Interactions of microorganisms, both prokaryotic and eukaryotic, with the metal antimony were studied. Of particular interest was the process of biomethylation. Volatilisation of trimethylantimony from inorganic antimony substrate by mixed inoculum (of environmental source) enrichment cultures was demonstrated to occur. Trimethylantimony was the sole volatile antimony species detected in incubations designed to promote the growth of clostridia, no stibine or other volatile methylated species were detected. Two Clostridium sp. were isolated from environmental enrichment incubations and three characterised Clostridium sp. were demonstrated to possess a biomethylating capability. Up to 21 μg. 1-1 involatile methylantimony species were detected in the culture medium of monoseptic incubations of the characterised Clostridium sp. The relative quantities of involatile mono-, di- and trimethylantimony species produced during the course of the cultivation period is consistent with trimethylantimony oxide being a final product of antimony biomethylation, with monoand dimethylantimony species appearing transiently in the cultures as intermediates of an antimony biomethylation pathway. The fungi Cryptococcus humicolus, Candida boidinii, Candida tropicalis, Geotrichum candidum and Saccharomyces cerevisiae were all demonstrated to possess a similar antimony biomethylating capability. Volatile and involatile methylantimony species were detected, with involatile species being the predominant form. Both stibine and trimethylantimony were detected in culture headspace gases of fungal incubations. Levels of trimethylantimony were higher in incubations supplied with antimony III substrate, whilst stibine was the predominant volatile antimony species in incubations supplied with V valency substrate. S. cerevisiae demonstrated the highest stibine generating capability with up to 0.3% substrate being transformed. Regardless of substrate, overall antimony biomethylation efficiency (to both volatile and involatile species) was low, indicating that this biotransformation does not form the primary mode of resistance to the metal. Less than 0.1% of antimony III substrate was biomethylated by C. humicolus, the most productive species in terms of formation of methylantimony compounds. The intracellular accumulation of methylated antimony species further belies the theory that antimony biomethylation constitutes a resistance mechanism. Study of C. humicolus revealed the biomethylation process to be enzymatic and inducible by arsenic but not by antimony. This may indicate that the enzymes of the arsenic biomethylation pathway are the likely biocatalysts for the biomethylation of antimony. The low efficiency of antimony biomethylation indicates that this is most likely a fortuitous process. A number of Gram-positive cocci isolated from soil and sediment were demonstrated to bioreduce antimonate to an unknown inorganic antimony III compound concurrently with lactate oxidation and biomass formation (as measured by protein). Up to 48% of the supplied antimonate was bioreduced. The demonstration of dissimilatory antimonate respiration adds this metal to the increasing list of known "unusual" electron acceptors such as uranium, arsenic, selenium, iron and manganese. These studies reveal some of the microbial interactions of microorganisms with the metal antimony, demonstrating the potential that microorganisms have to contribute to the biogeochemical cycling of antimony through biotransformation processes

Thesis (PhD (Microbiology))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy in Microbiology at the University of Stellenbosch. / ENGLISH ABSTRACT: The interaction between a soil yeast, Cryptococcus laurentii and a medicinal plant, Agathosma betulina (Berg.) Pillans (Rutaceae), was studied. Cryptococcus laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina and liquid chromatographytandem mass spectrometry analysis revealed that the yeast was capable of producing polyamines such as cadaverine and spermine. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when A. betulina seedlings grown under axenic and low nutrient conditions were inoculated with C. laurentii CAB 578 and cultivated for five months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. Furthermore, fluoromicroscopy, in combination with the fluorescent probes Calcofluor White and Fungolight revealed the presence of metabolic active yeast colonies on the rhizoplane. The first part of the study thus provided evidence for a symbiosis between A. betulina and C. laurentii CAB 578. During the second part of the investigation, the effect of this symbiosis on quantitative elemental distribution in A. betulina roots grown under axenic, nutrient-poor conditions was assessed using micro-particle-induced x-ray emission spectrometry. To aid in the interpretation of heterogeneous elemental distribution patterns, apoplastic barriers and endophytic C. laurentii CAB 578 in root tissues were located using fluorescence microscopy and transmission electron microscopy, respectively. The results showed that the average concentrations of iron, manganese and phosphorus were significantly (P < 0.05) higher within roots of yeast-inoculated plants, compared to