Rozprawy doktorskie na temat „Candia albicans”

Mestrado em Biologia Aplicada
The genetic code establishes the rules that determine the transfer of genetic information from nucleic acids to proteins. The importance of the genetic code in genome decoding and its high conservation suggests that its evolution is highly restricted or even frozen. Despite this, various prokaryotic and eukaryotic genetic code alterations have been found, showing that the code is surprisingly flexible. For instance, the human pathogen Candida albicans contains an ambiguous tRNACAG that decodes a CUG codon as Ser (97%) and as Leu (3%). To further study ambiguity in other amino acid codons, we have engineered 8 mutant tRNASer that misincorporate Ser at 8 different codons belonging to distinct amino acids families (Glu, Arg, Asn, Cys, Phe, Gln, His and Pro) in Candida albicans. The wild-type tRNA was subjected to site-directed mutagenesis in order to change its anticodon to CUC, CCU, GUU, GCA, GAA, CUG, GUG and GGG. The tRNA stability, the cellular changes and the stress response of the resulting mistranslating strains were evaluated through northern blot analysis, cell transformation efficiency, growth rate and expression of a HSP104-GFP reporter system. A phenotypic screening probing various environmental stress conditions was performed in order to further characterize these strains. Experimental data suggest that these genetic code ambiguities affect fitness negatively in standard growth conditions and introduce growth advantages in presence of stress conditions. Thus, stress response triggered by codon ambiguity increase adaptation potential.
O código genético estabelece regras que determinam a transferência de informação genética a partir dos ácidos nucleicos para proteínas. A importância do código genético na descodificação do genoma e sua alta conservação sugere que a sua evolução é altamente restrita. Apesar disso, várias alterações no código genético dos procariotas e eucariotas têm sido encontradas, mostrando que o código é surpreendentemente flexível. Por exemplo, o patogénico humano Candida albicans contém um tRNACAG ambíguo que descodifica o codão CUG como Ser (97%) e como Leu (3%). Para continuar o estudo da ambiguidade noutros codões, induzimos 8 tRNASer mutantes, que incorporam incorretamente o aminoácido serina a 8 codões diferentes, pertencentes a distintas famílias de aminoácidos (Glu, Arg, Asn, Cys, Phe, Gln, His e Pro), em Candida albicans. O tRNA não mutado foi submetido a mutagénese dirigida, a fim de modificar o seu anticodão UGA para CUC, CCU, GUU, GCA, GAA, CUG, GUG e GGG. A estabilidade do tRNA, as alterações celulares e resposta ao stress das estirpes mutantes resultantes foram avaliadas através da análise de Northern blot, da eficiência de transformação das células, da taxa de crescimento e da expressão do sistema repórter HSP104-GFP. Além disso, a caracterização fenotípica em determinadas condições de stress foi realizada com o intuito de caracterizar melhor essas estirpes. Os dados experimentais sugerem que essas ambiguidades ao código genético afetam negativamente a aptidão das células em condições de crescimento normais e introduzem vantagens no crescimento na presença de condições de stress. Assim, a resposta ao stress provocada pela ambiguidade dos codões pode aumentar o potencial de adaptação.

We assembled the genome of the human fungal pathogen Candida albicans into eight chromosomes, and annotated each of its genes. A genome comparison with Saccharomyces cerevisiae revealed an increased number of C. albicans superoxide dismutase genes. We analyzed the expression patterns and the function of one of these genes, SOD5, whose role is to protect the pathogen against extracellularly produced, neutrophil-generated superoxide radicals. Comparative genomics also showed that although many of the C. albicans transcription factors, such as Gal4p and Gcn4p, have homologues in S. cerevisiae, the sequence similarities occur only in the DNA binding motifs of those proteins. Deletion analysis of CaGcn4 and CaGal4 proteins show that the N' and C' termini respectively are needed for their transactivation ability. These two transactivation regions show no sequence similarity to the equivalent domains in their S. cerevisiae homologues, and the two C. albicans transactivatiog domains themselves show little similarity. A comparative analysis of the transcriptional machinery between C. albicans and S. cerevisiae showed low sequence similarity of the mediator complex that bridges activation domains of transcription factors to the RNA polymerase II complex. We performed a comparison of intergenic DNA regions to identify the cis-regulatory elements from Candida and Saccharomyces species to examine the organization of the transcriptional regulatory networks between these two organisms. We observed that the C. albicans GAL genes lack Gal4p binding sites, but that such sites are found upstream of telomeric genes and genes involved in glycolysis, and we show that CaGal4p regulates the expression of those genes. We identified the regulatory DNA sequences in the promoters of GAL genes, including a GAL-specific palindrome necessary for GAL10˛ expression. Cph1p, the C. albicans homolog of the Ste12p transcription factor controlling pheromone-induced gene expression in yeast, acts through this GAL-specific palindrome, functioning as an activator in the presence of galactose. This shows C. albicans and S. cerevisiae can regulate the same process by different regulatory circuits.

