the permeabillity of the membrane resulting in osmotic and oxidative stress sensitivity and changing antifungal drugs susceptibility. Furthermore, it was also observed that the ability to grow in medium with cell wall stressor was altered. About the virulence factor, C. neoformans was unable to grow at 37°C, but it had not affected the production of melanin or capsule. However, virulence tests in vitro with macrophages and in vivo with Galleria mellonella caterpillars showed a decrease in the virulence of ERG6 mutants. Finally, the membrane sterols analysis demonstrated a change in the membrane sterol composition. Thus, being an enzyme found especially fungi, the Erg6 may be a potential molecular target for antifungal drugs.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ceilândia, Programa de Pós-Graduação em Ciências e Tecnologias em Saúde, 2014.
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As proteínas Velvet compreendem quatro membros altamente conservados entre ascomicetos e basidiomicetos, os quais compartilham o domínio Velvet. Eles regulam diferentes vias de sinalização em resposta a estímulos ambientais e também coordenam o metabolismo secundário e diferenciação assexual e sexual em diferentes espécies de fungos. Recentemente, as proteínas Velvet foram implicadas na virulência de alguns agentes patogênicos de plantas. Buscou-se avaliar o papel das proteínas Velvet no fungo patogênico humano Cryptococcus neoformans, causador da criptococose, importante micose sistêmica e a terceira doença mais prevalente em indivíduos portadores do vírus HIV. Entre as micoses sistêmicas, ela é classificada como tendo a maior incidência em pacientes imunocomprometidos. A infecção por C.neoformans ocorre pela inalação de células infecciosas e é considerada uma infecção pulmonar primária, o que pode levar a uma infecção disseminada e meningoencefalite. O objetivo geral do trabalho foi avaliar o papel do gene VELB na patogenidade e morfogênese de C. neoformans. O primeiro passo realizado no estudo foi a deleção do gene VELB em Cryptococcus neoformans. O cassete foi transformado em células leveduriformes haploides de C. neoformans diretamente através de biobalística. Confirmou-se a deleção por PCR e Southern Blot. Após confirmação, os transformantes foram submetidos a diferentes condições de crescimento para determinação de seus fenótipos e virulência. Foram realizados testes de estresse osmótico (NaCl), estresse na parede celular (CongoRed, SDS, cafeína) e estresse oxidativo com peróxido de hidrogênio. Foram avaliados os fatores de virulência de C. neoformans como fosfolipase, urease, melanina e cápsula. Os testes fenotípicos não apresentaram alterações entre a linhagem e mutante indicando que o gene VELB parece não ter nenhum envolvimento em virulência e resposta a agentes estressores. Para avaliação da capacidade de acasalamento foram realizados co-cultivos no escuro em diferentes meios de cultura (Filament, SLAD e MS), além disto foi testada a capacidade de fusão dos mutantes. Estes testes demonstraram que a deleção de VELB bloqueia a produção de hifas no acasalamento entre mutantes e que estes apresentam também defeito no processo de fusão celular. Estes resultados gerados neste trabalho sobre o papel de VELB corroboram os dados anteriormente obtidos pelo nosso grupo de pesquisa de que os membros da família velvet estão diretamente envolvidos na regulação do ciclo sexual de C. neoformans. _________________________________________________________________________________ ABSTRACT
The Velvet proteins consist of four highly conserved members in ascomycetes and basidiomycetes, sharing the Velvet domain. They regulate different signaling ways in response to environmental stimulation and also coordinate secondary metabolism and asexual to sexual differentiation in different species of fungi. Recently, Velvet proteins have been implicated in the virulence of some pathogenic agents of plants. It was evaluated the role of Velvet proteins in the human pathogenic fungus Cryptococcus neoformans that causes cryptococcosis, an important systemic mycosis and the third most prevalent disease in HIV positive individuals. Compared to other systemic mycoses, it is classified as having the highest incidence in immunocompromised patients. C.neoformans infection is by inhalation of infectious cells and is considered a primary pulmonary infection, which can lead to widespread infection and meningoencephalitis. The overall objective of this study was to evaluate the role of the VELB gene in morphogenesis and pathogenicity of C. neoformans. The first step was performed at study deletion of the gene VELB in Cryptococcus neoformans. The cassette was transformed into haploid yeast cells of C. neoformans directly through biolistic. The deletion was confirmed by PCR and Southern blot. After confirmation, the transformants were submitted to different growth conditions to determine their phenotypes and virulence. Tests of osmotic stress (KCl, NaCl, sorbitol), stress in the cell wall (Congo Red, Calcofluor, SDS, caffeine) and oxidative stress with hydrogen peroxide were performed. Virulence factors of C. neoformans as phospholipase, urease, melanin and capsule were evaluated. Phenotypic tests showed no change between the strain and mutant indicating that the VELB gene seems to have no involvement in virulence and response to stressors. To evaluate the ability of mating co-cultures were performed in the dark in different culture media (Filament, SLAD and MS), in addition it was tested the ability of fusion of mutants. These tests showed that the deletion of VELB blocks the production of hyphae in mating and that these mutants also exhibit defective cell fusion process. These results generated in this paper about the VELB function corroborate data previously 13 obtained by our research group that the members of the velvet family are directly involved in the regulation of the sexual cycle of C. neoformans.

Cryptococcus neoformans e Cryptococcus gattii são leveduras encapsuladas e agentes etiológicos da criptococose. Estes microrganismos são patogénos intracelulares facultativos os quais interagem com macrófagos do hospedeiro durante o processo de infecção. C. neoformans é capaz de se replicar no interior de células fagocíticas utilizando uma estratégia única que envolve permeabilização do fagolisossoma e produção de polissacarídeos. No presente trabalho, a metodolologia da Análise da Diferença Representacional (RDA) foi aplicada para identificar genes diferencialmente expressos por C. neoformans e C. gattii durante o desenvolvimento intracelular em macrófagos peritoneais de ratos. C. neoformans internalizados por macrófagos expressaram genes relacionados ao tráfego vesicular, transporte de membrana, atividade de chaperona, regulação da meiose, metabolismo de monossacarídeo e resposta ao estresse. Em C. gattii, foram induzidos transcritos relacionados à respiração aeróbica, endocitose, tráfego vesicular, metabolismo de monossacarídeo e nitrogênio, sinalização celular e resposta ao estresse. Nossos resultados revelaram novos genes envolvidos no parasitismo intracelular de C. neoformans e C. gattii.

Cryptococcus neoformans and C. gattii are pathogenic basidiomycetous yeasts and the commonest cause of fungal infection of the central nervous system. Cryptococci are typically haploid but several hybrids belonging to different sero/mating type allelic patterns have been reported. In this study seven unisexual mating intra-varietal VNI/VNII (αAAα), six inter-varietal VNII/VNIV (aADα), and four inter-species comprising of one VNI/VGI (αABa) and three VNII/VGII (αABa) novel hybrid isolates were identified within the C. neoformans/C. gattii species complex using conventional and molecular techniques. This study demonstrated that protein profiles using MALDI-TOF can identify and differentiate hybrids. The minimal inhibitory concentrations (MICs) of nine drugs was measured by micro-broth dilution and compared with published data on haploid strains. MICs were similar amongst hybrids and haploid parental strains. All strains similarly expressed the major virulence factors – capsule, melanin and phospholipase B – and grew well at 37°C. Virulence of hybrids was investigated using the Galleria mellonella (wax moth) model. Results showed that hybrids are virulent in an experimental model, except for the VNII/VNIV (aADα) hybrids. Inter-species VNI/VGII hybrids are more virulent than the rest of the hybrids. Hybrids recovered from larvae manifested a significant increase in capsule and total cell size and produced a low proportion (5-10%) of giant cells. Hybrid strains showed similar phenotypic characteristics with regard to the virulence traits and antifungal susceptibility despite their different genotype. This study provided insights in the overall population structure of the C. neoformans/C. gattii species complex and showed that hybrid strains comprise an important component of this important human/animal pathogen.

Cell surface properties of the basidiomycete yeast Cryptococcus neoformans were investigated with a combination of novel and well proven approaches. Non-specific cell adhesion forces, as well as exposed carbohydrate and protein moieties potentially associated with specific cellular interaction, were analysed. Experimentation and analysis employed cryptococcal cells of different strains, capsular status and culture age. Investigation of cellular charge by particulate microelectrophoresis revealed encapsulated yeast forms of C. neoformans manifest a distinctive negative charge regardless of the age of cells involved; in turn, the neutral charge of acapsulate yeasts confirmed that the polysaccharide capsule, and not the cell wall, was responsible for this occurrence. Hydrophobicity was measured by MATH and HICH techniques, as well as by the attachment of polystyrene microspheres. All three techniques, where applicable, found C. neoformans yeast to be consistently hydrophilic; this state varied little regardless of strain and culture age. Cell surface carbohydrates and protein were investigated with novel fluorescent tagging protocols, flow cytometry and confocal microscopy. Cell surface carbohydrate was identified by controlled oxidation in association with biotin hydrazide and fluorescein-streptavidin tagging. Marked amounts of carbohydrate were measured and observed on the cell wall surface of cryptococcal yeasts. Furthermore, tagging of carbohydrates with selective fluorescent lectins supported the identification, measurement and observation of substantial amounts of mannose, glucose and N-acetyl-glucosamine. Cryptococcal cell surface protein was identified using sulfo-NHS-biotin with fluorescein-streptavidin, and then readily quantified by flow cytometry.

A criptococose é uma doença fúngica invasiva causada majoritariamente pelas espécies patogênicas Cryptococcus neoformans e Cryptococcus gattii. Estudos epidemiológicos recentes sugerem que a infecção causada por C. gattii desenvolve mais frequentemente criptococomas pulmonares, enquanto que C. neoformans estabelece principalmente quadros de meningoencefalite. Embora ocorra essa associação, o mecanismo de disseminação dessas espécies não está completamente elucidado, instigando a utilização de diferentes abordagens na caracterização da infecção. O desenvolvimento de microrganismos bioluminescentes tem permitido o monitoramento em tempo real da infecção em modelos animais. No presente trabalho, diferentes estratégias foram utilizadas para a construção de linhagens de C. neoformans e de C. gattii expressando o gene repórter luciferase Renilla, sem sucesso. Paralelamente, em uma segunda abordagem, bactérias bioluminescentes (BL) foram isoladas de amostras de água marinha coletadas nas adjacências da zona estuarina do rio Tramandaí, com o objetivo de selecionar o cassete lux (CDABE) para a utilização como gene repórter. Todos os isolados apresentaram luminescência a 28°C, mas, quando incubados a 37°C, somente o isolado BL6 permaneceu com uma atividade luminescente consideravelmente reduzida. Portanto as bactérias bioluminescentes pertencentes aos gêneros Vibrio, Photobacterium e Enterovibrio isoladas do rio Tramandaí não apresentaram níveis de emissão de luminescência adequados à temperatura fisiológica humana para a utilização do cassete lux como gene repórter para monitoramento de infecções in vivo.
Cryptococcosis is an invasive fungal disease caused mainly by pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Recent epidemiological studies have suggested that infection with C. gattii develops more frequently pulmonary cryptococcomas, while C. neoformans establishes mainly neurological damages. Although exists this association, the mechanism of dissemination of these species is not completely understood, instigating the use of different approaches for the characterization of the infections. Alternatively, the development of bioluminescent organisms has allowed the real-time monitoring of infection in animal models. In this study, different strategies were used to construct strains of C. neoformans and C. gattii expressing Renilla luciferase reporter gene without success. In a second approach, bioluminescent bacteria (BL) were isolated from seawater samples collected in the vicinity of the river Tramandaí estuarine zone, in order to select the lux cassette (CDABE) for use as a reporter gene. All isolates showed luminescence at 28°C, but when incubated at 37°C, only the isolate BL6 remained with a considerably reduced luminescent activity. Thus, bioluminescent bacteria belonging to the genera Vibrio, Photobacterium and Enterovibrio isolated from Tramandaí river estuary zone presented unsuitable luminescence emission levels at human physiological temperature, preventing the use of lux cassette as a reporter gene in experiments to monitoring cryptococcosis infection.