control plants. It was shown that the yeast was not a root endophyte and that elemental enrichment in the epi/exodermal-outer cortical tissues correlated with the presence of Casparian bands in the exodermal cells of both treatments. This was the first report describing the role of a soil yeast as a plant nutrient-scavenging microsymbiont. In the final part of the investigation, the effect of C. laurentii CAB 578 on the photosynthetic nitrogen, phosphorus and water-use efficiencies, as well as the carbon economy of A. betulina was studied. Agathosma betulina plants inoculated with C. laurentii CAB 578, as well as controls, were grown under axenic conditions and the following parameters measured: Apparent photon yield, foliar nitrogen and phosphorus concentrations, leaf dark respiration, maximum photosynthetic rate, photosynthetic nitrogen-use efficiency, photosynthetic phosphorus-use efficiency, photosynthetic wateruse efficiency, root construction cost, stomatal conductance, substomatal CO2 and transpiration rate. The data showed that the higher photosynthetic resource-use efficiencies in yeast-inoculated plants were a consequence of higher maximum rates of CO2 assimilation, which was not related to foliar nitrogen and phosphorus content. We hypothesize that photosynthetic stimulation in yeast-inoculated plants was a result of the increased demand for photosynthates of the yeast-root symbiosis. In summary, the study revealed that a symbiosis exists between A. betulina and the soil yeast C. laurentii CAB 578. This interaction has a significant effect on the size of the yeast population as well as on the physiology of the plant. / AFRIKAANSE OPSOMMING: Die interaksie tussen ‘n grondgis, Cryptococcus laurentii, en ‘n medisinale plant, Agathosma betulina (Berg.) Pillans, is ondersoek. Cryptococcus laurentii CAB 578 is vanuit die risosfeer van A. betulina in sy natuurlike omgewing geisoleer en vloeistof chromatografie tandem massa spektrofotometriese analise het bewys dat die gis poliamiene insluitend kadaverien en spermien produseer. Dit is bevind dat die eksogene aanwending van poli-amiene wortelgroei bevorder. Hierdie bevinding staaf die waargenome 51% verhoging in wortelgroei van mikroob-vrye A. betulina saailinge geinokuleer met C. laurentii CAB 578 en gekweek vir vyf maande onder lae nutriënt kondisies in ‘n glashuis. Met gebruik van die grond verdunningsplaat-metode, is dit verder bewys dat gisgetalle hoër was in die teenwoordigheid van wortels as in die omliggende grond. Dit is ook bewys met die gebruik van die fluoressente peilers Calcofluor White en Fungolight, in kombinasie met fluoressensie-mikroskopie, dat metabolies aktiewe giste die wortels se oppervlak gekoloniseer het. Die eerste deel van die studie het dus bewys dat ‘n simbiose tussen A. betulina en C. laurentii CAB 578 bestaan. Tydens die tweede deel van die ondersoek is die effek van C. laurentii CAB 578 op die konsentrasie en verspreiding van elemente binne A. betulina wortels, gekweek onder lae-nutriënt, mikroob-vrye kondisies, bepaal met behulp van mikro-partikel geinduseerde X-straal emissie spektrofotometrie. Om die interpretasie van heterogene verspreidingspatrone van die onderskeie elemente te ondersteun, is daar met behulp van fluoressensie en transmissie-elektron-mikroskopie vir apoplastiese versperrings en endofitiese C. laurentii CAB 578 in die wortelweefsel getoets. Dit is bevind dat die gemiddelde konsentrasie van fosfaat, mangaan en yster beduidend (P < 0.05) hoër was in gis-geinokuleerde plante, as in kontrole plante. Die gis was nie ‘n wortel endofiet nie en elementale verryking in die epi/eksodermale-buitenste korteks weefsels het gekorreleer met Casparian bande in die eksodermale selle van beide behandelings. Hierdie was die eerste verslag wat die rol van ‘n grondgis as ‘n nutriënt-bekommende mikrosimbiont vir plante beskryf het. In die laaste gedeelte van hierdie ondersoek is die effek van C. laurentii CAB 578 op die fotosintetiese fosfaat, stikstof en water-verbruiksdoeltreffendheid, asook die koolstof ekonomie in mikroob-vrye Agathosma betulina plante geinokuleer met C. laurentii CAB 578 asook kontrole plante bestudeer. Die volgende parameters is getoets: Blaar donker respirasie, blaar fosfaat en stikstof konsentrasies, fotosintetiese fosfaatverbruiksdoeltreffendheid, fotosintetiese stikstof-verbruiksdoeltreffendheid, fotosintetiese water-verbruiksdoeltreffendheid, huidmond konduktansie, huidmond CO2 konsentrasie, klaarblyklike foton opbrengs, maksimum fotosintetiese spoed, wortel konstruksie-koste, en transpirasie spoed. Die resultate het getoon dat die hoër maksimum fotosintestiese spoed in gis-geinokuleerde plante gelei het tot ‘n hoër fotosintetiese verbruiksdoeltreffendheid van fosfaat, stikstof en water en dat dit nie verband gehou het met blaar fosfaat en stikstof konsentrasies nie. Dit word voorgestel dat die stimulasie van fotosintese in gisgeinokuleerde plante ‘n gevolg is van die verhoogde aanvraag na fotosintaat deur die giswortel simbiose. Om op te som, die bevindings van hierdie studie het bewys dat ‘n simbiose tussen A. betulina en C. laurentii CAB 578 bestaan. Hierdie simbiose het ‘n beduidende effek op die populasie grootte van die gis sowel as die fisiologie van die plant.

