Unlike their bacterial counterparts, diseases caused by fungal pathogens are harder to treat due to a lack of discrete targets. Current antifungals are very broad spectrum and fall into three major classes: polyenes which target the cell membrane, azoles which target sterol biosynthesis and the echinocandins which target the cell wall. Recently a novel macrolide antibiotic produced by WAC 2288 was discovered in a co-culturing screen between various actinomycetes and pathogenic fungi. The active compound, a large type I polyketide compound called ibomycin, was specifically able to inhibit the growth of Cryptococcus neoformans but not Candida albicans. A combination of traditional and genetic approaches were used to identify the mode of action of ibomycin. Despite having characteristics associated with membrane perturbing agents such as fungicidal activity, causing hemolysis and even membrane localization in vivo, it does not seem that ibomycin disrupts the membrane in a sterol-dependent manner. We found evidence to suggest that ibomycin is not involved in disruption of cell wall biosynthesis based on localization in vivo and absence of viability rescue in presence of sorbitol. The results of haploinsufficiency and homozygous profiling of yeast deletion strains suggest that is no single protein target for ibomycin, but rather that membrane perturbation of ibomycin leads to downstream effects that impair vesicular trafficking and protein transport. Based on preliminary evidence, it is predicted that C. albicans is able to bind ibomycin but evades the induced toxic effects by barring access to its cell membrane. / Thesis / Master of Science (MSc)

The accuracy of the BioFire FilmArray Meningitis/Encephalitis (ME) panel for the identification of Cryptococcus has recently been called into question. The primary objective of this study was to assess the agreement between the BioFire ME polymerase chain reaction (PCR) and other markers of cryptococcal infection. This retrospective review identified five patients with cryptococcal meningoencephalitis, 4 of whom had a negative ME panel for Cryptococcus. All five cases had positive serum cryptococcal antigens, and three of five had a positive cerebrospinal fluid (CSF) culture for Cryptococcus. The BioFire ME panel does not appear to be reliable for ruling out Cryptococcus meningoencephalitis; multiple testing methods are recommended.

Cryptococcus neoformans is among the most common human pathogenic fungi and the leading cause of fungal meningitis. It consists of two divergent haploid serotypes A and D, as well as their hybrid serotypes AD. Hybridization between the two serotypes A and D is a widespread phenomenon in natural populations of this species. Studies have shown that serotype AD strains possess unique properties in several medically important traits: they are more virulent in animal models, more tolerant to antifungal drugs, more tolerant to UV radiation and other stresses than one or both parental serotypes. Despite the potential medical and evolutionary importance, relatively little is known about the phenotypic and genotypic consequences of the hybridization in C. neoformans. In my thesis, I present a series of studies that show: (1) recombination occurs at a very low frequency during hybridization between serotypes A and D; (2) serotypes A and D in C. neoformans have diverged from each other not only at the DNA sequence level, but also at the chromosomal structure level, both of which contributed to suppressed recombination and limited genetic introgression between the two serotypes; (3) though there is widespread hybridization, the hybrids seemed largely distinct and the genomes of the parental serotypes A and D populations are relatively unaffected by the hybrids in natural populations; and (4) the fixation of these serotype specific chromosomal rearrangements in the two serotypes, together with evidences of selective sweeps observed at both inversion junctions and genes located close to chromosomal inversions suggest that these rearranged regions (or genes surrounding these regions) were likely involved in the diversification between the two serotypes. My studies provide a genetic framework for future quantitative analyses of medically important traits of C. neoformans. / Thesis / Doctor of Philosophy (PhD)