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A criptococose é uma infecção fúngica causada por Cryptococcus neoformans, que possui tropismo pelo sistema nervoso central. O propósito deste estudo foi diagnosticar criptococose, identificar variedades de C. neoformans e avaliar a suscetibilidade a anfotericina B, ciclopirox olamina e β-lapachona em amostras da Micoteca URM-UFPE e recém isoladas de Líquor de imunocomprometidos de ambos os sexos atendidos em hospitais de Recife-PE, Brasil. Para o diagnóstico de criptococose, exame direto foi realizado com tinta da China e o semeio em ágar Sabouraud. As variedades foram detectadas com meio L-Canavanina-azul bromotimol. A concentração inibitória seguiu o Clinical and Laboratory Standards Institute. Dezenove casos de criptococose foram diagnosticados com obtenção de 16 culturas. Da Micoteca, foram obtidas 14 amostras de C. neoformans. O exame direto mostrou leveduras capsuladas. C. neoformans var. neoformans ocorreu em 50% dos casos. O CIM (μg/mL) desta variedade foi 0,25 - 4 (anfotericina B), 0,125 - 1 (ciclopirox olamina), 4 e 8 (β-lapachona). Os isolados de C. gattii apresentaram-se 0,25 - 4 (anfotericina B), 0,25-1 (ciclopirox olamina), 4 e 8 (β-lapachona). A concentração fungicida da β-lapachona foi 64 μg/mL. Criptococose predominou no sexo masculino com faixa etária de 25-46 anos e apresentando AIDS como fator de risco predominante. Detectaram-se 4 isolados resistentes a anfotericina B (3 isolados de C. gattii e 1 isolado de C. neoformans var. neoformans). A eficácia do ciclopirox olamina e da β-lapachona, comparada a resistência a anfotericina B, mostra a importância da susceptibilidade associados à variedade para monitorar desenvolvimento de resistência possibilitando descoberta de novas alternativas terapêuticas para criptococose.

Cryptococcus neoformans é uma levedura basidiomicética e patógeno humano oportunista, que causa infecção tanto em indivíduos imunocomprometidos quanto em imunocompetentes. A habilidade de sobrevivência e proliferação na temperatura do corpo humano é um fator de virulência essencial deste microrganismo. Análise da Diferença Representacional (RDA) foi aplicada com o intuito de delinear o perfil de expressão gênica durante desenvolvimento de C. neoformans a 37ºC ou 25ºC. Análises de 300 seqüências de cDNA permitiram a identificação de proteínas que podem ser críticas para processos de interação patógeno-hospedeiro. Produtos gênicos envolvidos em integridade da parede celular, resposta ao estresse, filamentação e metabolismo oxidativo são induzidos a 37ºC. Genes relacionados à silenciamento de cromatina e transporte de fosfolipídios possuem maior expressão em cultivo de C. neoformans a 25ºC. A metodologia de RDA mostrou-se viável no intuito de identificar genes regulados pela temperatura neste organismo, os quais podem representar alvos potenciais para estudos de inibição do desenvolvimento de C. neoformans no hospedeiro.

A expressão de determinantes de virulência por um patógeno é amplamente controlada pelo ambiente encontrado no hospedeiro. Neste contexto, a descrição de genes essenciais para o processo de interação patógeno-hospedeiro é fundamental para o melhor entendimento dos mecanismos de virulência utilizados durante a infecção. Cryptococcus neoformans é uma levedura encapsulada que causa meningite em pacientes imunocomprometidos. A pandemia de AIDS contribuiu significativamente para o aumento das pesquisas científicas relacionadas à biologia de C. neoformans. Estudos funcionais de três genes deste patógeno, VCX1, GAT1, e GRASP, envolvidos, respectivamente, no transporte de cálcio (Ca2+), no metabolismo de nitrogênio e no processo de secreção não convencional são apresentados e discutidos na presente Tese. A habilidade de desevolvimento a 37°C é um fator de virulência essencial de C. neoformans, o qual é controlado por uma via de sinalização regulada por Ca2+ e calcineurina. O gene VCX1 de C. neoformans codifica um transportador de cálcio vacuolar, requerido para o desenvolvimento intracelular em macrófagos e para a virulência em camundongos. O fator de transcrição Gat1 de C. neoformans regula a expressão de genes sensíveis ao processo de repressão catabólica por nitrogênio (NCR). Genes envolvidos na biossíntese de ergosterol, metabolismo de ferro, integridade da parede celular e síntese da cápsula também são regulados por Gat1. Entretanto, este fator de transcrição não é necessário para a sobrevivência durante a interação com macrófagos e para a virulência em modelo de infecção experimental. Uma estratégia de virulência essencial de C. neoformans é a secreção de glicuronoxilomanana (GXM), um polissacarídeo capsular com propriedades imuno-modulatórias. Nossos resultados demonstram que GRASP (golgi reassembly and stacking protein) está envolvida na secreção de GXM, formação da cápsula e virulência em C. neoformans. É importante ressaltar que o transportador de cálcio Vcx1 e o fator de transcrição Gat1 também participam do processo de secreção deste polissacarídeo, o que representa a sua complexidade em C. neoformans.
The expression of pathogen virulence determinants is highly controlled by the host milieu. In this context, the description of crucial genes for hostpathogen interaction is fundamental to better understand the virulence mechanisms utilized during infection. Cryptococcus neoformans is an encapsulated yeast, that causes a life-threatening meningoencephalitis in immunocompromised individuals. The AIDS pandemic has contributed significantly to the increase of scientific research concerning the C. neoformans biology. Functional analyses of three genes, VCX1, GAT1, and GRASP, related to calcium (Ca2+) transport, nitrogen metabolism and unconventional secretion in C. neoformans, respectively, are presented and discussed in this thesis. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this pathogen. This trait is controlled in part by the Ca2+- calcineurin pathway, which senses and utilizes cytosolic calcium for signaling. C. neoformans VCX1 gene encodes a vacuolar calcium exchanger, which is required for intracellular growth in macrophages and for full virulence in mice. The C. neoformans transcription factor Gat1 regulates the expression of Nitrogen Catabolite Repression (NCR) sensitive genes. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis are also Gat1-regulated in C. neoformans. However, Gat1 is not required for survival during macrophage infection and for virulence in a mice model of cryptococcosis. One essential virulence strategy of C. neoformans is the release of glucuronoxylomannan (GXM), a capsular immune-modulatory polysaccharide. Our results demonstrate that GRASP, a golgi reassembly and stacking protein, is involved in GXM secretion, capsule formation and virulence in C. neoformans. Interestingly, the vacuolar calcium exchanger Vcx1 and the transcription factor Gat1 are also involved in GXM secretion, which represents the complexity of this important process in C. neoformans.

The enzyme phospholipase Bl (PLBl), secreted by the pathogenic fungus Cryptococcus neoformans, has an established role in virulence. Although the three PLBl enzyme activities, phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase transacylase (LPTA) have been well characterized, nothing is known of the amino acid residues involved in, or the mechanism of, PLBl catalysis. In a previous study, it was demonstrated that treatment of PLBl with the N—glycosidase PNGase F abolished almost all enzyme activity, suggesting the N-linked glycans are important for enzyme activity. Therefore, in this study, the importance of individual N-linked glycosylation sites in catalysis of PLBl was investigated. Three sites, namely Asn56, Asn430 and Asn550 were altered to alanine by site-directed mutagenesis in both wild-type PLBl (full length) and a glycosylphosphatidylinositol (GPI) anchorless version (PLBIGPI') that is hypersecreted due to lack of membrane association. Each mutated PLBI cDNA was expressed heterologously in Saccharomyces cerevisiae. Alteration of Asn56 and Asn550 in both PLBl and PLBIGPI‘ abolished enzyme activity and secretion. Mass spectrometry (MS) analysis confirmed that both of these sites are occupied by a sugar moiety. Alteration of Asn430 reduced activity and secretion by approximately 50-70%, demonstrating that this site is also important but not as critical as the other two sites. Reduced secretion coincided with reduced enzyme in membranes and cell walls, confirming a reduction in the rate of PLBl transport to the cell surface. Thus, it was concluded that individual N—linked glycans are required for optimal transport of PLBl to the cell surface and optimal secretion of active PLBl and PLBIGPI'. This work also revealed that altering carbohydrate attachment sites as a means of improving crystallization efficiency for structural studies is not an appropriate strategy as N—linked glycans are essential for secretion of active enzyme. Previous evidence has indicated the possibility of two separate active sites to account for the three enzyme activities of PLBl, therefore, the structure of the catalytic centre was investigated. Examination of the protein sequence reveals the presence of lipase, subtilisin protease aspartate and phospholipase motifs containing the putative catalytic residues Serl46, Asp392 and Arg108, respectively. These amino acids are conserved in almost all fungal PLB enzymes and are essential for human cytosolic phospholipase A2 (cPLAz) catalysis. To determine the role of these residues in catalysis of cryptococcal PLBl, each was substituted with alanine and the altered cDNAs expressed in S. cerevisiae. All three altered PLB enzymes were deficient in all three activities in both the cell-associated and secreted fractions, and exhibited altered trafficking; PLB1(Sl46A) alone was transported to the plasma membrane but not to the cell wall or culture supernatant. Using a three-dimensional model of the PLBl active site, based on the X-ray structure of cPLAz, it was revealed that the catalytic residues are located in positions identical with those of cPLAz. It was concluded that the Arg108, Serl46 and Asp392 are part of a single active site, with Serl46 and Asp392 forming a catalytic dyad. Additionally, similar to the residues involved in the attachment of N—linked sugars, these residues at the catalytic centre were found to also have a role in PLBl secretion. In this thesis, unique structural information for a PLB enzyme from a pathogenic fungus is presented, and this may lead to the development of better antifungal strategies.

Nos últimos anos tem ocorrido um aumento de infecções fúngicas, devido à elevação do número de pacientes imunossuprimidos, principalmente pelo surgimento da epidemia da AIDS (Acquired Immunodeficiency Syndrome). O uso aumentado de antifúngicos tem levado ao surgimento de cepas resistentes a estes agentes, já verificado com o azólico fluconazol, o agente antifúngico mais utilizado na terapia de manutenção de pacientes com AIDS, que desenvolvem criptococose. Neste trabalho, foram determinadas as Concentrações Inibitórias Mínimas (CIMs) de fluconazol para isolados clínicos de Cryptococcus neoformans e Cryptococcus gattii, utilizando-se o teste de microdiluição em caldo padronizado pelo National Commitee for Clinical Laboratory Standards (NCCLS), o método padrão para testar susceptibilidade antifúngica de leveduras. Foram testados 173 isolados clínicos de C. neoformans e C. gattii, sendo que as CIMs para fluconazol variaram de 0,125 a 16 μg/mL, com uma CIM50% de 2 e uma CIM90% de 4 μg/mL. Todos os isolados foram considerados sensíveis a fluconazol. Também foi realizado um teste de indução de resistência a fluconazol, por exposição de um isolado sensível, a concentrações crescentes da droga, para verificar se apenas a exposição, levaria ao desenvolvimento de resistência. No entanto, verificou-se apenas uma resistência transitória, em uma das subpopulações, sendo que o fenótipo de sensibilidade foi restaurado. Os dados obtidos neste trabalho estão em concordância com estudos nacionais e internacionais, que também utilizaram o teste padrão de microdiluição em caldo. A resistência entre isolados clínicos de C. neoformans e C. gattii ainda tem se mostrado rara em termos mundiais comparada a resistência adquirida por outras leveduras. No entanto, devido aos relatos de desenvolvimento de resistência durante a terapia de manutenção da criptococose, é importante alertar quanto à possibilidade do surgimento e da propagação de cepas resistentes.
In the last years have occurred an increase of fungal infections due to rise of number of immunocompromised patients, particularly by appearance of the Acquired Immunodeficiency Syndrome (AIDS). The incresing use of antifungal agents has determined the appearance of resistants strains to this agents, already verified with the fluconazole azole, the antifungal agent more used in the maintenance therapy of AIDS patients that developing criptococosis. In this work were determined the Minimuns Inibitories Concentrations (MICs) of fluconazole to Cryptococcus neoformans and Cryptococcus gattii clinical isolates, using the National Commitee for Clinical Laboratory Standards (NCCLS) broth microdilution method. This is the test standard to test antifungal susceptibility to yeasts. Were tested 173 clinical isolates of C. neoformans and C. gattii. The MICs to fluconazole ranged of 0.125 to 16 μg/mL and the MIC50% was 2 and MIC90% was 4μg/mL. All isolates were considered susceptibles to fluconazole. A test of fluconazole resistance inducing was carry out by exposure to drug incresing concentrations of one susceptible isolate, to verify if only the exposure can to result in the resistance development. However occurred only a temporary resistance in one of the subpopulations, since the susceptibility profile was restored. The data obtained in this work are in agreement with national and international studies that also used the standard broth microdilution method. The resistance between C. neoformans and C. gattii clinical isolates has been rare in the world when compared with others yeasts. Nonetheless due to references to resistance development during the maintenance therapy of cryptococosis, it’s important to remain alert as for possibility of appearance and spread of resistants strains.