It has been envisaged that lytic enzymes may be crucial in determining the morphology and growth of fungi, and may therefore represent a target for antifungal agents. The chitinolytic system of Candida albicans was investigated using a range of 4-methylumbelliferyl glycosides as model substrates, and its potential as a target for antibiotics has been assessed. Maximum hydrolysis was obtained with substrates having monomer and tetramer chain lengths, being attributed to N-acetylglucosaminidase and endochitinase respectively. Activities were investigated in cell fractions, vacuoles, and also in whole cell preparations. The characteristics of both chitinase and N-acetylglucosaminidase were examined, including pH and temperature optima and Km values. Chitinase was semi-purified on Fast Protein Liquid chromatography system and activity could be located after native polyacrylamide gel electrophoresis. Analysis of chitinase activity during the growth of the yeast morphology of C.albicans revealed maximal activities during the logarithmic phase, suggesting a relationship of chitinase levels to active growth of the pathogen. It was found that the antibiotic allosamidin was a potent inhibitor of chitinase activity of C.albicans, but not of N-acetylglucosaminidase. Conversely, an analogue of N-acetylglucosamine was found to be a potent inhibitor of N-acetylglucosaminidase but not of chitinase. Treatment of yeast suspensions with allosamidin resulted in an increased chain length. No cell death, or discernible pattern of change in the radiolabelling was observed in the presence of either allosamidin or N-acetylglucosamine analogue. Similarly no consistent change in the optical density of cultures was observed in the presence of either inhibitor. Even in the presence of the membrane permeabilising agent amphotericin no effects were observed above those achieved with amphotericin alone. Comparative studies were carried out upon the chitinolytic activity of Kluyveromyces lactis toxin and bovine serum. The chitin synthetic system of Benjaminiella poitrasii was compared to that of C.albicans.

Azole-resistance in Candida albicans is becoming common and is associated with the widespread prophylactic use of azoles. Resistance to one azole is usually associated with resistance to other structurally dissimilar azoles. C.albicans is also inherently resistant to a wide range of eukaryotic inhibitors such as cycloheximide and gentamycin. Certain studies have shown that azole-resistance in some strains of C.albicans is associated with alterations in the cell membrane. This project has sought to determine whether azole-resistance in C.albicans strain 3302 was due, at least in part, to a multidrug resistance mechanism. An assay was developed using the fluorescent dye Rh123 to measure P-glycoprotein like activity. Active efflux of Rh123 has been shown to correlate with P-glycoprotein activity in a number of organisms. Results from this assay suggest that an energy-dependent efflux mechanism for Rh123 is present in azole-resistant strain 3302 but not in azole-sensitive strain 3153. The P-glycoprotein inhibitor, reserpine, inhibited Rh123 efflux. However, azoles did not appear to compete with Rh123 for efflux in the azole-resistant strain 3302, suggesting that azole-resistance in this strain is not mediated by a P-glycoprotein like mechanism. Southern analysis showed that sequences homologous to MDR genes existed in C.albicans. A PCR strategy was used to clone gene fragments containing the Walker motif which is found in MDR genes.