In the majority of sexual eukaryotes, mitochondria are inherited predominantly from a single, usually the female, parent Like the majority of higher plants and animals, the pathogenic yeast Cryptococcus neoformans has two mating types (sexes), however, these two sexes are morphologically similar. In this study, I examined the distribution of the mating types and how mating types influence the inheritance of mitochondria in C. neoformans. My survey of mating type alleles in 358 isolates collected from four geographic areas in the US showed a biased distribution of mating type alleles with most isolates containing mating type a alleles. To characterize the role of mating type locus on mitochondrial inheritance, I constructed two pairs of congenic strains that differed only at the mitochondrial genome and mating type locus. Mating between these two pairs of strains demonstrated that uniparental inheritance in C. neoformans was controlled by the mating type locus and progeny predominantly inherited mitochondria from the mating type a parent. Specifically, we identified two genes within the mating type locus, SXIIa in mating type a strain and SXI2a in mating type a strain, that control mitochondrial inheritance. Disruption of these two genes resulted in biparental mitochondrial inheritance in sexual crosses. These two genes are the first ones identified capable of controlling uniparental mitochondrial inheritance in any organism. In addition, we determined that the deletion of the SXIIa gene enhanced the spread of mitochondrial introns in sexual crosses. This discovery is consistent with the hypothesis that uniparental lnheritance might have evolved to prevent the spread of selfish cytoplasmic elements. / Thesis / Doctor of Philosophy (PhD)

Orientador: João Claudio Thomeo / Banca: Daniela Alonso Bocchini Martins / Banca: Tatiane Iembo / Resumo: A criptococose é uma micose sistêmica que afeta principalmente o sistema nervoso central, causando meningite. Causada pela inalação de propágulos de fungos do gênero Cryptococcus sp, C neoformans acomete hospedeiros com imunodepressão celular, cujo maior contingente é representado por indivíduos com AIDS. A fonte principal de aquisição desta levedura são fezes de aves, principalmente pombos (Columba livia). C. gattii infecta indivíduos aparentemente imunocompetentes, sendo isolado principalmente em espécies de eucaliptos. C. albidus e C. laurentii aparece em casos de meningites, fungemias, pneumonias, abscessos pulmonares e infecções cutâneas. C.uniguttulatus é encontrado no trato gastrointestinal, fezes de pássaros e contaminante de leitos de animais e habitats de roedores. Termo-tolerância, parede celular, cápsula, adesão, e produção de enzimas são fatores importantes de virulência do gênero. São José do Rio Preto é a 5ª cidade entre os 100 municípios do estado de São Paulo em de casos de AIDS. Analisou-se morfologicamente e bioquimicamente 48 amostras clínicas e 155 ambientais. Amostras de líquor resultaram em 30 isolados da espécie C. neoformans, 17 de C. gattii e um de C. luteolus. Foram identificados 26 isolados de C.albidus, 22 de C. laurentii, 9 de C. neoformans, 5 de C. gattii e um de C.uniguttulatus nas amostras ambientais. A concentração inibitória mínima para antifúngicos foi determinada para todos os isolados por meio do teste de microdiluição em caldo padronizado pelo NCCLS. Os resultados obtidos mostraram resistência à anfotericina B e itraconazol nas cepas clínicas de C. neoformans e C. gattii, e as cepas de C.albidus e C. laurentii apresentaram resistência a 5-fluorcitosina, itraconazol e anfotericina B / Abstract: Cryptococcosis is a systemic mycosis that mainly affects the central nervous system, causing meningitis. Caused by inhalation of seedlings of fungi of the genus Cryptococcus sp, C. neoformans affects hosts with immunosuppression cellular, whose largest contingent is represented by individuals with AIDS. The main source of acquiring this yeast is feces of birds, especially pigeons (Columba livia). C. gattii infects individuals apparently immunocompetent, being isolated mainly in species of eucalyptus. C. albidus and C. laurentii appear in cases of meningitis, nosocomial fungemia, pneumonia, lung abscesses and