The fungal infections are becoming an important cause of morbidity and mortality in immunocompromised patients. Here we have studied the activity of new antifungal agents as ravuconazole, albaconazole, micafungin as well as fluconazole tpathogenic yeasts as Candida albicans, Candida dubliniensis, Candida tropicalis, Candida lusitaniae, Candida glabrata and Candida krusei. We have also included clinical and environmental isolates of Cryptococcus neoformans. The susceptibilities tests indicated that the majority of the strains were sensible to the antifungal agents studied but Candida albicans (6.6%) and C.tropicalis (3.3%) were classified as resistant to fluconazole. These results show the importance of monitoring of the susceptibility in yeast like fungi from clinical source and emphasize that new and constant susceptibility studies deserve be applied to Candida and Cryptococcus neoformans to reduce the mortality among immunocompromised patients.
As infecções fúngicas têm-se tornado uma importante causa de morbidade e mortalidade em pacientes, principalmente os imunocomprometidos. Na tentativa de se transpor esta realidade, têm-se buscado estabelecer e avaliar novas drogas antifúngicas eficazes e com melhor tolerabilidade e segurança. O presente estudo foi objetivado a verificar a atividade dos triazólicos fluconazol, ravuconazol e albaconazol e da micafungina, frente a um banco de fungos leveduriformes incluindo as espécies C.albicans, C. dubliniensis, C. tropicalis, C. lusitaniae, C. glabrata e C. krusei, todas de origem clínica e Cryptococcus neoformans de origens clínica e ambiental. De modo geral, as espécies de Candida spp. e Cryptococcus foram sensíveis aos antifúngicos testados, com exceção da Candida albicans e Candida tropicalis frente ao fluconazol, onde 6,6% e 3,3%, respectivamente dos isolados, foram resistentes a este azólico. Estes resultados demonstram a necessidade de uma constante vigilância da suscetibilidade dos fungos e maiores pesquisas em relação às drogas antifúngicas, pois, desta forma, estaremos contribuindo para a menor morbidade e maior sobrevida dos pacientes.

As micoses estão incluídas entre as doenças infecciosas mais ubíquas em todo o mundo, afectando todos os estratos sociais e todos os grupos etários numa variedade de manifestações superficiais, cutâneas, subcutâneas e sistémicas. Um diagnóstico rápido e eficaz destas doenças, com uma correcta identificação da espécie fúngica responsável pela infecção, é essencial para um planeamento do tratamento mais eficaz para o doente infectado. Tem-se registado um aumento da incidência das infecções fúngicas também em consequência do aumento do número de casos de doentes imunodeprimidos, devido particularmente à crescente utilização de terapêuticas imunossupressivas, procedimentos médicos invasivos, prescrição de tratamentos prolongados, entre outros aspectos. O aparecimento da epidemia da Imunodeficiência Humana no início da década de 80, causada pelo vírus VIH, contribuiu também decisivamente para o aumento das infecções fúngicas oportunistas. A Criptococose é uma infecção fúngica, predominantemente oportunista e com uma distribuição epidemiológica mundial, causada por leveduras encapsuladas do género Cryptococcus. A espécie clinicamente mais relevante é Cryptococcus neoformans, cujas estirpes têm sido tradicionalmente classificadas em cinco serotipos relacionados com os antigénios da respectiva cápsula polissacárida: A (C. neoformans var. grubii); D (C. neoformans var. neoformans); B e C (actualmente reconhecidos como uma espécie distinta mas filogeneticamente próxima, C. gattii); e AD (estirpes híbridas). O presente trabalho teve como principal objectivo determinar retrospectivamente os tipos moleculares de uma colecção alargada de estirpes de C. neoformans, isoladas e mantidas durante os últimos 18 anos no Laboratório de Micologia do IHMT/UNL. A maioria das estirpes foi isolada de doentes imunodeprimidos com criptococose, existindo também algumas estirpes de origem ambiental. Foi utilizada a técnica de PCR-RFLP do gene URA5 para diferenciar as estirpes de Cryptococcus neoformans, tendo sido detectados quatro tipos moleculares: VN1 e VN2 (relacionados com C. neoformans var. grubii, serotipo A); VN3 (relacionado com as estirpes híbridas de serotipo AD); e VN4 (relacionado com C. neoformans var. neoformans, serotipo D). Não foram encontrados entre os isolados de origem clínica perfis de restrição correspondentes aos tipos moleculares VG1, VG2, VG3 e VG4 (relacionados com C. gattii, serotipos B e C). O tipo molecular VN1 foi o mais abundante entre os isolados de origem clínica (45% dos isolados), seguindo-se o grupo de 5 estirpes híbridas do tipo molecular VN3 (31%). Os tipos moleculares menos abundantes entre os isolados foram o VN2 (12%) e VN4 (12%). A estandardização do método de tipagem molecular utilizado neste trabalho permite comparar os resultados obtidos com os de outros estudos epidemiológicos semelhantes, realizados noutras regiões do globo e publicados em anos recentes, contribuindo para um conhecimento melhorado da epidemiologia global deste importante fungo patogénico. Em Portugal obteve-se uma percentagem mais elevada de isolados dos grupos moleculares VN1 e VN3 em relação a outros países da Europa e América Latina, em que os tipos mais abundantes são VN1 e VN2. Nestas mesmas regiões, o tipo molecular VN4, relacionado com as estirpes de C. neoformans var. neoformans do serotipo D, é muito raro. Esta variedade é mais comum em zonas mediterrâneas e está muito associada a casos clínicos de infecções cutâneas associadas a infecções do Sistema Nervoso Central. Este tipo molecular parece ser também significativamente mais abundante no nosso país.
Mycoses are amongst the infectious diseases that are very dangerous affecting all social status in several ways all over the world. These infectious diseases can be superficial, cutaneous, subcutaneous and invasive or systemic, spreading all over the body. A fast and efficient diagnosis of these diseases, including a correct identification of the respective etiological agents, is essential to plan the best treatment to be applied to the patients. An increase in the number of people infected by fungal infections is registered worldwide, as a consequence of an increase in the number of cases of immunocompromised patients and of immunosuppressive therapies, invasive medical procedures and prolonged prescribed treatments, among other aspects. The outcome of the human immunodeficiency epidemics at the beginning of the 80’s, caused by HIV, has also contributed to the increase of the occurrence of opportunistic mycoses. Cryptococcosis is a fungal infection caused by several species of Cryptococcus and presents a worldwide epidemiologic distribution. The most clinically relevant species is Cryptococcus neoformans, whose strains have been traditionally classified into five serotypes related with the antigens of the respective polysaccharide capsules: serotype A (C. neoformans var. grubii), D (C. neoformans var. neoformans); B and C (presently recognised as a distinct but closely related species, C. gattii); and AD (hybrid strains). The main objective of this work was to retrospectively determine the molecular types of a large group of C. neoformans strains that were isolated and maintained for the last 18 years in the Mycology lab of IHMT/UNL. Most of these strains were isolated from immunocompromised patients with Cryptococcosis. The PCR-RFLP of the URA5 gene was used to differentiate the strains. Four molecular types were detected among the isolates: VN1 and VN2 (related to C. neoformans var. grubii, serotype A); VN3 (related with hybrid strains of the serotype AD); and VN4 (related with C. neoformans var. neoformans, serotype D). Molecular types VG1, VG2, VG3 and VG4, related with C. gattii, were not found. VN1 was the most abundant molecular type among clinical isolates (45% of the isolates), followed by VN3 (31%) and VN2 and VN4, both corresponding to 12% of the clinical isolates. The standardized typing method used in this work allowed comparing our results with other recent epidemiological studies on the same subject. In Portugal we have found a greater percentage of C. neoformans molecular types VN1 and VN3 whilst in other European and Latin American countries the most abundant types were 7 VN1 and VN2. Type VN4 was also found to be more abundant in our country. This type seems to be common in Mediterranean areas and is associated to cutaneous manifestations of infections of the central nervous system.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2013.
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Cryptococcus neoformans é um fungo encapsulado patogênico humano que afeta principalmente indivíduos imunossuprimidos. Sua cápsula é um dos mais importantes fatores de virulência, e está envolvida com os processos de evasão da resposta imune e disseminação no hospedeiro. Neste trabalho foi investigado a ativação do inflamassoma por C. neoformans e o papel de sua cápsula nesse processo. Observou-se que o mutante acapsular de C. neoformans induziu elevados níveis da secreção de IL-1β e ativação da protease caspase-1. A secreção de IL-1β foi analisada em macrófagos derivados de medula deficientes para as proteínas ASC, caspase-1, NLRP3 e NLRC4, demonstrando que essa secreção depende do inflamassoma NLRP3, mas não de NLRC4. Além disso, os componentes do inflamassoma se mostraram dispensáveis para fagocitose e atividade antifúngica frente a infecção. Os mecanismos envolvidos no processamento de IL-1β foram dependentes de catepsina B frente ao dano lisossomal e sinalização via quinase Syk. Foi demonstrado também que a sinalização por IL-1β não aumenta a atividade antifúngica de macrófagos. No entanto, ela participa na restrição da taxa de infecção por mecanismos desconhecidos. Estes resultados mostram pela primeira vez que C. neoformans ativa o inflamassoma NLRP3 de forma dependente de caspase-1 através da liberação de catepsina B e sinalização via Syk, levando a produção de IL-1β e restrição da taxa de infecção. Adicionalmente, esses dados demonstram que os isolados encapsulados conseguem diminuir a ativação do inflamassoma. ______________________________________________________________________________ ABSTRACT
Cryptococcus neoformans is an encapsulated human pathogenic fungus that affects primarly immunocompromised individuals. It has a prominent capsule that is an important virulence factor, which is involved in immune response evasion and fungal dissemination. In the present study we investigated whether C. neoformans was able of triggering inflammasome activation and the role of its capsule in this event. The results showed that the acapsular mutant induced high levels of IL-1β secretion and caspase-1 activation. IL-1β -/- -/-secretion induced by C. neoformans was assessed in WT, Asc , Casp1 , -/- -/- Nlrp3 and Nlrc4 bone marrow-derived macrophages, showing IL-1β secretion to be dependent on NLRP3, but independent on NLRC4 inflammasome. In addition, inflammasome components were dispensable for C. neoformans uptake or killing. The mechanisms underlying IL-1β processing and release were dependent on cathepsin B release following lysosomal destabilization and Syk tirosine kinase signaling. We also demonstrate that IL- 1β signaling is not required to restrict intracellular yeast, but limited the rate of infection by an unknown mechanism. Together, our data show for the first time that acapsular C. neoformans activates the NLRP3 inflammasome and IL-1β secretion in a caspase-1 dependent manner and that the mechanisms involved in this event rely on cathepsin B released during lysosomal damage and Syk signaling, leading to restriction of infection rate. Furthermore, our results show that encapsulated C. neoformans is able to dimish inflammasome activation.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ceilândia, Programa de Pós-Graduação em Ciências e Tecnologias em Saúde, 2013.
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O basidiomiceto Cryptococcus neoformans, é um fungo oportunista, que comumente infecta pacientes portadores do vírus da imunodeficiência humana (HIV) e imunocomprometidos em geral. É o agente etiológico da criptococose, doença potencialmente fatal e cosmopolita cuja incidência mundial vem se aproximando da de doenças como a tuberculose. O gene VOSA pertence à família velvet, exclusiva de fungos e primeiramente descrita em Aspergillus, e está envolvido na viabilidade dos esporos e na regulação das fases assexuada e sexuada. Proteínas dessa família são fatores transcricionais altamente conservados entre ascomicetos e basidiomicetos que regulam diferentes processos em resposta a estímulos ambientais. A análise in silico do gene VOSA de C. neoformans mostrou sua conservação entre os fungos. Construiu-se um cassete de deleção do gene VOSA contendo o gene de resistência à higromicina B por PCR double joint. O cassete foi transformado por biobalística nas linhagens selvagens KN99a/α (soroA) que permitiu a obtenção de mutantes vosAaΔ e vosAαΔ. Da mesma forma, o fragmento contendo o locus VOSA foi transformado para obtenção do reconstituído tipo a (vosAaΔ::VOSA). Confirmou-se a deleção e a reconstituição de VOSA por PCR comum. Os mutantes vosAaΔ e vosAαΔ foram submetidos aos testes de fenótipo: crescimento a 30°C e 37°C, formação de cápsula, produção de urease e fosfolipase, alterações de parede celular e produção de melanina, no entanto não foram observadas quaisquer alterações no fenótipo quando comparados com a linhagem selvagem. Quanto à capacidade de realizar acasalamento, os mutantes vosAaΔ e vosAαΔ foram submetidos a acasalamento com linhagem selvagem de tipo sexual oposto e apresentaram diminuição na formação de hifas e alteração morfológica significativa de sua estrutura. Mutantes de tipo sexual opostos (vosAaΔ vs. vosAαΔ) também foram submetidos a acasalamento e não apresentaram qualquer formação de hifas. Acredita-se que VOSA seja um regulador positivo da reprodução sexuada, e que controle a formação de estruturas sexuais. A expressão de genes velvet foi analisada no mutante vosAaΔ em comparação à cepa selvagem sem indicação de qualquer alteração transcricional. Os resultados até agora obtidos em C. neoformans colaboram para um melhor entendimento do papel de VOSA na patobiologia deste patógeno. Pretende-se identificar em detalhe o papel deste gene no ciclo de vida de C. neoformans, em especial na produção de esporos, que são as formas infectivas e de disseminação deste patógeno. Os dados moleculares gerados neste trabalho servem como ponto de partida para elucidar a complexa regulação do ciclo sexual de C. neoformans e pela primeira vez descreve o envolvimento de um gene velvet na biologia de um basidiomiceto. ______________________________________________________________________________ ABSTRACT
The basidiomycete fungus Cryptococcus neoformans is an opportunistic organism which commonly infects HIV and immunocompromised patients. It is the etiological agent of cryptococcosis, a potentially fatal and cosmopolitan disease, whose world’s incidence is approaching from diseases such as tuberculosis. VOSA gene belongs to the velvet family, fungal exclusive and primarily described in Aspergillus, involved in the regulation of sexual and asexual development as well as in the viability of the spores. Proteins of this family are transcriptional factors highly conserved among ascomycetes and basidiomycetes regulating different process in response to environmental stimuli. In silico analysis of VOSA gene showed its conservation among the fungi. A VOSA disruption cassette containing the hygromycin reistance gene was assembled by Double Joint PCR. The cassette was transformed by biolistic in KN99a/α (soroA) wild type strains that allowed obtaining the vosAaΔ and vosAαΔ mutants. Likewise, the fragment containing the VOSA locus was transformed to obtain a mating type a reconstituted strain (vosAaΔ::VOSAa). Both disruption and reconstitution were confirmed by traditional PCR. The mutants vosAαΔ and vosAaΔ were tested for various phenotypes: growth at 30 and 37°C, capsule formation, urease and phospholipase production, changes in cell wall and melanin production, however no phenotypic changes were observed compared to the wild type strains. Regarding the ability to mate, vosAaΔ and vosAαΔ mutants were crossed with wild type strain of opposite mating type and the crosses showed decreased hyphae formation and significant morphological alteration of its structure. Mutants of opposite mating type (vosAaΔ vs. vosAαΔ) were subjected to mating and showed no formation of hyphae. VOSA is believed to be a positive regulator of sexual reproduction, and that controlling the formation of sexual structures. Expression of velvet genes was analyzed in vosAaΔ mutant in comparison with wild type strain and any significant transcriptional changes were noticed. The results obtained so far in C. neoformans collaborate for a better understanding of its role in the pathobiology of this pathogen. It is intended to identify in detail the role of VOSA in C. neoformans lyfe cycle, in particular spore production, the infective and dissemination form of the pathogen. The molecular data generated in this study are starting point for a research to elucidate the molecular mechanism of the sexual cycle regulation on C. neoformans and for the first time describ a velvet gene involved in the biology of a basidiomycete.