Introdução: A meningite criptocócica causa elevada mortalidade, sobretudo em pacientes acometidos de alguma condição imunossupressora. O objetivo deste estudo foi identificar fenômenos de baixa suscetibilidade a antifúngicos e outros preditores clínicos que possam explicar falha terapêutica e recidiva da neurocriptococose Metodologia: Foram avaliados 96 casos com coleta de dados clínicos epidemiológicos e laboratoriais. Os isolados foram identificados quanto a genótipo molecular, suscetibilidade de anfotericina B (AMB) e fluconazol (FCZ) pela determinação da concentração inibitória mínima (Minimal Inhibitory Concentration, MIC), nível de heteroresistência ao FCZ (NHF) e determinação do tempo de morte frente AMB (Time-Kill, TK). Foram selecionados isolados heterorresistentes para análise quantitativa de DNA por PCR em tempo Real, expressão de bombas de efluxo por citometria de fluxo e isolados tolerantes a AMB para estudo de resistência ao estresse oxidativo. Foi realizada análise univariável e múltipla usando regressão logística para identificar preditores de óbito hospitalar e de um desfecho composto definido pelo óbito, encaminhamento para unidade de terapia intensiva ou recidiva 6 meses após alta hospitalar. Resultados: A maioria dos pacientes eram imunodeprimidos, com CD4 de 2 a 722 cel./mm3 e 96,7% eram portadores do HIV. Foram identificados 93 isolados de C. neoformans, sendo 76 do genótipo VNI e 17 VNII e 3 C. gattii, todos VGII. MIC de AMB variou de 0,012 a 0,94 mg/L e MIC de FCZ estiverem entre 0,12 e 64 mg/L. Resistência a FCZ (MIC>16mg/L) foi maior em VNI do que em VNII (p=0,03). Dentre os isolados VNI, 64,5% sofreu atividade fungicida até as 24h (TK24) de exposição à AMB e 6 cepas VNI não sofreram ação fungicida (TK>72). A maioria dos isolados VNII (64,7%) apresentou TK24. Os 3 isolados VGII sofreram atividade fungicida a partir de TK24. A maioria dos isolados VNI, VNII e todos os isolados VGII apresentaram alto NHF (>32mg/L). Diferença no NHF de acordo com os genótipos foi observada (p=0,005). No modelo múltiplo, as variáveis associadas significativamente ao óbito foram: idade em anos (OR=1,08;IC95%=1,02-1,15), contagem de leveduras no líquido cefalorraquidiano em logaritmo (LCR) (OR=1,66;IC95%=1,21-2,28) e uma variável composta por hipertensão arterial sistêmica ou diagnóstico de edema cerebral ou dilatação ventricular por tomografia (OR=35,68;IC95%=4,97-256,31). Para o desfecho composto, as variáveis associadas foram: contagem de leveduras do 1D em logaritmo (OR=1,50; IC95%=;1,20-1,86; p=<0,001), cultura de sangue positiva para Cryptococcus spp. (OR=3,30; IC95%=0,86-12,59; p=0,08) e descrição de neurotoxoplasmose (OR=18,62; IC95%=1,85-187,5; p=0,01). As associações foram consistentes em modelos de sobrevida. Conclusão: Foi possível descrever genótipos mais frequentes e identificar fatores genéticos, como aumento da expressão de genes e bombas de efluxo, relacionados à resistência aos fármacos. Nenhum dos testes de suscetibilidade esteve associado com os desfechos. Variáveis obtidas nos primeiros dias de internação mostraram utilidade para predizer o prognóstico em pacientes com meningite criptocóccica. Estes preditores podem ajudar a identificar os casos com maior potencial de óbito e que necessitam da otimização dos recursos terapêuticos. / Background: Cryptococcal meningitis causes high mortality in immunocompromised patients. The objective of this study was to identify the phenomena of low susceptibility to antifungal and other clinical predictors that may explain therapeutic failure and relapse of neurocryptococcosis Methodology: It was analyzed 96 cases with clinical and epidemiological data. The respective isolates were identified for genotype, susceptibility profile by Minimal Inhibitory Concentration (MIC), FCZ heteroresistance level (NHF), and time to death determination against 1 mg / L BMA (Time-Kill, TK). We isolated heteroresistant DNA expression analysis by real-time PCR, expression of efflux pumps by flow cytometry and, some isolates tolerant to AMB were selected to study resistance to oxidative stress. Univariable and multiple analyses using logistic regression were performed to identify predictors of in-hospital mortality and a composed outcome defined by death, referral to the intensive care unit and relapse 6 months after hospital discharge. Results: Most of the patients were immunocompromised, with CD4 range from 2 to 722 cells/mm3 and 96.7% patients HIV-positive. It was analyzed 93 strains of Cryptococcus neoformans of which 76 were genotype VNI and 17 were VNII and 3 were C. gattii, all were VGII. AMB MIC ranged from 0.012 to 0.94 mg/L and FCZ MIC were between 0.12 and 64 mg/L. Resistance to FCZ (MIC>16mg/L) was higher to VNI than VNII (p=0.03). Among the VNI strains, 64.5% had fungicidal activity up to 24h (TK24) of exposure to AMB and 6 VNI did not present this activity until 72h (TK> 72). Most VNII strains (64.7%) had TK24. The 3 VGII strains presented fungicidal activity from TK24. According to the MIC, all strains were susceptible to AMB. The majority of VNI strains (93.4%) and VNII (76.5%) and 3 VGII strains showed high NHF (>32mg/L) and it was observed statistical difference according to the genotypes VNI and VNII (p=0.005). At the multiple analysis, the variables significantly associated with the death were the age in years (OR=1.08,95%CI=1.02-1.15), the cerebrospinal fluid (CSF) yeasts count-log (OR=1.66,95%CI=1.21-2.28), and a variable composed of systemic arterial hypertension or diagnosis of cerebral edema or ventricular dilatation by tomography (OR=35.68,95%CI=4.97-256.31). At the composed outcome, the variables associated were: CSF yeasts count-log (OR=1,50; IC95%=;1,20-1,86; p=<0,001), positive blood culture for Cryptococcus spp. (OR=3,30; IC95%=0,86-12,59; p=0,08) and neurotoxoplasmosis (OR=18,62; IC95%=1,85-187,5; p=0,01). The associations were consistent at survival models. Conclusion: It was possible to describe more frequent genotypes and to identify genetic factors, such as increased gene expression and efflux pumps, related to drug resistance. The antifungal susceptibilities were not associated with the outcomes. were not associated with outcomes. Variables available in the first days of hospitalization showed utility to predict the prognosis in patients with cryptococcal meningitis. These predictors can help to identify the cases with higher potential of death and that require the optimization of the therapeutic resources.