skin infections. C. uniguttulatus is found in the gastrointestinal tract and feces of birds and contaminant of beds of animals and habitats of rodents. Thermo-tolerance, cell wall, capsule, accession, and production of enzymes are important factors in the virulence of the genus. São José do Rio Preto is the 5th city among the 100 cities in the state of São Paulo in cases of AIDS. We analyzed morphologically and biochemically 48 clinical samples and 155 environmental. Liquor samples resulted in 30 isolates of the species C. neoformans, 17 of C. gattii and one of C. luteolus. We identified 26 isolates of C. albidus, 22 of C. laurentii, 9 of C. neoformans, 5 of C. gattii and one of C. uniguttulatus in environmental samples. The minimal inhibitory concentration for antifungal agents was determined for all isolates by means of the test of broth microdilution standardized by the NCCLS. The results obtained showed resistance to amphotericin B and itraconazole in clinical strains of C. neoformans and C. gattii, C. albidus and C. laurentii exhibited resistance to 5-fluorocytosine, itraconazole and amphotericin B / Mestre

O objetivo desta tese foi desenvolver um modelo farmacocinético-farmacodinâmico (PK-PD) aplicável a avaliação de esquemas posológicos de antifúngicos sistêmicos no tratamento de infecções cerebrais associadas ao Cryptococcus neorformans. Inicialmente um modelo de infecção cerebral em ratos Wistar machos imunocompetentes foi estabelecido. Os animais foram inoculados a partir da administração iv de 1.106 UFC/mL na veia lateral caudal, de uma cepa de Cryptococcus neoformans var neoformans (ATCC 28957). A presença da levedura em cérebro, pulmão, fígado, rins e coração foi avaliada após 7, 10 e 14 dias. Paralelamente foram investigados os parâmetros bioquímicos (contagem de leucócitos, TGO, TGP, uréia, creatinina, albumina e CK) e a permeabilidade vascular cerebral com azul de Evans. Após 10 dias de inoculação foi produzida uma infecção com características semelhantes a doença em humanos. C. neoformans esteve presente em todos os tecidos investigados pelas análises histológicas e microbiológicas e diferenças nos níveis de albumina, ureia, TGP e CK, alteração no número de leucócitos (monócitos e neutrófilos) e elevação da permeabilidade cerebral ao azul de Evans foram observadas nos animais infectados. Após estabelecida e caracterizada a infecção, foi avaliada a farmacocinética plasmática e tecidual cerebral através da técnica de microdiálise, do fluconazol (FLU) (20 mg/kg, i.v. bolus) e do voriconazol (VRC) (5 mg/kg, i.v. bolus) em ratos Wistar sadios (n = 13) e infectados (n = 13). De posse dos dados das concentrações plasmáticas e teciduais vs tempo dos grupos sadios e infectados construiu-se um modelo farmacocinético populacional (PopPK) para cada fármaco investigado. A penetração cerebral do VRC demonstrou-se elevada nos animais infectados (fTsadios = 0,85 vs fTinfectados = 1,86). O modelo PopPK de dois compartimentos e eliminação por Michaelis Menten descreveu o perfil de concentrações versus tempo de VRC em plasma e tecido, simultaneamente. A covariável infecção foi incluída em V2 e VM. Observou-se o grande potencial do VRC para tratar meningite associada a C. neoformans, pois os níveis alcançados em tecidos infectados foram superiores aos valores descritos para CIM de VRC contra C. neoformans (0,03 - 0,5 μg/mL). A farmacocinética do FLC foi descrita através de um modelo PopPK de dois compartimentos com eliminação linear incluindo dados de concentrações plasmáticas e livres cerebrais para ambos os grupos investigados. Nesse modelo a covariável infecção foi atribuída ao parâmetro k21 e covariável peso foi atribuída aos parâmetros V1 e V2. De posse desse modelo popPK, foram investigados os desfechos farmacodinâmicos considerando o nível de exposição cerebral nas doses de 125 e 250 mg/kg para ratos e 400-2000 mg para humanos observado em tecido sadio e infectado através da probabilidade de atingir o alvo terapêutico (PTA - fASC/CIM = 389) do FLC usando simulações de Monte Carlo. Essas simulações demonstraram um uso limitado de fluconazol em monoterapia para o tratamento de meningite por C. neoformans. Após a etapa farmacocinética procederam-se os estudos farmacodinâmicos através da metologia de curvas de morte