Cryptococcus neoformans is an encapsulated yeast responsible for severe meningoencephalitis. The importance of epidemiological studies on cryptococcosis has increased since the beginning of the AIDS epidemic. C. neoformans exists in two varieties containing four serotypes, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Locally C. neoformans var. neoformans has been associated with pigeon feces during those months having an average temperature of 64.2°F j(17.8°C) and above. Clinical and environmental isolates of C. neoformans obtained from regional hospitals and environmental samplings, respectively, have been grouped into their variety status utilizing canavanine-glycine-bromthymol blue agar. Polyclonal antisera against C. neoformans serotypes A, B, C and D were isolated from challenged rabbits. Serotyping C. neofromans isolates using the polyclonal antisera resulted in 57% (20 of 35) of the serotypes confirmed with a direct immunofluorescent assay utilizing a single monoclonal antibody (E1). Data from the immunofluorescence assay suggest all C. neoformans obtained from regional hospitals (26 of 26) and those isolated from the environment (9 of 9) belong to the A serotype group. These data have provided information leading to the origin of infection for cryptococcosis in our region, which may be beneficial to immunocompromised individuals.

The fungal pathogen Cryptococcus neoformans variety grubii is an opportunistic pathogen of immunocompromised people while the related species, Cryptococcus gattii, appears to infect people regardless of their immune status. The objective of this study was to investigate the differences between C. neoformans and C. gattii infections in a mouse model of cryptococcosis in order to better understand why C. gattii is able to cause disease in immunocompetent hosts. Normally, protective inflammation mediated by neutrophils and an adaptive Th1-type immune response is required for clearance of C. neoformans infections, whereas a Th2-type immune response is inefficient. Furthermore, neutrophils act as important first-responders in innate immunity by initiating the adaptive Th1 immune response during the early stage of infection. We hypothesized that while C. neoformans infections are cleared because they elicit strong protective inflammatory immune responses, C. gattii infections persist because they do not, thus enabling this species to cause disease in immunocompetent hosts. The results support the hypothesis because we found that C. gattii infections induce less protective inflammation than C. neoformans infections with respect to leukocyte recruitment to the sites of infection and cytokine induction. Mice infected with the C. gattii strains tested had less neutrophil migration into their lungs and had a reduced protective cytokine profile, suggesting that C. gattii strains may be able to skew the immune response towards a less efficient response, but not necessarily a Th2 allergic immune response. However, we also observed that the C. gattii strains tested varied in virulence, indicating that their ability to limit protective inflammation is not the only factor involved in their pathogenicity. Overall, these results provide important new insights into the virulence of Cryptococcus species; this information may be useful in understanding the outbreak of C. gattii infections that is occurring in British Columbia.

No período de 1981-2010, foram estudados, retrospectivamente, 54 casos de criptococose em pacientes com transplante de órgão sólido, identificados no Laboratório de Micologia da Santa Casa Complexo Hospitalar, Porto Alegre, RS. A criptococose ocorreu em 31 transplantados de rim, 13 de fígado, 7 de pulmão, 2 de pâncreas e rim, e 1 de coração. A idade média foi de 47,91 ± 13,98 (12-76 anos). Um total de 38 pacientes do sexo masculino (70,4%). As manifestações clínicas mais frequentes (54 pacientes) foram febre, cefaléia, vômito, tosse e estado mental alterado. Os achados radiográficos mais comuns no tórax, em 27 pacientes, foram nódulo, consolidação, cavitação e derrame pleural, sendo 10 com comprometimento pulmonar comprovado. Trinta e quatro apresentavam acometimento do sistema nervoso central, 7 tinham envolvimento cutâneo, e 4 em outros locais. O liquor, sangue e urina, respectivamente, contribuíram para o diagnóstico microbiológico com maior frequência. A maioria das infecções, nesta série de pacientes com criptococose, foi causada por Cryptococcus neoformans (92,7%). Pela primeira vez na literatura, documentamos C. gattii em pacientes com transplante de pulmão. Finalmente, quanto ao regime imunossupressor primário utilizado, houve maior mortalidade entre os pacientes que usaram o regime terapêutico baseado em ciclosporina e menor naqueles que usaram tacrolimus.
In the period of 1981 to 2010, 54 cases of cryptococcosis in patients with solid organ transplantation indetified at Mycology Laboratory in Santa Casa Hospital Complex, Porto Alegre, RS, were retrospectively studied. Cryptococcois occured in 31 kidney, 13 liver, 7 lung, 2 kidney-pancreas, and 1 heart transplant. The mean age was 47.3 years old (range, 12-76; SD 13.98). A total of 38 patients were male (70.4%). The most frequent clinical manifestation (54 patients) was fever, headache, vomiting, cough and altered mental status. The most common chest radiographic fidings, in 27 patients, were nodules, masses, consolidation, cavitation, and pleural effusion, 10 with proved pulmonary involvement. Thirty four patients had central nervous system involvement, 7 with cutaneous involvement, and 4 at other sites. The cerebospinal fluid, blood, and urine had the highest yield for the microbiologic diagnosis, respectivelly. Nearly all infections in this series of patients with cryptococcosis involved Cryptococcus neoformans (92.7%). By the first time in the literature, we documented C. gattii in lung transplant patients. Finally, considering the type of primary immunosupressive agent used, there was a higher mortality rate on patients with cyclosporine based therapy, and lowest in those with tacrolimus.

Objetivando estudar o perfil fenotípico de leveduras mantidas em banco de microrganismos de Cryptococcus neoformans, 40 cepas de origem clínica e 44 de origem ambiental foram escolhidas aleatoriamente. As cepas passaram por tipagem bioquímica para diferenciação em C. neoformans e C. gattii, e dentre as cepas clínicas 90% foram tipadas como C. neoformans e 10% como C. gattii, já dentre as cepas ambientais 95,5% foram C. neoformans e 4,5% C. gattii. As cepas cepas que foram positivas no teste bioquímico para C. gattii passaram por tipagem molecular (PCR-RFLP) e verificou-se que apenas quatro cepas eram realmente C. gattii (VGII), e duas outras C. neoformans (VNI e VNIII). Quando estudado os fatores relacionados a virulência, todas as cepas tanto clínicas quanto ambientais foram produtoras de fosfolipase, sendo que cepas de origem clínica produziram essa enzima em maior quantidade. Todas as cepas tanto clínicas quanto ambientais foram produtoras da enzima protease e todas também apresentaram intensidade de cor da colônia ( melanização). Quando avaliado a espessura capsular in vitro todas apresentaram cápsula, das cepas clínicas, 67% apresentaram cápsula média e das ambientais 70%. Quando avaliado a sensibilidade aos antifúngicos pelo métodos E-test todas as cepas clínicas e ambientais foram sensíveis aos antifúngicos anfotericina B, cetoconazol, fluconazol, voriconazol e posoconazol. O itraconazol também foi testado e apresentou cepas sensíveis à droga, porém uma cepa clínica e duas ambientais tiveram classificação dose dependente. Destacamos que a fosfolipase poderia ser usada como um marcador fenotípico para o complexo Cryptococcus neoformans e uma correta identificação das culturas mantidas em banco de microrganismos é necessário.
Aiming to study the phenotypic profile of yeasts maintened in stock culture identified as Cryptococcus neoformans, 40 clinical and 44 environmental strains, were chosen ran domly. The strains had undergone biochemical typing for differentiation as C. neoformans and C. gattii. Among the clinical strains 90% were typed as C. neoformans and 10% as C. gattii, already among the environmental strains were 95,5% C. neoformans and 4,5% C. gattii. The strains thah were positive in biochemical test for C. gattii underwent molecular typing (PCR-RFLP) and found that only four strain were actually C. gattii (VGII) and two other C. neoformans (VNI and VNII). When the factors related to virulence were studied, both the clinical and environmental strains were phospholipase positive but the clinical strains produced a greater amount of this enzyme. All strains were both clinical and environmental production of protease and also showed an intensity colony color (melanization). When the thickness of the capsule was evaluated, all of it showed a capsule, from the clinical strains 67% had an average capsule and from environmental strains 70% had an average capsule. When assessed by sensitivity to antifungal by E-test method all clinical and environmental strains were sensitive to the anphotericine B, ketoconazole, fluconazole, voriconazole and posoconazole. Itraconazole was tested and the samples showed drug sensitive-strains. We point out that phospholipase production would be used as marked to C. neoformans complex and a correct identification of strains maintened in stock culture is necessary.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2015.
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O Cryptococcus neoformans é um fungo patogênico humano que afeta majoritariamente indivíduos imunossuprimidos. Ele é o agente causador da meningite criptocócica e tem como principal fator de virulência uma cápsula polissacarídica que recobre sua parede celular. Os componentes desta cápsula estão envolvidos em diversos mecanismos de escape e de supressão da resposta imune desenvolvidos pelo fungo, o que leva a uma resposta não adequada da imunidade inata frente à infecção com consequente disseminação do patógeno. Dentre estes componentes, a glucoronoxilomanana (GXM) se destaca devido à grande variedade de modulações que a mesma é capaz de exercer em diversos tipos celulares. Neste trabalho, demonstramos uma nova interação entre estes componentes capsulares secretados e macrófagos: a inibição da secreção de IL-1β e da piroptose em macrófagos previamente ativados. Primeiramente verificamos que estes componentes possuíam a capacidade de inibir a produção de IL-1β, sem interferir na ativação fornecida pelo primeiro sinal – necessária para a produção da dada citocina. Caracterizamos parcialmente a molécula responsável por esta inibição, chegando a uma molécula pequena (menor que 1kDa), resistente ao calor (121 °C) e polar. Também verificamos que a piroptose dos macrófagos estimulados era prevenida por estes componentes capsulares, confirmando a interferência na via do inflamassoma destas células. Posteriormente realizamos ensaios para definir o mecanismo de ação pelo qual esta interferência estava ocorrendo. Verificamos que existiam depósitos da citocina intracelular pró-IL-1β e também que a enzima caspase-1 estava ativada nos macrófagos em contato com os componentes capsulares do C. neoformans. Por último, realizamos ensaios de interação entre os macrófagos e leveduras de C. neoformans, observando uma replicação intracelular exacerbada do fungo nas células tratadas com os componentes capsulares. Estes resultados demonstram a presença de uma molécula com caráter anti-inflamatório, acrescentando mais um mecanismo de modulação ao já extenso arsenal de fatores de virulência expressos pelo C. neoformans.
Cryptococcus neoformans is a human pathogenic fungus that affects mainly immunocompromised individuals. It is the causative agent of the cryptococcal meningitis and it´s major virulence factor is a polysaccharide capsule that covers it´s cell wall. The components of this capsule are involved in various mechanisms of evasion and suppression of the immune response developed by the fungus, which leads to an inadequate answer of the innate immunity to the infection and consequent dissemination of the pathogen. Among these components, the glucuronoxylomannan (GXM) stands out due to the great variety of modulations that it is able to perform in several cell types. In this study, we demonstrated a novel interaction between these capsular components secreted by the fungus and macrophages: the inhibition of IL-1β secretion and pyroptosis in previously stimulated macrophages. First, we verified that these components had the ability to inhibit the production of IL-1β without interfering in the activation led by the first signal – necessary for the production of this given cytokine. Then we partially characterized the molecule responsible for this inhibition, discovering that it was a small molecule (smaller then 1kDa), heat resistant (121 °C) and polar. We also found that the pyroptosis was being prevented in the stimulated macrophages, confirming the interference in the inflammasome pathway of these cells. Later we performed tests to define the mechanism of action by which these interferences were occurring. We found deposits of the intracellular cytokine pro-IL-1β and also activated caspase-1 enzyme in macrophages that were in contact with C. neoformans capsular components. Ultimately, we performed interaction tests between macrophages and yeast cells of C. neoformans, observing an exacerbated intracellular growth of the fungus inside the cells treated with the capsular components. These results demonstrated the presence of a molecule with anti-inflammatory character, adding another modulatory mechanism into the already extensive arsenal of virulence factors expressed by the C. neoformans.