A radiação solar é um dos principais fatores responsáveis pelo controle das populações de fungos no meio ambiente. A inatividade de estruturas de dispersão e de infecção pela radiação UVA e UVB é ainda mais importante no controle de fungos que se dispersam pelo ar e que podem infectar o hospedeiro, quando inalados, como é o caso de espécies do gênero Cryptococcus. C. neofarmans é capaz de produzir melanina na presença de substratos exógenos, como a L-dopa. A melanização é capaz de proteger o fungo contra diversos fatores ambientais bióticos e abióticos. Neste trabalho, foi avaliado o efeito de exposições a uma intensidade ambiental de radiação UVB (I.000mW m-2) na sobrevivência de células melanizadas e não melanizadas de quatro linhagens de Cryptococcus neoformans e de quatro linhagens de Cryptococcus laurentii. Foi determinada a sobrevivência relativa (sobrevivência das células expostas em relação à das células não expostas à radiação de células com 2, 4, 6 e 8 dias, crescidas em meio com e sem L-dopa, após exposição às doses de 1.8 e 3.6 kJ m-2. Tanto a irradiância como as doses, são observadas no meio ambiente, mesmo em regiões temperadas. Foram observadas diferenças na tolerância à radiação UVB, tnato entre as linhagens de C. neoformans, como entre as linhagens de C. laurentii. As linhagens de C. neoformans, foram observbadas diferenças na tolerância à radiação ao longo do desenvolvimento, tanto nas células melanizadas como nas não melanizadas. Na maioria das situações (linhagem, tempo de crescimento, dose de UVB), não houve diferença significativa entre a tolerância das células melanizadas e não melanizadas e, quando isso ocorreu, a diferença foi menor do que a observada anteriormente com UVC. E, C laurentii, não houve diferença na tolerância à radiação ao longo do desenvolvimento das células melanizadas e não melanizadas. Também não foram observadas diferenças significativas entre a tolerância das células melanizadas e não melanizadas em nenhuma das linhagens. / Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. The inactivation of dispersal and infection structures by UVA and UVB radiation is even more important for the control of fungi that disperse through the air and that can produce melanin in the presence of oxogeneous substrates such as L-dopa. Melanization can protect the fungus against various biotic and abiotic factors. In the present study, we investigated the effect of exposure to an environmental intensity of UVB radiation of 1,000 mWm-2 on the survival of melanized and non-melanized cells of four strains of Cryptococcus neoformans and of four strains of Cryptococcus laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of 2, 4, 6 and 8 day cells grown on medium with and without L-dopa was determinated after exposure to doses of 1.8 and 3.6kJ m-2. Both irradiance and these doses are observed in the environment even in temperate regions. Difference in tolerance to UVB radiation were observed both in the C. neoformans and C. laurentii strains. The C. neofarmans strains were more sensitive to radiation than the C. laurentii strains. In C. neoformans, diferences in tolerance to radiation were observed during development both in melanized and non-melanized cells. For most features (strains, time of growth and UVB dose) there was no difference in tolerance between melanized and non-melanized cell and when differences occurred they were smaller than those previously observed with UVC. In C. laurentii, there was no difference in tolerance to radiation during development between melanized and non-melanized cells., Also, no significant differences in tolerance were observed between melanized and non-melanized cells of any strain

Made available in DSpace on 2014-07-29T15:26:23Z (GMT). No. of bitstreams: 1 TESE ORIONALDA.pdf: 2127265 bytes, checksum: 6265bfc95f48ea602e76fd036eed1634 (MD5) Previous issue date: 2010-10-25 / Diferentes características fenotípicas como formação de cápsula polissacarídica, a expressão de lacase determinando a melanização em substratos fenólicos e produção de enzimas é prováveis fatores de virulência presentes nas espécies do complexo Cryptococcus neoformans. Neste trabalho foi avaliada a espessura da cápsula e da parede celular de isolados de C. neoformans. A atividade da fosfolipase e a suscetibilidade in vitro de C. neoformans à anfotericina B, voriconazol e ao extrato bruto da planta Pimenta pseudocaryophyllus foram também realizados para estes isolados. Os constituintes da célula como cápsula e parede celular foram ainda analisados quando sob a ação dos antifúngicose do extrato bruto da planta. A melanização das células foi obtida usando-se o meio mínimo adicionado de L-DOPA, a produção de fosfolipase foi feita em meio contendo gema de ovo, enquanto a suscetibilidade in vitro do microrganismo foi realizada pelo método de microdiluição em caldo seguindo o protococlo do CLSI. As células extraídas de um meio indutor de cápsula foram submetidas a uma concentração correspondente a 0,5 vezes da CIM obtida no teste de suscetibilidade in