em função do tempo do fluconazol e voriconazol frente a C. neoformans. Os dados da curva de morte foram modelados adequadamente com o modelo PK-PD de Emax modificado incluindo um termo de atraso de crescimento. A CIM foi determinada para ambos os fármacos por microdiluição e os valores foram de 0,03 μg/mL para voriconazol e 0,5 μg/mL para fluconazol, indicando que esta cepa ATCC 28957 é sensível a ambos os fármacos. Os valores de k, EC50 e kmax foram determinados para vários múltiplos das CIM de cada fármaco (0,03×, 0,06×, 0,25×, 0,5×, 1× 4×, 16×, 32× e 64×). O valor médio de k foi de 0,38 h-1, EC50 foi de 1,26 ± 0,18 μg/mL e 0,32 ± 0,06 μg/mL e kmax foi de 0,95 ± 0,21 h-1 e 0,64 ± 0,12 h-1 para FLC e VRC, respectivamente. Por fim, de posse dos parâmetros calculados através do modelo PK-PD foram realizadas simulações dos desfechos de tratamento para meningite criptocócica no cenário clínico para ambos os fármacos após administração das doses 200 e 400 mg de voriconazol e 800 e 2000 mg de fluconazol por dez semanas. Através das simulações conclui-se que para fluconazol há 25% de insucesso na dose de 800 mg e 10% na dose de 2000 mg com um tempo médio de 3 semanas para erradicação da levedura. Para o voriconazol, o EC50 teve pouco impacto sobre a erradicação do fungo e, em todos os cenários foi observada uma erradicação completa do fungo em curto espaço de tempo (1 - 2 semanas). Os resultados incentivam o uso de voriconazol nos pacientes com meningite criptocócica e uma reavaliação do uso de fluconazol. / The aim of this thesis was to develop a pharmacokinetic-pharmacodynamic (PK-PD) model for the evaluation of systemic antifungal dosing regimens for the treatment of brain infections associated with Cryptococcus neorformans. Firstly a model of brain infection in immunocompetent male Wistar rats was established. The animals were inoculated by intravenously administration of 1. 106 CFU/mL of Cryptococcus neoformans var neoformans (ATCC 28957) into the tail lateral vein. The presence of yeasts in the brain, lung, the liver, kidneys and the heart was evaluated after 7, 10 and 14 days. The biochemical parameters (leucocytes counting, GOT, GPT, urea, creatinine, albumin and CK) and cerebral vascular permeability with Evans blue were investigated. After 10 days post inoculation an infection with characteristics similar in humans was produced. C. neoformans was present in all tissues investigated by histological and microbiological analyzes and differences in albumin, urea, GPT and CK levels, alterations in the number of leukocytes (monocytes and neutrophils), and elevation of cerebral permeability to Evans blue were observed in infected animals. After establishing and characterizing the infection, the plasma and cerebral tissue pharmacokinetics were evaluated by microdialysis after administration of fluconazole (FLU) (20 mg/kg, iv bolus) and voriconazole (VRC) (5 mg/kg, iv bolus) in healthy (n = 13) and infected Wistar rats (n = 13). A population pharmacokinetic model (PopPK) was build for each drug, based on data from plasma and tissue concentrations vs. time of healthy and infected groups. The brain penetration of voriconazole was shown to be high in infected animals (fThealthy = 0.85 vs fTinfected = 1.86) than in healthy ones. The two-compartment model with Michaelis Menten elimination best described the concentration of VRC in plasma and tissue. The covariate infection was included in V2 and VM. The great potential of voriconazole to treat meningitis associated with C. neoformans was observed, as the levels reached in infected tissues were higher than the values described for MIC against C. neoformans (0.03 - 0.5 μg/mL). The pharmacokinetics of FLC was described using a two-compartment model with linear elimination including data from plasma and brain free concentrations for both groups investigated. In this model the covariate infection was attributed to parameter k21 and covariate weight was assigned to parameters V1 and V2. With this popPK model, the pharmacodynamic outcomes were investigated considering the level of brain exposure at doses of 125 and 250 mg/kg for rats and 400 - 2000 mg for humans observed in healthy and infected tissue through the probability of attaining the target (PTA - fAUC/MIC = 389) of fluconazole