Thesis (Ph.D.)--Boston University
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The pathogenic yeast, Cryptococcus neoformans , is a leading cause of death among individuals with compromised T cell function. Mannoproteins (MP) are a set of functionally heterogeneous, heavily mannosylated glycoforms found in many fungal species including C. neoformans. Cryptococcal MP have been shown to stimulate cell-mediated immunity and pro-inflammatory cytokines, both vital for the clearance of this yeast. As it is unclear how MP elicit immunity, it was hypothesized that the extensive conjugated carbohydrate backbone present on MP plays an essential role in immune stimulation. Mannose receptors (MR), including the macrophage mannose receptor (MMR) and dendritic cell-specific ICAM-3-grabing nonintegrin (DC-SIGN), both present on antigen-presenting cells (APC) and known to recognize mannosylated pathogens were shown to bind MP. Deglycosylation of MP or MR blockade with competitive mannosylated ligands inhibited T-cell activation. The immunodominant APC responsible for immune stimulation was determined by incubating T cells with purified splenic dendritic cells (DC), B cells, and peritoneal macrophages. T cells responded to MP only in the presence of DC. In addition, whole splenocyte populations, but not DC-depleted splenocytes, stimulated MP-specific T cells. Comparing APC populations, only DC captured fluorescent MP in a MR-dependent process. The kinetics of MP capture appear to involve 2 processes, a major, rapid, saturable receptor-mediated process that is inhibited with mannan, and minor pinocytic uptake. Impressively, DC pulsed for short periods with MP were still able to functionally stimulate T cells in a MR-dependent mechanism. Confocal microscopy of MP in DC at early time points indicated co-localization with transferrin, MMR and DC-SIGN suggesting MP enters early endosomes via MR. Subsequently, MP enter degradative perinuclear compartments. In vivo, MP-immunization resulted in increased survival, as well as a decrease in organ fungal load in response to live C. neoformans challenge. This partial protection was dependent on T cells, and not B cells. Moreover, analysis of tissue pro-inflammatory cytokine levels showed an increase in tumor necrosis factor-α and interferon-γ in infected MP-immunized mice. Collectively, these studies begin to provide both a molecular and cellular basis for the immunogenicity of cryptococcal MP and support T cell vaccination strategies that target MR and DCs.
2031-01-01

Inteins are autocatalytic protein domains that splice out of the nascent polypeptide shortly after translation, requiring no co-factors to facilitate splicing. There is an intein coding sequence within the Prp8 gene of Cryptococcus neoformans, a human pathogen that causes cryptococcosis in immunocompromised people. The intein, Cne PRP8, is a drug target as Prp8 is a central component of the spliceosome and thus believed to be essential to the fungus. Improved knowledge of the intein and its requirements for splicing can contribute to design of a screening system and the search for an inhibitor of intein splicing. Purification of Cne PRP8 for crystallisation was performed using either an N- or a C-terminal His�Tag�, where the N-terminal His�Tag� was removed by 3C protease prior to crystallisation trials. C-terminally His�Tagged� Cne PRP8 formed the largest crystals. The crystals were triangular plates with stepped faces. A 2.8 Å data set was collected with an R[merge] of 0.151 and a mosaicity of 2.1�. A smaller crystal gave a 3.6 Å data set with an R[merge] of 0.085 and a mosaicity of 1.5�. Molecular replacement was not sufficient to solve the structure, likely because the data were weak and the molecules in the asymmetric unit too numerous. Purified Cne PRP8 was additionally shown by circular dichroism to lack regular secondary structure, suggesting that regions of Cne PRP8 could be natively unstructured. Cne PRP8 was expressed as a fusion protein between Haemophilus influenzae trigger factor (HiTF) and a chitin binding domain (CBD). Antibodies to the different parts of the fusion protein facilitated the observation of splicing ability by western blotting. From this it was determined that Cne PRP8 is capable of splicing in a foreign protein context. Context is important, with maximum splicing occurring when Cne PRP8 has two native N-terminal extein residues and one native C-terminal extein residue. The first residue and the last two residues of Cne PRP8 are essential for splicing; additionally the conserved threonine (T62) and histidine (H65) were shown to be catalytically important. Also required for splicing are arginine 154, tyrosine 162, and aspartate 166. Leucine 161 undergoes ~50% splicing when mutated to alanine, and tryptophan 151 undergoes limited C-terminal cleavage, but no splicing, when mutated to alanine. Tryptophan 151 was identified as a potentially crucial residue, which may function to prevent C-terminal cleavage before the N-terminal rearrangements have taken place. Overall it appears that Cne PRP8 residues that are more diverged from the general intein consensus are less essential for splicing. Wild type Cne PRP8 is insensitive to zinc inhibition in vivo. It is also unresponsive to cadmium, calcium, cobalt, lithium, magnesium, manganese and nickel. However, a partially splicing-deficient mutant exhibited further inhibition in response to zinc and cadmium. This mutant also showed a limited increase in splicing efficiency in response to temperatures lower than 37�C. This study has identified critical residues, in addition to those at the splice junctions necessary for catalysis, which participate in splicing intermediates.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
A criptococose à a infecÃÃo causada a partir da inalaÃÃo de leveduras do gÃnero Cryptococcus. A doenÃa costuma acometer mais freqÃentemente pacientes com alguma imunodepressÃo e evoluir de forma subaguda ou crÃnica. Tem predileÃÃo por sistema nervoso central, manifestando-se como meningite, mas tambÃm pode apresentar-se como doenÃa pulmonar ou de forma disseminada, com envolvimento de outros ÃrgÃos. A atividade proteolÃtica em leveduras do gÃnero Cryptococcus tem sido associada a sua patogenicidade. A atividade da enzima fosfolipase tambÃm se faz importante para este gÃnero, uma vez que està relacionada à nutriÃÃo e à capacidade invasiva do microrganismo. Outro mecanismo de virulÃncia de maior relevÃncia para a patogenicidade de Cryptococcus spp. à a cÃpsula polissacarÃdica presente em sua superfÃcie, responsÃvel principal pelo escape aos mecanismos de defesa do hospedeiro. Esta pesquisa teve como objetivo traÃar um perfil clÃnico, epidemiolÃgico e laboratorial dos pacientes com criptococose entre os anos de 1985 e 2010, bem como investigar os efeitos dos inibidores de protease Saquinavir, Darunavir, Ritonavir e Indinavir sobre o crescimento, espessura da cÃpsula, atividade de protease e expressÃo de fosfolipase em cepas de Cryptococcus neoformans isoladas de humanos. A comparaÃÃo entre grupo soropositivo para o HIV com grupo soronegativo mostrou que o primeiro apresenta maiores mÃdias de idade e maior freqÃÃncia de sexo masculino acometido; por outro lado, o grupo soropositivo demonstra menores proteinorraquia e celularidade no lÃquor, bem como menores valores de leucometria e plaquetometria em sangue perifÃrico. ApÃs a realizaÃÃo do teste de sensibilidade por microdiluiÃÃo em caldo, os fungos foram expostos a trÃs concentraÃÃes distintas dos fÃrmacos e avaliados com relaÃÃo à expressÃo das duas enzimas e espessura da cÃpsula. Os resultados mostraram inibiÃÃo significativa (p<0,05) da atividade de protease pelo menos na maior concentraÃÃo testada para todas as drogas, bem como estreitamento da cÃpsula em algumas combinaÃÃes de drogas e cepas. Por outro lado, quanto à atividade de fosfolipase, observou-se um aumento na expressÃo dessa enzima, especialmente nas concentraÃÃes mais elevadas das quatro drogas. Conclui-se, portanto, que apesar de estes antirretrovirais nÃo inibirem o crescimento de Cryptococcus neoformans, sÃo capazes de alterar mecanismos de virulÃncia destas leveduras.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação para a Ciência e a Tecnologia (FCT)
Criptococose é uma importante micose sistêmica que acomete principalmente pacientes imunocomprometidos e é classificada como a infecção fúngica com maior mortalidade entre indivíduos portadores de HIV. Cryptococcus neoformans é uma levedura capsulada e o principal agente etiológico da criptococose; encontra-se dispersa no meio ambiente na forma de basidiósporos, os quais são responsáveis pela infecção em humanos e animais. As principais manifestações clínicas estão associadas à infecção pulmonar e meningite. A terapia se dá basicamente com a administração de anfotericina B (AMB) na terapia de ataque associada ou não a 5-fluocitosina e como manutenção é indicado ao longo do tratamento o fluconazol (FCZ). Apesar de esta terapêutica ser eficiente, é constatado elevado número de casos de reincidência e desenvolvimento de resistência aos azóis, além de problemas de toxicidade. Diante desta problemática, este estudo propõe o desenvolvimento de novas alternativas terapêuticas para o tratamento da criptococose, visando duas abordagens distintas. A primeira, aplicando novo composto antifúngico e a segunda, o estudo de microRNAs (miRNAs) envolvidos na interação da levedura e células de glioblastoma humano (U87-MG), com a finalidade de usá-los como reguladores da infecção, sendo esta uma estratégia recentemente divulgada em uma variedade de doenças. Para tanto, inicialmente moléculas análogas de resveratrol foram avaliadas quanto a atividade antifúngica e o derivado orto,orto-HDZ (HDZ) mostrou forte atividade fungicida contra isolados de C. neoformans, com valores de concentração fungicida mínima (CFM) variando entre 0,97 a 7,81 µg/mL. A associação entre HDZ e FCZ mostrou sinergia com potencialização do efeito do FCZ em até 64 vezes para um isolado clínico resistente. Baixa toxicidade foi observada em ensaios in vitro e in vivo (Galleria mellonella e camundongos), apesar de mostrar alterações em embriões de Zebrafish. Além disso, este estudo mostrou a capacidade da associação HDZ + FCZ em inibir a interação da levedura e células hospedeiras (pneumócitos, macrófagos e glioblastoma) por citometria de fluxo e imunofluorescência. Ensaios in vivo (modelo murino) da atividade antifúngica de HDZ em combinação com FCZ mostrou que após 15 dias de tratamento houve redução significativa das unidades formadoras de colônias (UFC) no fígado, baço, cérebro, pulmões e lavado broncoalveolar, assim como o aumento da sobrevida dos animais em 198 dias e redução da morbidade, a qual foi avaliada por ensaio de comportamento (SHIRPA). Diante da segunda proposta deste trabalho, foram encontrados 10 miRNAs super-expressos na interação de C. neoformans e células U87-MG e foi observado que os miRNAs regulam principalmente vias de sinalização TGF-β, MAP quinase (MAPK) e interação receptor e matriz extracelular (MEC). Tais dados foram cruzados com transcritos que mostraram diferencialmente expressos na interação fungo-célula e as proteínas GTPase-3 e COP-1 foram selecionadas como importantes nesse processo. Tais proteínas estão associadas à via Rho-GTPase a qual é primordial para a ligação da levedura em células hospedeiras e mostrou aumento da expressão na infecção, assim como ligeira redução após o tratamento com HDZ. A indução da apoptose de células de glioblastoma por C. neoformans, foi observada através da técnica TUNEL e da expressão da proteína pró-apoptótica Bak e o tratamento com HDZ foi capaz de reverter esse processo. Nossos resultados indicam HDZ como uma interessante molécula para avançar nos estudos de desenvolvimento de um protótipo antifúngico contra C. neoformans e a inibição da expressão dos miRNAs durante a interação fungo-célula, pode ser interessante para controle da infecção fúngica, pois acreditamos que uma vez inibidos, podem reduzir a invasão da levedura em células hospedeiras, sendo então os miRNAs interessantes novos alvos terapêuticos e que o tratamento com HDZ são promissores para o desenvolvimento de um novo antifúngico.
Cryptococcosis is an important systemic mycosis that mainly affects immunocompromised patients and is classified as the fungal infection with higher mortality among individuals with HIV. Cryptococcus neoformans is an encapsulated yeast and the main etiologic agent of cryptococcosis. This yeast is dispersed into the environment in the form of basidiospores, which are responsible for infection in humans and animals. The main clinical manifestations are associated with pulmonary infection and meningitis. The treatment is basically the administration of amphotericin B (AMB) as therapy consolidation with or without the use of 5-flucytosine and, for the maintenance therapy, fluconazole (FCZ) is indicated. Although this therapy is effective, it is observed high number of cases of relapses, development of resistance to azoles and toxicity problems. In front of these problems, this study proposes the development of new therapies for the treatment of cryptococcosis, targeting two distinct approaches. The first aims a new antifungal compound and the second the study microRNAs (miRNAs) involved in the interaction between yeast and human glioblastoma cells (U87-MG), in order to use them as regulators of infection, since this is a recently disclosed strategy in a variety of diseases. For this purpose, initially, analogs of resveratrol molecule were evaluated for antifungal activity and the derived ortho,ortho-HDZ (HDZ) showed a strong fungicidal activity against isolates of C. neoformans with values of minimum fungicidal concentration (MFC) ranging between 0.97 to 7.81 µg/mL. The association between HDZ and FCZ showed synergy with potentiation of the effect of FCZ in up to 64 times for a resistant clinical isolate to FCZ. Low toxicity was observed in in vitro and in vivo assays (Galleria mellonella and mice), despite showing changes in Zebrafish embryos. In vivo assay (murine model) of the antifungal activity of HDZ in combination with FCZ showed that after 15 days of treatment, a significant reduction in colony forming units (CFU) in the liver, spleen, brain, lungs and bronchoalveolar lavage was observed, as well as, increased survival of the animals in 198 days with the reduction of the morbidity, which was evaluated by behavioral assay (SHIRPA). Considering the second purpose of this study, 10 miRNAs were over-expressed during the interaction of C. neoformans and U87-MG cells and it was noted that these miRNAs regulate the signaling pathway of TGF-β, MAP kinase (MAPK) and the receptor and matrix extracellular components (MEC) interaction. These data was cross with transcripts that showed differentially expressed in fungus-cell interaction and the GTPase-3 and COP-1 protein were selected as important in this process. Such proteins are associated to Rho-GT pathway Rho-GTPase which is essential for the yeast adhesion into host cells and showed increased expression in infection, as well as a slight reduction after treatment with HDZ. Induction of apoptosis in glioblastoma cells by C. neoformans was observed by TUNEL technique and the expression of pro-apoptotic protein Bak and treatment with HDZ was able to reverse this process. Our results indicate HDZ as an interesting molecule to advance the development of studies of a prototype antifungal against C. neoformans and that the inhibition of expression of miRNAs during fungus-cell interaction, may be interesting to control fungal infection, because we believe that once inhibited, the yeast can reduce invasion of host cells, being the miRNAs interesting new therapeutic targets and the treatment with HDZ is promising for the development of a new antifungal drug.
CNPq: 403586/2012-7
FCT: 345/13