vitro, sendo medido o tamanho da cápsula e parede de 30 células de cada isolado e a média destes valores foi calculada. Os valores da espessura da cápsula de isolados de C. neoformans melanizados variaram de 1,20 a 1,90 &#956;m e de 2,63 a 5,12 &#956;m em não melanizados, quando submetidos ao meio de indução de cápsula. Com relação à parede, a variação foi de 0,80 a 1,09 &#956;m em células melanizadas e de 0,40 a 0,49 &#956;m em não melanizadas. A diferença para as medidas de cápsula, como de parede mostrou-se com valores estatisticamente significantes (p <0,05). Todos os isolados de C. neoformans foram capazes de produzir fosfolipase, sendo que os isolados melanizados apresentaram atividade desta enzima em 85,3%, enquanto nos não melanizados o percentual foi de 79,5%. A suscetibilidade do microrganismo melanizado ou não aos antifúngicos e ao extrato da planta mostrou-se equivalente, com a variação de CIM para voriconazol de 0,062-0,125 &#956;g/mL, 0,125-0,5 &#956;g/mL para anfotericina B e de 64 a 128 &#956;g/mL para o extrato bruto de P. pseudocaryophyllus. Os antifúngicos vorioconazol e anfotericina B e extrato bruto de P. pseudocaryophyllus, induziram redução de espessura da cápsula e parede celular de C. neoformans, quando comparados com o controle (ausência do fármaco ou da extrsto de planta) independente da presença ou não da melanina. As alterações verificadas na cápsula e parede celular de C. neoformans neste estudo poderão servir de guias para outras pesquisas direcionadas aos efeitos de antifúngicos na patogenia de criptococose.

Ureases (EC 3.5.1.5) são metaloenzimas que hidrolisam uréia para produzir amônia e dióxido de carbono. Estas enzimas, que são amplamente encontradas em fungos, bactérias e plantas, compartilham de estruturas similares. A presença de urease em várias bactérias patogênicas (Helicobacter pylori e Proteus mirabilis, p.e) está fortemente correlacionada com a patogênese em doenças humanas. Muitos fungos de importância médica possuem atividade ureásica, entre eles citamos Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii, e espécies de Trichosporon e Aspergillus. C. neoformans é uma levedura que produz vários fatores de virulência conhecidos, como presença de cápsula polisacarídica, produção de melanina e capacidade de desenvolvimento a 37ºC. A maioria de isolados clínicos produz grandes quantidades de urease e muitos autores sugerem que a urease de Cryptococcus exerça uma função importante na patogênese, porém com mecanismos ainda não esclarecidos. Cryptococcus gattii – sorotipo B, tipo molecular VGII, linhagem R265, com capacidade de infectar pacientes imunocompetentes, causou uma epidemia na Ilha de Vancouver (Canadá) entre 1999 e 2003. Neste trabalho desenvolvemos um procedimento de purificação e apresentamos a caracterização físico-química e cinética da urease de C. gattii, cepa R265, após ter sido purificada na razão de 539 vezes. A massa molecular estimada foi de 120 kDa, Km 2,0 mM para uréia, pH ótimo 8,0. O ácido acetohidroxâmico demonstrou ser um bom inibidor em concentrações micromolares, enquanto ρ-hidroximercuriobenzoato causou inibição em concentrações mais altas, comparado a outras ureases. Espera-se que estudos adicionais com essa urease purificada permitam investigar propriedades biológicas independentes da atividade ureolítica e estabelecer sua contribuição para a patogênese da criptococose. / Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea to produce ammonia and carbon dioxide. These enzymes, which are found in fungi, bacteria, and plants show very similar structures. The presence of urease in many pathogenic bacteria (Helicobacter pylori and Proteus mirabilis) is strongly correlative with pathogenesis in human diseases. Many medically important fungi have urease activity, among which are Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii, and species of Trichosporon and Aspergillus. C. neoformans is a heterothallic yeast with several known virulence factors, including a polysaccharide capsule, melanin production, and the ability to grow at 37°C.The majority of clinical isolates produce large amounts of urease and several authors suggest that Cryptococcal urease play an important role in pathogenesis but with unclear mechanisms but probably by different routes dependent and independent of ureolytic activity, althought many questions are unclear. We wanted to further investigate the role of urease in biological properties by using Cryptococcus gattii as a pathogen model. C. gatti – serotype B, molecular type VGII, R265 strain, which has capacity to infect immunocompetent individuals, caused an outbreak on Vancouver Island in Canada from 1999 to 2003. This work presents the physicochemical characterization of a urease from C. gatti strain R265 after a 539 fold purification. The estimated native molecular mass was 120 kDa, Km 2.0 mM urea, pH optimum 8.0. Aceto hydroxamic acid was a strong inhibitor at low concentration while ρ-hidroxy mercuribenzoate needed higher concentrations for inhibition compared to other ureases.