using Monte Carlo simulations. These simulations demonstrated limited use of fluconazole in monotherapy for the treatment of C. neoformans meningitis. After the pharmacokinetics modeling, the pharmacodynamic studies were carried out using the methodology of time-kill curves of fluconazole and voriconazole versus C. neoformans. The kill curves data were suitably modeled with the modified Emax PK-PD model including a growth delay term. MIC was determined for both drugs by microdilution and values were 0.03 μg.mL-1 for voriconazole and 0.5 μg.mL-1 for fluconazole, indicating that this ATCC 28957 strain is sensitive to both drugs. The values of k, EC50 and kmax were determined for several MIC multiples of each drug (0.03 ×, 0.06 ×, 0.25 ×, 0,5×, 1 × 4 ×, 16 ×, 32 × and 64 ×). The mean value of k was 0.38 h-1, EC50 was 1.26 ± 0.18 μg.mL-1 and 0.32 ± 0.06 μg.mL-1 and kmax was 0.95 ± 0.21 h -1 and 0.64 ± 0.12 h-1 for FLC and VRC, respectively. Finally, the parameters obtained using the PK-PD model were used to simulate treatment outcomes for cryptococcal meningitis in the clinical setting for both drugs after administration of 200 and 400 mg of voriconazole and 800 and 2000 mg of fluconazole for 10 weeks. By the simulations it is concluded that for fluconazole there is a 25% rate of failure at the dose of 800 mg and 10% at the dose of 2000 mg with an average time of 3 weeks for eradication of the yeast. For voriconazole, the EC50 had little impact on fungus eradication and, in all scenarios complete eradication of the fungus was observed in a short time (1 - 2 weeks). The results encourage the use of voriconazole in patients with cryptococcal meningitis and a reassessment of fluconazole use.

Infecções fúngicas em humanos vem aumentando nos últimos anos e acometem principalmente pacientes imunocomprometidos, portadores do vírus HIV, transplantados ou com câncer. Os antifúngicos empregados no tratamento pertencem a poucos grupos de fármacos e o aparecimento de resistência antifúngica em muitos patógenos leva à necessidade de desenvolvimento de novos agentes antifúngicos. As cepas Trichosporon japonicum QU139 e Candida catenulata LV102 apresentam atividade killer sobre diversas leveduras patogênicas, apresentando bom potencial para desenvolvimento de novos agentes antimicrobianos. O objetivo do trabalho foi a otimização das condições para produção e detecção dos compostos antimicrobianos, para seu futuro uso terapêutico, e sua purificação. O efeito killer da cepa T. japonicum QU139 foi avaliado pelo método dos poços contra células sensíveis de Cryptococcus gattii C20 nos meios GYP, YM e Queijo em diferentes pH e temperaturas. A máxima atividade killer foi encontrada no meio GYP, pH 4,5 à 25°C após 24 horas de incubação para T.japonicum QU139 e C. catenulata LV102. Não foi possível isolar o composto antimicrobiano produzido pela levedura T.japonicum QU139 pelos métodos de isolamento de proteína/glicoproteína, corroborando a hipótese de que a toxina seja um glicolipídeo. / Human fungal infections have increased in the last years and affect mainly immunocompromised patients, carriers of HIV vírus, transplanted or with cancer. The antifungal agents used in treatment belong to a few groups of drugs and the increase of antifungal resistance in many pathogens leads to the necessity of developing new antifungal agents. Strains Trichosporon japonicum QU139 and Candida catenulata LV102 showed killer activity against several pathogenic yeasts, having a good potential for the development of new antimicrobial agents. The objective of the work was the optimization of conditions for production and detection of the antimicrobial compounds, aiming their future terapeutic use, and their purification. The killer effect of T. japonicum QU139 strain was evaluated by the well method against sensitive cells of Cryptococcus gattii C20 in media GYP, YM and Cheese in different pH and temperatures. The maximum killer activity was found in media GYP, pH 4.5, 25°C after 24 hours of incubation for T.japonicum QU139 and C. catenulata LV102. The isolation of the antimicrobial compound produced by the yeast T.japonicum QU139 was not possible by the methods for isolation of proteins/glicoproteins, corroborating the hypothesis that the toxin is a glycolipid.