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2016.
Texto liberado parcialmente pelo autor. Conteúdo restrito: Apêndices.
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Fatores de transcrição da família Gti1/Pac2 ocorrem exclusivamente em fungos e foram cooptados pela evolução para desempenhar papéis reguladores distintos de acordo com a espécie, papéis esses que vão do controle da transição micélio-levedura e virulência em fungos termodimórficos, à regulação do acasalamento em Candida e do metabolismo secundário em patógenos de plantas. Como essas são funções-chaves para patogênese, levantamos a hipótese de que o homólogo em Cryptococcusneoformans poderia também controlar a virulência, no caso em questão de um importante patógeno fúngico de humanos, que causa uma doença que vitima centenas de milhares de pessoas todo ano. O mutante para o gene que caracterizamos retém a capacidade de secretar diversas substâncias associadas à virulência – melanina, urease e fosfolipase – mas é hipocapsular e tem um defeito de citocinese e de crescimento a 37 °C. É avirulento em camundongos e hipovirulento em Galleria a 30 °C, o que indica que a temperatura do hospedeiro não é a única razão para o fenótipo observado. Além disso, a análise de RNA-Seq mostra que na ausência da proteína correspondente, a expressão da maioria das ORFscodantes do locus MAT é parcial ou completamente reprimida a 37 °C num meio de cultura de células. Entretanto, o mutante não apresenta um defeito na iniciação do acasalamento. Estes resultados dão suporte à idéia de que acasalamento e a resposta a estresses ambientais são mecanismos que evoluíram paralelamente, e é o primeiro relato de um fator de transcrição que controla o locus MAT de um patógeno fúngico num contexto independente do acasalamento. A proteína foi, portanto, nomeada MAT locuscontroller 1 (“controladora do locus MAT 1”, Mlc1).
Gti1/Pac2 transcription factors occur exclusively in fungi and have been co-opted during evolution to perform distinct regulatory roles according to species, ranging from yeast-to-mycelium transition and virulence in dimorphic fungi, to mating in Candida and secondary metabolism in plant pathogens. As these roles are important in causing disease, we hypothesized that the Cryptococcus neoformans homologue could also control virulence in this important fungal pathogen, which causes a disease that kills hundreds of thousands of people each year. The mutant for this gene retains the ability to secrete several substances associated with virulence – melanin, urease and phospholipase – but is hypocapsular and has a cytokinesis and a growth defect at 37 °C. It is avirulent in mice and hypovirulent in Galleria at 30 °C, which indicates that host temperature is not the only reason. Furthermore, RNA-Seq analysis shows that in the absence of the protein, the expression of most protein-coding ORFs of the MAT locus is partial or completely repressed at 37 °C in a cell culture medium. However, the mutant does not have a defect in mating initiation. These results support the idea that mating and the response to environmental stress are co-evolved mechanisms, and is the first report of a transcription factor that controls the MAT locus of a fungal pathogen in a mating-unrelated context. The protein has thus been named MAT locus controller 1 (Mlc1).

A criptococose acomete pacientes imunocomprometidos, principalmente pacientes com a resposta imune mediada por células T debilitada, sendo portanto, suscetíveis a patógenos oportunistas. C. neoformans possui um repertório de fatores de virulência que permitem o estabelecimento da infecção e sua disseminação para o sistema nervoso central, causando meningoencefalite. O tratamento da criptococose é baseado apenas na utilização prolongada de antifúngicos e provoca muitos efeitos colaterais nos pacientes. Por estes motivos, novas terapias estão sendo abordadas, com o intuito de promover a ativação do sistema imune do hospedeiro para debelar a infecção. Para que tais terapias possam ser desenvolvidas se faz necessário identificar moléculas que estimulem a resposta imune celular. Proteínas de choque térmico de patógenos têm sido utilizadas como alvo no tratamento de muitas doenças infecciosas em seres humanos. Estas proteínas estimulam a resposta imune mediada por células T e apresentam resultados promissores em tratamentos contra infecções fúngicas. Da mesma forma, proteínas que fazem parte do sistema de defesa antioxidante, como as catalases, por exemplo, desempenham um papel importante no combate a radicais livres produzidos pelo hospedeiro e parecem ter outras funções no processo da infecção. O entendimento da biologia destas proteínas bem como a sua participação na interação com células do sistema imune do hospedeiro é fundamental para a validação da sua aplicabilidade no tratamento da criptococose. Nesse contexto, apresentamos aqui um estudo das funções biológicas de uma das proteínas de choque térmico (Hsp70) e de uma das enzimas envolvidas na detoxificação de espécies reativas de oxigênio (catalase 2) do fungo C. neoformans. Também foram avaliados os efeitos terapêuticos destas proteínas no tratamento da criptococose. Demonstramos que a proteína Hsp70 de C. neoformans está localizada na cápsula polissacarídica, sendo importante no processo de adesão da levedura a células epiteliais A549. Demonstramos também que Cn_rHsp70 co-localiza com o principal componente da cápsula polissacarídica a glucuronoxilomanana (GXM) na superfície de macrófagos. A Cn_rHsp70 quando em contato com células do fungo, diminui sua viabilidade celular e não altera o tamanho da cápsula, mas interfere na secreção de GXM. Em relação à catalase 2, foi demonstrado que a proteína é localizada na parede celular do fungo e possui atividade de degradação de peróxido de hidrogênio quando associada à superfície celular. Em ensaios de infecção experimental em animais imunizados com construções de DNA contendo as sequências de Hsp70 e catalase 2, nenhuma das proteínas conferiu proteção, mas ocorreu estímulo da resposta mediada por células Th1 e Th2. O mecanismo pelo qual estas proteínas estimulam o sistema imune ainda precisa ser elucidado.
Cryptococcus neoformans affects immunocompromised patients, especially patients with impaired T cell-mediated immune response, and therefore are susceptible to opportunistic pathogens. C. neoformans has a repertoire of virulence factors that allows the establishment and dissemination of the infection in the central nervous system, causing meningoencephalitis. The virulence factors of Cryptococcus has been subject of many studies that have showed the involvement of gene products in the infection process. However, the treatment of cryptococcal meningoencephalitis consists of antifungal therapy and displays many side effects. For these reasons, new therapies are being addressed, in order to promote the activation of the host immune system to overcome the infection. To develop these therapies, it is necessary to identify molecules that stimulate cell-mediated immune response. Heat shock proteins from many pathogens have been used as a target for the treatment of many humans infectious diseases. These proteins stimulate the T cell-mediated immune response and show promising results in treatment against fungal infections. Similarly, proteins from antioxidant system such as catalase for example, play an important role against free radicals produced by the host and appear to have other functions in the infection process. The understanding of these proteins biology and their involvement with host immune system cells is essential for the validation of its applicability in the treatment of cryptococcosis. In this context, we present here a study of the biological function of a heat shock protein (Hsp70) and one of the enzymes involved in detoxification of reactive oxygen species (catalase 2) of the fungus C. neoformans. We evaluated the therapeutic effects of these proteins in the treatment of cryptococcosis. We have demonstrated that the protein Hsp70 from C. neoformans is located in the polysaccharide capsule, and it is important in the adhesion process of the yeast to epithelial cells A549. Also demonstrated that Cn_rHsp70 co-localizes with the main component of the polysaccharide capsule, glucuronoxilomanana (GXM), on the surface of macrophages. When Cn_rHsp70 are in contact with fungal cells, the cellular viability decreased and does not change the capsule size however, interferes with the GXM secretion. Catalase 2 is deposited on the fungal cell wall and showed activity by hydrogen peroxide degradation. In vivo experiments using animals infected and treated with Hsp70 DNA vaccines and catalase 2, no protection were observed, but there are immune response mediated by Th1 and Th2 cells. The mechanism by which these proteins stimulate the immune system has yet to be elucidated.