La criptococosis es una micosis profunda que puede afectar al ser humano y a diversos animales. Tiene un curso crónico ó subagudo. El agente causal es una levadura capsulada llamada Cryptococcus neoformans. Este proceso puede presentarse en personas sanas, pero afecta sobre todo a sujetos inmunodeprimidos. La infección se inicia por la inhalación de las levaduras y diseminación por vía hematógena al sistema nervioso central.El objetivo principal de este trabajo ha sido de disponer de datos para conocer la epidemiología de la criptococosis en España.- Se han recopilado 184 cepas de 8 diferentes áreas geográficas de España, 128 de orígen clínico humano ( 95 % VIH + y ADVP), 13 de orígen animal, y 43 de muestras ambientales. - Se han estudiado muestras de polvo doméstico de 79 domicilios para comprobar si podían ser una fuente de infección. Seis muestras provenían de domicilios de pacientes con SIDA y criptococosis, 11 de pacientes VIH(+) sin criptococosis, y 62 de personas sanas. No se ha aislado C. neoformans en niguna muestra. El polvo doméstico no parece ser un reservorio de importancia para la transmisión de la criptococosis.- El estudio de las biovariedades mediante el crecimiento en medio de L-canavanina-glicina-azul de bromotimol (CGB) junto con la asimilación de D-prolina y D-triptófano ha permitido demostrar por primera vez, la presencia en nuestro país de la variedad gattii de C. neoformans. Esta variedad produjo 5 brotes epidémicos de criptococosis en cabras en la provincia de Cáceres, con un alto porcentaje de animales afectados. Todas sufrían de síntomas respiratorios severos asociados a caquexia y síntomas neurológicos, y con un 100 % de mortalidad en los animales enfermos. Todas las cepas pertenecían al serotipo B.- Se obtuvieron sueros policlonales de los diferentes serotipos mediante inmunización de conejos. Se procedió por técnica de aglutinación al estudio del serotipado de todas las cepas. La distribución de los serotipos no es homogénea, varía según las regiones geográficas. El serotipo A es predominante, sin embargo hay una elevada tasa del serotipo D en las muestras clínicas y ambientales.- El estudio de las concentraciones inhibitorias mínimas (CIM) mediante la técnica de microdilución ha mostrado que el nivel global de resistencias es muy bajo. No se ha hallado ninguna cepa con CIM>1 mg/ml para anfotericina B. Las cepas del serotipo A mostraron menor sensibilidad. La 5-fluorocitosina ha resultado el antifúngico menos activo. El fluconazol ha mostrado menor actividad que el itraconazol. Tres cepas tenían CIM>64 mg/ml. El serotipo D mostró menor sensibilidad a los dos triazoles que el resto de los serotipos (P<0.00001). Las cepas de la var. gattii mostraron una sensibilidad uniformemente baja, y las ambientales tenían sensibilidades similares a las clínicas.- El método colorimétrico Sensititre utilizado para el estudio de las sensibilidades de 42 cepas de C. neoformans fué útil por su fácil ejecución y lectura de puntos de corte, sin embargo es un sistema que proporciona valores más bajos de CIM que el método de referencia.- Se determinaron las CIM de un nuevo antifúngico derivado de las sordarinas (GM 237354) en 190 cepas de C. neoformans. Se obtuvieron 5 cepas con valores de 2 mg/ml. La var. gattii es la que mostró menor sensibilidad. Se obtuvieron diferencias significativas entre los serotipos A y AD con respecto al serotipo B.- La aplicación de la técnica molecular del RAPD ha permitido demostrar que por lo menos coexisten 2 genotipos diferentes de la var. gattii aislada en cabras. Uno de los patrones parece ser el dominante, se ha hallado en todas los brotes epidémicos, mientras que el otro sólo se detectó en una zona. Con la misma técnica se ha podido discriminar entre diferentes aislamientos de un mismo paciente con criptococosis. / Cryptococcosis is a deep mycosis that can affect human beings and several types of animals. It can have either a chronic or a subacute course. Its causal agent is an encapsulated yeast called Cryptococcus neoformans. This process can appear in healthy humans although it mainly affects immunodepressed individuals. The infection begins by inhalation of the yeast and dissemination to the central nervous system by the haematogenous route.The main purpose of this study was to obtain information on the epidemiology of cryptococcosis in Spain.- One hundred and eighty-four strains were collected from eight different geographical areas in Spain. One hundred and twenty-eight strains were of human clinical origin (95% HIV + IDU), 13 of animal origin and 43 were environmental samples. - Samples of household dust proceeding from 79 homes were studied to check whether they could be a source of infection. Six samples proceeded from the homes of patients with AIDS and cryptococcosis, 11 from HIV(+) patients without cryptococcosis and 62 from healthy individuals. C. neoformans was not isolated in any of the samples. Domestic dust does not appear to be a reservoir of importance in the transmission of cryptococcosis.