A criptococose clinica foi observada por volta do primeiro dia da inoculação e a sobrevida dos animais 15 dias após-inoculação (PI). Isolamos C. neoformans no cérebro a partir do 5º dia (PI) e no pulmão a partir 11º dia (PI). No 1º dia (PI) observamos por meio do tecido cerebral que já havia um quadro inicial de infecção, presença de edema, que evoluiu durante todo o período. C. neoformans foi visualizado primeiramente nos vasos capilares, o que nos leva a sugerir que seja esta rota importante para a entrada da levedura no órgão. A meningite aguda aconteceu por volta do 7º dia quando visualizamos o microrganismo na meninge e com um discreto infiltrado inflamatório. A partir do 13º dia a doença evoluiu para crônica permanecendo até o óbito dos animais. A monoterapia com anfotericina B (AMB) reduziu a sobrevida dos animais, enquanto que, o tratamento isolado com fluconazol (FLC) prolongou a mesma. A associação AMB-FLC foi eficaz, quando iniciamos o tratamento 24 horas (PI), enquanto que, o mesmo tratamento iniciado a partir do 7º dia quando já tínhamos um quadro compatível de meningite aguda não foi satisfatório. É importante ressaltar que os resultados descritos foram referentes à inoculação com isolado sensível in vitro ao fluconazol, enquanto que, o tratamento não resultou em êxito quando empregamos isolado resistente in vitro ao fluconazol. / Clinical cryptococcosis was observed on day 1 postinoculation (PI), and the animals survived until day 15 PI. C. neoformans was isolated from brain tissue starting on day 5 PI, and from lung tissue starting on day 11 PI. On day 1 PI, signs of infection were already observed in brain tissue, with presence of edema, which evolved throughout the period. C. neoformans was first seen in the capillaries, suggesting that this is an important route for the entrance of the yeast into this organ. Acute meningitis occurred around day 7 PI, when the microorganism was observed in the meninges, along with a discrete inflammatory infiltrate. From day 13 PI onward the disease became chronic, persisting until the death of the animals. Treatment with amphotericin B (AMB) alone shortened the animals\' survival, while treatment with fluconazole (FLC) alone lengthened it. Treatment with the two drugs in association was effective when treatment was begun at 24 hours PI, however, when treatment was begun at day 7 PI, already with signs of acute meningitis, the effectiveness was unsatisfactory. It is important to emphasize that these results relate to inoculation with an isolate susceptible in vitro to fluconazole, while the treatment was not effective for an isolate resistant in vitro to fluconazole.

Cytokines play an important regulatory role during immune responses against pathogens. The outcome of an induced cytokine pattern is determined by many factors. It strongly depends on the nature of the pathogen and the host’s ability to control the quality and strength of cytokine signals. In pulmonary infection with Cryptococcus neoformans T helper (Th) 1 and Th17 cell subsets and their associated cytokines confer protection, whereas a Th2-biased response with production of interleukin (IL) -4 confers susceptibility. Since inappropriate Th responses often lead to death in immunosuppressed human patients, especially HIV-1 infected patients, this work aimed to elucidate mechanisms of Th2 induction and regulation by assessing the Th2 hallmark cytokine IL-4 in an experimental model of cryptococcosis. Therefore, a kinetic study of IL-4 expression during 70 days after intranasal infection was performed in susceptible mice. The analyses included characterization of pulmonary leukocytes and Th cell cytokine profiling. IL-4 profiling revealed Cryptococcus-specific IL-4 production not before six weeks after infection. This unexpected finding was further validated by equal results observed in a kinetic study done in IL-4 reporter mice. These mice express a green fluorescent protein simultaneously to IL-4 expression in the same cell and this protein can be detected by flow cytometry. Two cellular sources of IL-4 were identified: Th2 cells were found as expected, but also, as shown for the first time, eosinophilic granulocytes could be demonstrated to secrete IL-4. Next, the influence of eosinophils on pulmonary inflammation and disease development was investigated using ΔdblGATA-1 mice constitutively devoid of eosinophilic granulocytes. Experiments with infected ΔdblGATA-1 mice revealed novel regulatory functions of eosinophils in cryptococcosis. In the absence of eosinophils pulmonary Th cell recruitment was significantly diminished. In addition, Th2 polarization was reduced in ΔdblGATA-1 mice as shown by reduced numbers of Th2 cells expressing the