Cryptococcus neoformans é um patógeno humano oportunista e uma das principais causas de infecções fúngicas relacionadas à óbitos, especialmente em pacientes imunocomprometidos. O processo de infecção inicia-se com a colonização do tecido pulmonar, e posteriormente a levedura se dissemina pela via hematogênica até alcançar o sistema nervoso central (SNC), provocando meningite. A fim de transmigrar e alcançar o SNC, as células fúngicas devem atravessar a barreira hematoencefálica (BHE). O estabelecimento adequado da doença é dependente de diferentes fatores de virulência como a cápsula polissacarídica, a capacidade de se desenvolver a 37 ºC, e outros fatores diretamente relacionados à transmigração como urease, fosfolipase B1 e ácido hialurônico sintase que são descritas como componentes essenciais para a habilidade de atravessar a BHE. O cálcio (Ca2+) é um segundo mensageiro celular que atua na via de sinalização da calcineurina. Essa via em C. neoformans é necessária e responsável pela virulência e adaptação no hospedeiro. Além disso, a regulação dos níveis intracelulares de Ca2+ é mediada pela ação de transportadores de cálcio como Pmc1 e Vcx1, os quais são foco de nossos estudos. Apesar de grandes avanços no entendimento dos mecanismos moleculares de infecção de C. neoformans, as opções terapêuticas ainda são escassas, por isso a elucidação de fatores que regulam a transmigração e a descoberta de novos alvos para fármacos é importante. Nesta dissertação foi demonstrado que os mutantes nulos para o transportador de cálcio Pmc1 (pmc1) e o duplo mutante pmc1vcx1 são avirulentos em um modelo murino de infecção sistêmica e incapazes de acessar o SNC. Além disto, a deleção do gene PMC1 causa alterações no perfil de expressão gênica da levedura, principalmente nas vias envolvidas com a transmigração e homeostase de Ca2+. Genes relacionados com a atividade de urease, e envolvidos com o mecanismo paracelular de transmigração tiveram alterações no seu perfil de expressão. Considerando os dados obtidos, é comprovada a participação do transportador Pmc1 no processo de transmigração através da BHE e para o estabelecimento adequado da doença criptococose.
Cryptococcus neoformans an opportunistic human pathogen, is the main cause of fungal infections related to death, especially in immunocompromised patients. The infection starts with the colonization of the lung tissue, thereafter the yeast disseminates hematogenously to reach the central nervous system (CNS), causing meningoencephalitis. In order to transmigrate to CNS the fungal cells must cross the blood brain barrier (BBB). For infection establishment, C. neoformans relies on virulence factors, such as polysaccharide capsule, the ability to grow at 37 ºC, and some factors directly related to transmigration. Enzymes as urease, phospholipase B and hyaluronic acid synthase are known as essential components to cross the BBB. The calcium (Ca2+) is a cellular second messenger that participates on calcineurin signaling pathway. This pathway in C. neoformans is crucial and responsible for virulence and host adaptation. Moreover, the regulation of Ca2+ intracellular levels is coordinated by Ca2+ transporters such as Pmc1 and Vcx1, which are the main foccus of our studies. Despite of many advances in understanding the molecular mechanisms of C. neoformans infection the available repertoire of antifungal drugs is still poor, therefore it is necessary to comprise the factors that regulate dissemination to CNS, and also unveil new targets for antifungal therapy. In the present study it was demonstrated through a murine model of systemic infection that the null mutants pmc1 and pmc1vcx1 are avirulent and unable to access the SNC. Besides, the disruption of PMC1 gene leads to alterations on the gene expression profile, mainly in pathways involved with transmigration and calcium homeostasis. Genes related to urease activity, and connected to paracellular mechanism of transmigration had alterations on their expression profile. Taken togheter the result shows the requirement of Pmc1 transporter to transmigration events through BBB and for proper cryptococcosis establishment.

FREITAS, Daniel Domingues. Avaliação in vitro da curcumina frente às cepas de Candida spp. e Cryptococcus neo¬formans resistentes ao fluconazol. 2015. 70 f. Dissertação (Mestrado em Microbiologia Médica) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015.
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Considering that some therapeutic properties of curcumin are well described, so the present study points to an antifungal effect of curcumin against strains of pathogenic yeasts resistant to fluconazole, strains of Candida spp. and Cryptococcus neoformans. All strains studied were inhibited by curcumin with different degrees of inhibition among species with MICs ranging from 8-64 mg / ml. After exposure strains of C. albicans, curcumin a decrease in the number of viable cells was observed, thus indicating damage to cell membranes, with possible compromise of their functions. Our results showed that after treatment with curcumin, found in more intense staining with PI (Propidium iodide), indicating changes in cell membranes. In this study, the mitochondrial function of C. albicans cells appears to have been affected after exposure to curcumin. The collapse in Δψm (Mitochondrial Transmembrane Potential) can lead to openings of transient pores in the mitochondrial membranes. In relation to the different mechanisms of action described for curcumin, there is evidence that DNA is one of the celular targets of this molecule. Our data suggest that after exposure to curcumin, cells of C. albicans showed total breaks in DNA strands. In conclusion, the compound curcumin has antifungal activity in vitro against strains of Candida spp. and Cryptococcus neoformans resistant to fluconazole. In addition to promoting DNA damage and externalization of phosphatidylserine, the respective compound seem to act at specific sites near mitochondria of C. albicans cells, leading to death by apoptosis.
Tendo em vista que algumas propriedades terapêuticas da curcumina já são bem descritas, dessa forma o presente estudo aponta para um efeito antifúngico da curcumina frente a cepas de leveduras patogênicas resistentes ao fluconazol, cepas de Candida spp. e Cryptococcus neoformans. Todas as cepas estudadas foram inibidas pela curcumina, com diferentes graus de inibição entre as espécies com CIMs variando entre 8-64 μg/mL. Após a exposição das cepas de C. albicans a curcumina, foi observado uma diminuição no número de células viáveis, indicando assim danos às membranas celulares, com possível comprometimento de suas funções. Os nossos resultados revelaram que após o tratamento com a curcumina, constatamos uma intensificação na marcação com PI (Iodeto de propídio), indicando alterações nas membranas celulares. No presente trabalho, a função mitocondrial das células de C. albicans parece ter sido afetada após exposição à curcumina. O colapso no Δψm (Potencial Transmembrana Mitocondrial) pode conduzir a aberturas de poros transientes nas membranas mitocondriais. Em relação aos diversos mecanismos de ação descritos para a curcumina, existem evidências de que o DNA é um dos alvos celulares desta molécula. Nossos dados sugerem que após a exposição à curcumina, as células de C. albicans, apresentaram quebras totais nas cadeias de DNA. Em conclusão, o composto curcumina apresenta atividade antifúngica in vitro contra cepas de Candida spp. e Cryptococcus neo-formans resistentes ao fluconazol. Além de promover danos ao DNA e externalização da fosfatidilserina, o respectivo composto parecem atuar em sítios específicos próximos a mitocôndria das células de C. albicans, levando à morte por apoptose.

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Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil
Cryptococcus neoformans (C. n) e Cryptococcus gattii (C. g) são agentes da criptococose. A carência de publicações sobre teste de suscetibilidade de C. n e principalmente de C. g é evidente. A maioria de isolados clínicos de C. g mostra-se suscetível in vitro a fluconazol e itraconazol; no entanto o surgimento de resistência a drogas antifúngicas incentivou os testes de suscetibilidade in vitro. Como conseqüência, Clinical and Laboratory Standadards Institute \2013 CLSI, anteriormente National Committee for Clinical Laboratory Standards \2013 NCCLS, publicaram metodologia padronizada M27-A2 para alcançar reprodutibilidade e permitir a comparação de resultados de suscetibilidade entre laboratórios, porém alguns problemas como o tempo de incubação e o padrão de leitura levaram à busca de técnicas alternativas, tais como a citometria de fluxo. O objetivo deste trabalho é padronizar a técnica de citometria de fluxo para o teste de suscetibilidade in vitro de C.n e C.g. visando a redução do tempo de incubação, a utilização de leitura automatizada e a obtenção de resultados rápidos e reprodutíveis A concentração inibitória mínima (CIM) de 20 isolados de C.n e 21 de C.g foi determinada por citometria de fluxo e os resultados comparados com o protocolo padrão proposto pelo CLSI/M27A-2. Após duas horas de incubação com anfotericina B utilizando FUN-1, C. gattii resultou em 100% de concordância entre as duas técnicas para diluição de 2\03BCg/mL e 95,2% para 1\03BCg/mL e C. neoformans resultou em 100% de concordância para 1 e 2\03BCg/mL. Para azólicos e flucitosina, foram obtidos resultados reprodutíveis com o fluorocromo Acridine Orange após 18 horas de incubação, que resultou em 78% de concordância entre as duas técnicas para fluconazol, 85% para itraconazol e 97% para flucitosina. Em ambas as metodologias, C. gattii foi menos suscetível do que C. neoformans frente ao itraconazol e flucitosina (p<0,05). A citometria de fluxo é uma ferramenta útil, com potencial para testes in vitro e determinação da CIM dos antifúngicos estudados, com apreciável redução do tempo mínimo para obtenção de resultados
Cr yptococcus neoformans ( C.n ) and Cryptococcus gattii ( C.g ) are the agents of cryptococcosis. The lack of publications on susceptibility tests of C.n and especially C.g is evident. Most clinical isolates of C.g proved to be susceptible in vitro to fluconazol e and itraconazole and yet the emergence of resistance to antifungal drugs encouraged susceptibility testing in vitro . As a consequence, Clinical and Laboratory Standadards Institute – CLSI ( National Committee for Clinical Laboratory Standards – NCCLS ) pub lished standardized methodology to achieve reproducibility and allow comparison of susceptibility results between laboratories (M27 - A2 document) , however some problems of methodology, like incubation time and standard reading, led to the search for alterna tive techniques, such as flow cytometry. The objective of this work is to standardize the technique of flow cytometry to test the in vitro susceptibility of C . n and C . g in order to reduce the incubation time, use of automated reading and obtain fast and re producible results . The minimum inhibitory concentration (MIC) of 20 isolates of C.n and 21 of C.g was determined by flow cytometry and the results compared with the standard protocol proposed by CLSI/M27A - 2. Flow citometry showed 100% agreement with CLSI/ M27A - 2 results for 2 μ g/mL and 95.2% for 1 μ g/mL dilution when C.g isolates were tested, and 100% agreement for 1 and 2 μ g/ml dilution when C.n were tested after two hours of incubation with amphotericin B using FUN - 1 . Reproducible results were obtained with fluorochrome Acridin e Orange for azoles and flucytosine after 18 hours incubation, resulting in 78% agreement for fluconazole, 85% for itraconazole and 97% for flucytosine. C.g was less susceptible than C.n against itraconazole and flucytosine (p <0.05) in both methodologies. Flow cytometry is a useful tool, with potential for in vitro susceptibility test s of the antifungal agents studied, with appreciable reduction in the minimum time for achieving results.

The fungal pathogen Cryptococcus neoformans is the leading cause of encephalitis in people with the acquired immunodeficiency syndrome (AIDS). The range of drugs available to treat C. neoformans is limited in number and efficacy; therefore, there is a dire need for new, more powerful therapeutics. To achieve this we need to better understand how the fungus can cause disease, which will lead to the identification of factors relating to virulence and ultimately may provide new drug targets. The plethora of emerging genomic resources has allowed for targeted biology to focus on key genes in important cellular processes that relate to virulence. The work described in this thesis contributes to the development of genomic resources for biological investigations in Cryptococcus neoformans. This work has three specific components: 1) physical mapping of the genomes for strains serotypes A and D; 2) analysis of the low iron transcriptome in a serotype A strain; and 3) characterization of a putative siderophore (iron) transporter gene, SIT1, that was identified by transcriptome analysis. The first component involved the hybridization of 125 markers to a set of 9,216 BAC clones from the strain JEC21 (serotype D) and to 6,528 clones from the strain H99 (serotype A). These data provided the first genome-wide comparison of gene synteny between two strains of the fungus, and linked contigs to specific karyotype bands. The second component of the work involved the analysis of the low iron transcriptome for the serotype A strain, H99 using serial analysis of gene expression (SAGE). A number of interesting genes were identified in the low iron transcriptome including those involved with the response to stress and mechanisms of iron uptake. A key finding was that the low iron transcriptome was remarkably similar to the in vivo library from cells grown in rabbit cerebral spinal fluid (CSF) and significantly distinct from the libraries grown in yeast nutrient broth (YNB). The third component of the work focused on the gene SIT1, which encodes a putative siderophore transporter. The gene was characterized in three strain backgrounds of varying virulence. This work showed that SIT1 was important for iron utilization in conditions of low iron or when a siderophore was provided as the sole iron source. Further, there were pleiotrophic phenotypes for a number of virulence-related attributes including melanization, cAMP signaling and cell wall integrity. Finally, throughout the entire body of work, multiple differences were identified between strains of the same or different serotypes on a genomic and biological level, and this variation may lend insight into differences in virulence between strains.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