- The study of biovarieties using growth in L-canavanine-glycine-bromothymol blue (CGB) medium together with D-proline and D-tryptophan assimilation made it possible to prove, for the first time, the presence in our country of the Cryptococcus neoformans variety gattii. In the province of Cáceres, this variety produced five epidemic cryptococcosis outbreaks in goats, with a high percentage of affected animals. All the animals had severe respiratory symptoms associated with cachexia and neurological symptoms, with 100% mortality in sick animals. All were serotype-B strains.- Polyclonal serums of the various serotypes were obtained by immunisation of rabbits. The agglutination technique was used to carry out a serotyped study of all the strains. The distribution of the serotypes was not homogeneous but varied depending on the geographical regions. Serotype A was predominant although there was a high rate of serotype D in the clinical and environmental samples.- The study of the minimal inhibitory concentrations (MIC) using the microdilution technique proved that the global resistance level was very low. No strain with a MIC > 1 mg/ml was found for amphotericin B. Serotype-A strains showed less sensitivity. 5-fluorocytosine proved to be the least active antifungal agent. Fluconazol proved to be less active than itraconazole. Three strains had a MIC > 64 mg/ml. Serotype D showed less sensitivity to the two triazoles than the rest of the serotypes (P < 0.00001). All the strains of the gattii variety showed uniformly low sensitivity and the sensitivity of the environmental strains was similar to that of the clinical ones.- The colorimetric method Sensititre used for sensitivity testing in 42 strains of C. neoformans was useful because of its ease of implementation and breakpoint reading. However, this system gave lower values of MIC than the reference method. - The MICs of the new antifungal agent derived from sordarins (GM 237354) were determined in 190 strains of C. neoformans. Five strains with values of 2 mg/ml were obtained. The variety gattii is that which showed least sensitivity. Significant differences were obtained between serotypes A and D with respect to serotype B.- Application of the molecular technique RAPD demonstrated the coexistence of at least two different genotypes of the var. gattii isolated in goats. One of the patterns appears to be dominant, it is found in all epidemic outbreaks, whereas the other was only detected in one area. It was possible to distinguish between different isolations of a same patient with cryptococcosis using the same technique.

Thesis (Ph. D.)--University of Nevada, Reno, 1999. / Includes bibliographical references. Online version available on the World Wide Web.

A radiação solar é um dos principais fatores responsáveis pelo controle das populações de fungos no meio ambiente. A inatividade de estruturas de dispersão e de infecção pela radiação UVA e UVB é ainda mais importante no controle de fungos que se dispersam pelo ar e que podem infectar o hospedeiro, quando inalados, como é o caso de espécies do gênero Cryptococcus. C. neofarmans é capaz de produzir melanina na presença de substratos exógenos, como a L-dopa. A melanização é capaz de proteger o fungo contra diversos fatores ambientais bióticos e abióticos. Neste trabalho, foi avaliado o efeito de exposições a uma intensidade ambiental de radiação UVB (I.000mW m-2) na sobrevivência de células melanizadas e não melanizadas de quatro linhagens de Cryptococcus neoformans e de quatro linhagens de Cryptococcus laurentii. Foi determinada a sobrevivência relativa (sobrevivência das células expostas em relação à das células não expostas à radiação de células com 2, 4, 6 e 8 dias, crescidas em meio com e sem L-dopa, após exposição às doses de 1.8 e 3.6 kJ m-2. Tanto a irradiância como as doses, são observadas no meio ambiente, mesmo em regiões temperadas. Foram observadas diferenças na tolerância à radiação UVB, tnato entre as linhagens de C. neoformans, como entre as linhagens de C. laurentii. As linhagens de C. neoformans, foram observbadas diferenças na tolerância à radiação ao longo do desenvolvimento, tanto nas células melanizadas como nas não melanizadas. Na maioria das situações (linhagem, tempo de crescimento, dose de UVB), não houve diferença significativa entre a tolerância das células melanizadas e não melanizadas e, quando isso ocorreu, a diferença foi menor do que a observada anteriormente com UVC. E, C laurentii, não houve diferença na tolerância à radiação ao longo do desenvolvimento das células melanizadas e não melanizadas. Também não foram observadas diferenças significativas entre a tolerância das células melanizadas e não melanizadas em nenhuma das linhagens. / Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. The inactivation of dispersal and infection structures by UVA and UVB radiation is even more important for the control of fungi that disperse through the air and that can produce melanin in the presence of oxogeneous substrates such as L-dopa. Melanization can protect the fungus against various biotic and abiotic factors. In the present study, we investigated the effect of exposure to an environmental intensity of UVB radiation of 1,000 mWm-2 on the survival of melanized and non-melanized cells of four strains of Cryptococcus