Th2-related surface marker T1/ST2 and reduced albeit not absent IL-4 production by Th cells. In addition to reduced IL-4 production, in the absence of eosinophils Th cells with enhanced interferon-γ and IL-17 production were observed. However, control of pulmonary fungal growth was only slightly enhanced in the absence of eosinophils and dissemination of cryptococci to the brain was unaltered. This may be related to the shared IL-4 production by not only eosinophils but also Th2 cells. Blocking more than one cellular source of IL-4 could be required to prevent immunopathology. To test the hypothesis of gradual IL-4-dependent immunopathology, experiments were conducted using mice expressing only one allele of the IL-4receptor (R) alpha (α) chain (+/-) instead of two (+/+). Indeed, mono-allelic expression of the IL-4Rα resulted in an intermediate expression of the IL-4R on the surface of myeloid and lymphoid cells indicating a gene-dosage effect for IL-4R expression. Infected IL-4Rα+/- mice displayed reduced susceptibility as compared with IL-4Rα+/+ mice, and IL-4Rα-/- mice completely lacking IL-4R expression were found to be protected with survival for the complete time period of the experiment (i.e. up to 275 days). Reduced susceptibility found in infected IL-4Rα+/- mice was associated with decreased serum levels of immunoglobulin E, reduced mucus production by airway epithelia, attenuation of airway hyper-reactivity, and reduced formation of alternatively activated macrophages in lung parenchyma – pathophysiological features, which are typically found in experimental models of asthma but also in asthma of humans and animals. Since no up-regulation of IL-4R by the infection with Cryptococcus neoformans was found, the experimental pulmonary infection model used appears to be a very sensitive low-level IL-4 system. This work highlights the outstanding role of IL-4 and its different cellular sources as well as its receptor in cryptococcosis and provides novel insights into pathogenesis. Moreover, a cellular (i.e. eosinophils) and a molecular (i.e. IL-4R) target for treatment of this mycosis and possibly of asthma is provided.

Infecções fúngicas em humanos vem aumentando nos últimos anos e acometem principalmente pacientes imunocomprometidos, portadores do vírus HIV, transplantados ou com câncer. Os antifúngicos empregados no tratamento pertencem a poucos grupos de fármacos e o aparecimento de resistência antifúngica em muitos patógenos leva à necessidade de desenvolvimento de novos agentes antifúngicos. As cepas Trichosporon japonicum QU139 e Candida catenulata LV102 apresentam atividade killer sobre diversas leveduras patogênicas, apresentando bom potencial para desenvolvimento de novos agentes antimicrobianos. O objetivo do trabalho foi a otimização das condições para produção e detecção dos compostos antimicrobianos, para seu futuro uso terapêutico, e sua purificação. O efeito killer da cepa T. japonicum QU139 foi avaliado pelo método dos poços contra células sensíveis de Cryptococcus gattii C20 nos meios GYP, YM e Queijo em diferentes pH e temperaturas. A máxima atividade killer foi encontrada no meio GYP, pH 4,5 à 25°C após 24 horas de incubação para T.japonicum QU139 e C. catenulata LV102. Não foi possível isolar o composto antimicrobiano produzido pela levedura T.japonicum QU139 pelos métodos de isolamento de proteína/glicoproteína, corroborando a hipótese de que a toxina seja um glicolipídeo. / Human fungal infections have increased in the last years and affect mainly immunocompromised patients, carriers of HIV vírus, transplanted or with cancer. The antifungal agents used in treatment belong to a few groups of drugs and the increase of antifungal resistance in many pathogens leads to the necessity of developing new antifungal agents. Strains Trichosporon japonicum QU139 and Candida catenulata LV102 showed killer activity against several pathogenic yeasts, having a good potential for the development of new antimicrobial agents. The objective of the work was the optimization of conditions for production and detection of the antimicrobial compounds, aiming their future terapeutic use, and their purification. The killer effect of T. japonicum QU139 strain was evaluated by the well method against sensitive cells of Cryptococcus gattii C20 in media GYP, YM and Cheese in different pH and temperatures. The maximum killer activity was found in media GYP, pH 4.5, 25°C after 24 hours of incubation for T.japonicum QU139 and C. catenulata LV102. The isolation of the antimicrobial compound produced by the yeast T.japonicum QU139 was not possible by the methods for isolation of proteins/glicoproteins, corroborating the hypothesis that the toxin is a glycolipid.