This thesis examines the functional and genetic factors that control the inbred mouse host response to Cryptococcus neoformans. The inbred mouse strains C57BL/6J and C3H/HeN were susceptible, and the inbred mouse strains CBA/J and SJL/J were resistant, to experimental cryptococcal pneumonia. This observation led to three general hypotheses: (1) early innate immune differences between inbred strains cause differential susceptibility to cryptococcal pneumonia; (2) natural variation in susceptibility among inbred strains is a complex trait under the control of specific genetic loci; and (3) allergic airway disease is a common phenotype among susceptible inbred strains. Functional characterization of the innate immune response in C57BL/6J and SJL/J mice revealed a heightened pro-inflammatory response in SJL/J as early as three hours post-infection. This polarization continued throughout the course of infection; C57BL/6J mice presented an allergic, Th2 immune response, and SJL/J mice presented a Th1 immune response. Intracellular signaling analysis in vitro revealed that the enhanced pro-inflammatory response observed in SJL/J mice was dependent on the prolonged activation of the NF-κB and phosphatidylinositol 3 kinase pathways. Next, a quantitative trait loci (QTL) analysis for cryptococcal pneumonia susceptibility in a segregating F2 population bred from the parental inbred strains C57BL/6J and CBA/J was performed. This revealed a sex-effect in the quantitative lung fungal burden that warranted a stratified QTL analysis approach. Two significant novel QTL were identified in the female F2 intercross: Cnes1 on chromosome 1, and Cnes2 on chromosome 17. Cnes3 was identified in the male F2 intercross as a unique QTL distal to Cnes2. Furthermore, a genome-wide pairwise analysis revealed significant QTL interactions in both the female and male F2 intercrosses that collectively explained 43.8 and 19.5% of the phenotypic variance in each sex, respectively. Finally, characte
Cette thèse étudie les facteurs fonctionnels et génétiques contrôlant la réponse de l'hôte aux infections fongiques causées par la levure Cryptococcus neoformans. Les lignées de souris consanguines C57BL/6J et C3H/HeN sont sensibles à la pneumonie expérimentale à cryptocoques, alors que les lignées CBA/J et SJL/J y sont résistantes. En se basant sur ces observations, trois hypothèses ont été formulées: (1) des différences du système immunitaire inné expliquent la sensibilité différentielle à la pneumonie à cryptocoques des lignées de souris consanguines; (2) la variation naturelle de la sensibilité entre lignées consanguines est un trait génétique complexe sous le contrôle de locus génétiques spécifiques; et (3) la maladie allergique respiratoire est un phénotype commun associé à une sensibilité à la pneumonie à cryptocoques progressive chez les lignées consanguines. La caractérisation fonctionnelle de la réaction immunitaire innée chez les souris C57BL/6J et SJL/J a révélé une réponse pro-inflammatoire accrue chez les souris SJL/J trois heures après l'infection. Cette polarisation a continué tout au long de l'infection, les souris C57BL/6J développant une réponse immunitaire allergique Th2, alors que les souris SJL/J ont présenté une réponse immunitaire Th1. Une analyse in vitro de la signalisation intracellulaire a révélé que la réaction pro-inflammatoire observée chez les souris SJL/J dépend de l'activation prolongée des cascades de signalisation NF-κB et PI3K (phosphatidylinositol 3 kinase). L'étude des liaisons de traits complexes (QTL) chez des souris F2 (C57BL/6J x CBA/J) a révélé un effet du sexe sur le phénotype, justifiant ainsi une analyse séparée. Deux locus de susceptibilité ont été identifiés chez les femelles: Cnes1 (chromosome 1), et Cnes2 (chromosome 17). Chez les mâles, seul le locus Cnes3 (chromosome 17, en aval de Cnes2) a été identifié. Une analyse QTL sur 2 locus a

Cryptococcus neoformans is an opportunistic fungal pathogen and a leading cause of fungal infection related fatalities in immunocompromised hosts. Compared to well-studied; Cryptococcus neoformans virulence factors like the polysaccharide capsule and melanin synthesis, very little is known about phospholipase B1 (Plb1). Plb1 is a phospholipid modifying enzyme that is implicated in multiple stages of cryptococcal pathogenesis. Herein I demonstrate that a Plb 1 deficient strain of C.neoformans has a profound defect in intracellular growth within macrophages. In addition, I show that the Δplb1 strain undergoes a novel morphological change during in vitro and in vivo infection, resulting in a sub-population of very large 'titan cells' that may arise as a result of the mutant's inability to cope within the macrophage. I go on to test whether these phenotypes are due to a reduction in eicosanoid production caused by Plb 1 deficiency. Finally, I present an addition project where I optimise a C. neoforman's intracellular proliferation assay for high throughput analysis via flow cytometry. This work provides a new insight into the function of this unappreciated virulence factor and helps to lay the foundation for new treatment strategies to combat cryptococcosis.

Known for over a hundred years, the human fungal pathogen, Cryptococcus neoformans causes cryptococcosis, a life threatening disease. Infection is acquired through inhalation of spores or dried yeast cells into the lungs from which the fungus can potentially transmit to all body parts of which the brain is the most affected organ. Once in the brain, the yeast C. neoformans causes meningoencephalitis, a fatal condition even with optimal treatment. The mechanism by which C. neoformans penetrates the normally impermeable blood brain barrier (BBB) to cause brain infection is not understood. This thesis presents two aspects of investigation: 1) the extent to which binding and uptake of cryptococci by brain microvascular endothelial cells (BMEC) explains transcytosis as a mechanism for cryptococcal traversal of the BBB and 2) the relationship between Cryptococcus – macrophage interaction and Cryptococcal meningoencaphalitis (CM) disease. We show that adherence and internalization of cryptococci by brain microvascular endothelial cells is a rare event characterized by a small number of cryptococci, an indication that C. neoformans most likely uses multiple routes to traverse the BBB. Secondly, by studying clinical isolates from cerebral spinal fluid (CSF) of HIV- associated CM patients, we demonstrate that high rate of cryptococci uptake by macrophages is associated with patient fungal burden whilst the intracellular proliferation rate is inversely associated with TNF- \(\alpha\) levels in the patient CSF. Interestingly, the high uptake – high fungal burden isolates were less encapsulated but more rapid melanin formers, traits known to modulate phagocytosis and protection from host-induced oxidative stress respectively. We therefore hypothesize that highly phagocytosed C. neoformans strains use phagocytes to disseminate faster to the brain resulting in high fungal burden.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Cryptococcosis is a fungal disease caused by yeasts belonging to the complex Cryptococus neoformans. The antifungal arsenal available for the treatment of this disease is still restricted and is related to the high toxicity and side effects of some drugs, causing great harm to the patients. In this context, it is necessary to discover new bioactives to quell the infections and reduce the adverse effects. The plants have rich source of secondary bioactive metabolites such as tannins, terpenoids, saponins, alkaloids, flavonoids and other compounds, registered with expressive antimicrobial properties. Therefore, the research of natural compounds and derivatives of natural products has been relevant in recent years, due to their relevance in the discovery of new drugs, in addition to which the association of drugs or compounds with different mechanisms of action has been used as an alternative in conventional therapy. We evaluated the mechanisms of action and antifungal effect of natural compounds, curcumin and morin, on Cryptococcus neoformans and Cryptococcus gattii isolates. The antifungal activity was determined across the minimum inhibitory concentration (MIC) and minimum fungicide (MFC). Polyphenols showed MICs ranging from 2 to 256 μg/mL. The compounds showed a synergistic interaction in 30% (6/20) and 15% (3/20) of the isolates when dealing with morin and curcumin, respectively, associated with fluconazole. In the interaction with amphotericin B morin presented synergism in 70% (14/20) of the isolates. Both compounds did not exhibit antagonism in any of the combinations. Changes in fungal cell morphology were observed, and in contact with red blood cells, presented low toxicity. The mechanisms of action revealed that both polyphenols act on the membrane of the fungal cell, inhibiting the synthesis of ergosterol and causing damage to it. These results demonstrate the potential of the compounds as natural bioactives, with great impact on the discovery of new drugs and their efficacy to be used in the treatment of fungal infections.
A Criptococose é uma doença fúngica causada por leveduras pertencentes ao complexo Cryptococus neoformans. O arsenal antifúngico disponível para o tratamento dessa doença ainda é restrito e está relacionado à elevada toxicidade e efeitos colaterais de alguns fármacos, causando grande prejuízo aos pacientes Neste contexto, se faz necessário descobrir novos bioativos para debelar as infecções e reduzir os efeitos adversos. As plantas apresentam rica fonte de metabólitos secundários bioativos como taninos, terpenóides, saponinas, alcalóides, flavonoides e outros compostos, registrados com expressiva propriedade antimicrobiana. Portanto, a pesquisa de compostos naturais e derivados de produtos naturais tem sido pertinente nos últimos anos, devido à sua relevância na descoberta de novos medicamentos, além do que, a associação de fármacos ou compostos, com diferentes mecanismos de ação tem sido utilizada como alternativa na terapia convencional. Nós avaliamos os mecanismos de ação e o efeito antifúngico dos compostos naturais, curcumina e morina, sobre isolados de Cryptococcus neoformans e Cryptococcus gattii. A atividade antifúngica foi averiguada através da determinação da concentração inibitória mínima (CIM) e fungicida mínima (CFM). Os polifenóis, apresentaram CIMs que variaram de 2 a 256 μg/mL. Os compostos apresentaram interação sinérgica em 30% (6/20) e 15%(3/20) dos isolados se tratando da morina e curcumina, respectivamente, associada ao fluconazol. Na interação com a anfotericina B a morina apresentou sinergismo em 70% (14/20) dos isolados. Ambos os compostos não apresentaram antagonismo em nenhuma das combinações. Alterações na morfologia da célula fúngica foram observadas, e em contato com as hemácias, apresentaram baixa toxicidade. Os ensaios de mecanismos de ação revelaram que ambos os polifenóis atuam na membrana da célula fúngica inibindo a síntese de ergosterol e causando danos a mesma. Estes resultados demonstram o potencial dos compostos como bioativos naturais, apresentando grande impacto na descoberta de novos fármacos e sua eficácia para serem utilizados no tratamento de infecções fúngicas.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.
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Os genes de histonas desacetilases (HDAC) são altamente conservados entre diferentes espécies e dirigem importantes processos epigenéticos na manutenção da estrutura e funcionamento do genoma. Entretanto, o papel das HDAC na regulação dos traços de virulência de patógenos permanece pobremente explorado. O fungo patogênico em humanos Cryptococcus neoformans passa por mudanças fenotípicas que promovem persistência e sobrevida dentro do hospedeiro ou em nichos ecológicos. Tais mudanças estão associadas à regulação diferencial da expressão gênica. Neste contexto, inicialmente foi avaliado o efeito de dois inibidores químicos de histona desacetilases (HDACi), o butirato de sódio (NaBut) e a tricostatina A (TSA), sobre as células de C. neoformans. Os resultados demonstraram que ambos inibidores foram capazes de afetar os principais traços de virulência de C. neoformans. Em seguida, foram identificados e deletados 8 genes de HDAC de Classe I e Classe II em C. neoformans. A maioria dos processos previamente associados à virulência em C. neoformans foi afetada pelo tratamento com HDACi eou pela deleção de genes: a capacidade de crescimento a 37 – 39 ºC, a formação da cápsula polissacarídica, a produção de melanina, as atividades de fosfolipase e de proteases, a formação de hifas de acasalamento e a integridade da parede celular. Os mutantes de HDAC também mostraram defeitos na sobrevivência intracelular quando co-cultivados com macrófagos murinos, assim como virulência comprometida nos modelos de infecção de Galleria mellonella e camundongos. A histona desacetilase Clr3 foi apontada como um importante regulador da virulência em C. neoformans. A linhagem de C. neoformans clr3mutante mostrou-se hipovirulenta em ambos os modelos de criptococose animal. Outrossim, a análise de RNA-seq indicou que a proteína Clr3 regula a expressão de vários genes importantes para a adaptação e sobrevivência de C. neoformans ao ambiente hospedeiro. Os resultados indicam que a remodelação da cromatina, por meio das conservadas HDAC, é um importante mecanismo envolvido na virulência de C. neoformans. _________________________________________________________________________________________________ ABSTRACT
Histone Deacetylase (HDAC) genes are highly conserved among different species, directing one of the most important epigenetic processes regulating gene expression – chromatin remodeling. However, the role of HDACs in the regulation of virulence traits in pathogens remains poorly explored. The human fungal pathogen Cryptococcus neoformans undergoes phenotypic changes to promote persistence and survival inside the host or in specific ecological niches. Very likely, these changes are associated with epigenetic regulation. In this context, we initially evaluated the effect of two chemical inhibitors of histone deacetylases (HDACi): Sodium butyrate (NaBut) and Trichostatin A (TSA). The results showed that both were able to impair the expression of the main virulence traits of C. neoformans. Based on these data, we identified and deleted eight genes encoding predicted class I/II HDACs in C. neoformans. In this way, we predicted that we would be able to assign specific function to each HDAC, especially in regards to virulence trait expression. Phenotypes of specific HDAC mutant strains indicate that individual proteins control non-identical but overlapping cellular processes associated with virulence. In the other hand, for some genes we also have observed an opposite regulation. Most processes previously related to virulence in C. neoformans were affected here, such as thermotolerance (growth at 37-39 oC); capsule, melanin and protease formation; and cell wall integrity. Additionally, defects in mating and hyphal development were observed for the clr3HDAC mutant strain. HDAC mutants also displayed defects in intracellular survival when co-cultured with activated macrophages, a finding highly correlated with altered virulence in vivo. Also, we tested the virulence in Galleria mellonella and mice. Overall our results support that the Clr3 histone deacetylase is a newly identified regulator of fungal virulence. The corresponding mutant was hypovirulent in both animal models of cryptococcosis. Furthermore transcriptional profiling shows that the Clr3 protein regulates the expression of many genes that are important for the adaptation of C. neoformans for survival inside the host. Our work displays that chromatin remodeling by the conserved histone deacetylases is an important mechanism behind the virulence of C. neoformans.