neoformans and of four strains of Cryptococcus laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of 2, 4, 6 and 8 day cells grown on medium with and without L-dopa was determinated after exposure to doses of 1.8 and 3.6kJ m-2. Both irradiance and these doses are observed in the environment even in temperate regions. Difference in tolerance to UVB radiation were observed both in the C. neoformans and C. laurentii strains. The C. neofarmans strains were more sensitive to radiation than the C. laurentii strains. In C. neoformans, diferences in tolerance to radiation were observed during development both in melanized and non-melanized cells. For most features (strains, time of growth and UVB dose) there was no difference in tolerance between melanized and non-melanized cell and when differences occurred they were smaller than those previously observed with UVC. In C. laurentii, there was no difference in tolerance to radiation during development between melanized and non-melanized cells., Also, no significant differences in tolerance were observed between melanized and non-melanized cells of any strain

Resumo: Nas últimas décadas, evidenciou-se aumento considerável de micoses de caráter invasivo e de difícil tratamento. A criptococose vem representando significativa causa de mortalidade, principalmente em indivíduos HIV positivos. É enfermidade causada por espécies de leveduras encapsuladas, Cryptococcus neoformans e Cryptococcus gattii, ambas as espécies constituindo-se nos agentes fúngicos mais frequentes a comprometer o sistema nervoso central (SNC). A disponibilidade de antifúngicos para a criptococose no mercado, atualmente é limitado, visto que em alguns casos, muitos deles são ineficientes e tóxicos. Além disso, adiciona-se também a diminuição de cepas sensíveis aos antifúngicos atuais, sendo um importante fator de complicação no tratamento dessas infecções. Neste estudo o gene ERG11 que codifica a proteína Lanosterol 14 C Desmetilase, alvo de fluconazol, de isolados clínicos seriados de C. neoformans recuperados de um paciente e um isolado de psitacídeo de C. gattii, que apresentaram valores de CIM elevadas (Concentração Inibitória Mínima) para o fluconazol, foi analisado. Para atingir este objetivo, os isolados clínicos sequenciais 26, 27 e 30 de um paciente do Rio de Janeiro, C. neoformans ATCC 90012 e o isolado do psitacídeo de C. gattii foram selecionados. O isolado C. gattii e os isolados 26, 27 e 30 de C. neoformans, caracterizados como sensível, intermediário e resistente in vitro a fluconazol, respectivamente, foram selecionados para análise de pontos de mutação na sequência do gene ERG11 através do sequenciamento dos produtos amplificados utilizando primers desenhados a partir da sequência do gene. A sequência do gene ERG11 do isolado 30 (resistente) apresentou um ponto de mutação e duas variações na sequência nucleotídica. A primeira, quando alinhada e comparada com a sequência do isolado 26 (sensível) e do gene de referência... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the last decades, there was a considerable increase in invasive fungal infections of difficult treatment. Cryptococcosis has represented a significant cause of morbidity and mortality, especially among HIV positive individuals. It is a disease caused by encapsulated yeasts species, Cryptococcus neoformans and Cryptococcus gattii, both species becoming the most common fungal agents to compromise the central nervous system (CNS). The availability of commercial antifungals is currently restricted and many of them are ineffective and toxic. In addition, there was the emergence of resistant strains to the current antifungals and this is an important complicating factor in treating these infections. In this study, the ERG11 gene that encodes the protein alpha Lanosterol 14-demethylase, target of fluconazole of serial clinical isolates of C. neoformans recovered from a patient and an isolate of C. gattii of a parrot, which showed higher MIC values (Minimum Inhibitory Concentration) for fluconazole was analyzed. To achieve this goal, the sequential clinical isolates 26, 27 and 30 of a patient from Rio de Janeiro, C. neoformans ATCC 90012 and the isolate of C. gattii of the parrot were selected. Isolates 26, 27 and 30 susceptible (S), intermediate (I) and resistant (R) in vitro, respectively, and the isolation of C. gattii were selected for analysis of mutation points in the ERG11 gene sequence by sequencing of amplified products using primers designed from the sequence of the gene. The ERG11 gene sequence of isolate 30 (resistant) showed a mutation point and two variations in the nucleotide sequence. The first, when aligned and compared with the sequence of isolate 26 (susceptible) and the reference gene was significant and the other two changes were silent. When the sequence was translated into Lanosterol 14-alpha demethylase protein, we found that the mutation led to an amino acid exchange... (Complete abstract click electronic access below) / Orientador: Ana Marisa Fusco Almedia / Coorientador: Maria José Soares Mendes Giannini / Banca: Marica de Souza Carvalho Melhem / Banca: Cleslei Fernando Zanelli / Mestre