Orientador: Altair Antoninha Del Bel Cury
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Candida albicans está associada com a etiologia da estomatite protética, patologia que acomete entre 11 a 67% dos usuários de próteses removíveis. Entretanto, mais recentemente, a Candida glabrata tem se destacado por apresentar hidrofobicidade e adesão à superfície de resina acrílica superior à da Candida albicans. Em acréscimo, características de superfície dos materiais como rugosidade (Ra) e energia livre de superfície (ELS) podem contribuir para a adesão de microrganismos. Desse modo, o objetivo desta pesquisa foi avaliar a rugosidade e energia livre de superfície dos rembasadores de próteses à base de poli - metilmetacrilato (Coe Soft e Kooliner) e silicone (Ufi Gel P) antes da contaminação com C. albicans ATCC 90028 e C. glabrata ATCC 2001, bem como verificar a eficácia dos limpadores químicos à base de peróxidos (Polident 3 minutes e Efferdent) e Hipoclorito de sódio a 0,5% na remoção desses microrganismos.Assim, para cada material rembasador foram confeccionadas 64 bases de resina acrílica Onda CryI (25 x 12 x 1mm), preparadas conforme as instruções do fabricante e, reembasadas constituindo dessa forma os corj'i>osde prova. Estes tiveram a rugosidade e energia de superfície determinadas. A seguir, foram separadas aleatoriamente em 2 grupos constituídos de 32 amostras cada, conforme o tipo de Candida. Em seguida, estes foram subdivididos em 4 grupos de 8 de acordo com os tratamentos: G1 - Água destilada (Controle); G2 - Polident ; 3 minutes; G3 - Efferdent; G4 - Hipoclorito de sódio 0,5%. Todas as amostras foram imersas em saliva humana durante'30 minutos para a formação da película adquirida. Posteriormente, foram submetidas ao teste de adesão durante período de 2 horas com uma das candidas, e então submetidos aos tratamentos nos tempos de: G1 - 15 minutos; G2 - 3 minutos; G3 -15 minutos e G4 -10 minutos. A contagem das células remanesce.ntes após o tratamento foi realizada em microscópio de luz (400x). Os dados foram submetidos à análise de variância e teste de Tukey (rugosidade de superfície e aderência fúngica) e ANOVA on Ranks para ELS, com nível de significância de 5%. O rembasador à base de silicone (Ufi Gel P) apresentou os menores valores de rugosidade comparados aos rembasadores à base de poli-imetil metacrilato (Coe 50ft e Kooliner), p<0,05. Entretanto, todos os materiais diferiram entre si para a energia de superfície (p<0,05), sendo que o Coe 50ft e o Ufi Gel P apresentaram os maiores e menores valores, respectivamente. Candida glabrata apresentou o maior número de células remanescentes aderidas, independente do rembasador utilizado (p<0,01). Dentre os limpadores, apenas o hipoclorito de sódio 0,5% diferiu do controle (p=0.001), apresentando um menor número de células remanescentes aderidas. Condui-se que o hipoclorito de sódio a 0,5% foi eficaz na remoção das células aderidas dos rembasadores, independente da espécie de Candida
Abstract: Candida albicans is associated with denture stomatitis etiology, pathology which affect about 11 to 67% of removable prostheses users. However, more recently, the Candida glabrata has been highlighted for presenting superior hydrophobicity and resin acrylic surface adherence when compared to the C. albicans. In addition, surface characteristics' materiais such roughness (Ra) and surface free energy (EL8) may contribute to the microorganisms' adhesion. Thus, the purpose of this study was to evaluate the surface roughness and surface free energy of methyl methacylate liners materiais (Coe 80ft and Kooliner) and silicone (Ufi Gel P) before contamination with C. albicans (ATCC 90028) and C. glabrata (ATCC2001), as well to verify the peroxide chemical denture cleansers efficacy (Polident 3 minutos and Efferdent) and 0.5% sodium hypochlorite - NaOCl, in the miaoorganisms' removing. Thus, 64 rectangular bases measuring 25 x 12 x 1 mm using microwave-polymerized acryli~ resins, following manufacturers' recommendations, to each material were made, then were relined and after surface roughness and surface free energy were measured. Next, the samples were randomly separated by lottery into two groups of 32 each, according to the fungus and these were subdivided into four groups of eight as the treatments: G1 - Distilled water (Control); G2 - Polident 3 min. utes; G3 - Efferdent; G4 - 0,5% NaOCI. Ali the samples rested in human whole saliva for 30 minutes to form an acquired pellicle. After, they were submitted to the adherence assay with one of the fungus for two hours, and then, treated, following these times: G1 - Distilled water (15 minutes); 2 - Polident 3 minutes (3 minutes); 3 - Efferdent (15 minutes); 4 - 0,5% sodium hypochlorite (10 minutes). The adhered cells were counted using a light microscope (Axiostar 2 Plus, Carl Zeiss, Jena, Germany) at 400 x magnification. The data were submitted to the analysis of variance and Tukey test (roughness and fungus adherence) and ANOVA on Ranks for EL8, with significance levei of 5%. The silicone-based liner (Ufi Gel P) showed lower values of roughness compared to the methyl methacrilate-based liners (Coe 80ft and Kooliner), (p<0.05). However, ali these materiais were different among them for surface free energy (p<0.05), where the Coe 80ft and Ufi Gel P showed the highest and lowest values, respectively. Candida glabrata showed the highest number of adhered cells for ali materiais (p<0.01). Among evaluated cleansers, only 0.5% sodium hypochlorite differed from the control (p=0.001), showing the lowest number of remaining cells adhered. The conclusion is that the 0.5% sodium hypochlorite was the only one chemical cleanser efficient in the adhered cells removing in ali denture liners independent of the Candida specie
Doutorado
Protese Dental
Doutor em Clínica Odontológica

Orientadores: Altair Antoninha Del Bel Cury, Wander José da Silva
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Os reembasadores para base de prótese, após a exposição à cavidade bucal, apresentam alterações de superfície facilitando a adesão e a colonização por micro-organismos. Para a limpeza de superfície desses materiais são indicados os limpadores químicos para evitar os danos mecânicos que podem ser provocados pelas cerdas das escovas dentais. Assim, o objetivo nesta pesquisa foi avaliar a eficácia de limpadores químicos na remoção de biofilme de Candida spp. desenvolvido sobre a superfície de um reembasador classificado como permanente à base de poli-metilmetacrilato e na prevenção da re-colonização dessa superfície, especialmente a Candida spp., comumente associada ao desenvolvimento da candidíase. Espécimes (10 mm diâmetro X 3 mm altura) de resina acrílica reembasada com um reembasador mais representativo disponível comercialmente teve sua rugosidade de superfície mensurada antes (baseline) e biofilme de C. albicans ATCC 90028 ou C. glabrata ATCC 2001 foi desenvolvido sobre os mesmos. Após a formação dos biofilmes os espécimes foram aleatorizados e submetidos aos tratamentos (n=16): AD - Água destilada (Controle), 15 min; POL - Polident 3 minutos, 3 min; EFF - Efferdent, 15 min; HPS - Hipoclorito de sódio a 0,5%, 10 minutos. Metade destes espécimes (n=8) foi utilizada para determinação da eficácia dos limpadores, utilizando contagem de células viáveis, enquanto os espécimes remanescentes (n=8), após os tratamentos, foram novamente colocados em meio de cultura estéril e incubados por mais 48 h a fim de determinar o efeito dos limpadores na prevenção da recolonização. Após os tratamentos os espécimes tiveram a rugosidade de superfície determinada, considerada pós-tratamento. Alguns espécimes de cada uma das espécies de Candida tiveram a superfície analisada após os tratamentos, por microscopia eletrônica de varredura (MEV). Os dados foram submetidos à análise de variância e teste de Tukey HSD em nível de significância de 5%. A rugosidade de superfície foi significantemente maior após os tratamentos (P<0,05). Quantos aos tratamentos, o HPS mostrou-se efetivo tanto para a desinfecção quanto na recolonização de ambas as espécies de Candida, pois houve ausência total de crescimento. Na avaliação da desinfecção, imediatamente após os tratamentos, quando C. albicans foi considerada, não houve diferença significativa entre os peróxidos alcalinos (p>0,05) e ambos diminuíram o número de células fúngicas (p<0,05) comparado ao tratamento com AD. Entretanto, para C. glabrata, os tratamentos com ADD e peróxidos alcalinos não se diferenciaram entre si (p>0,05). Na análise dos resultados para a recolonização foi observada que houve inversão no comportamento, pois enquanto, para C. albicans, os tratamentos com AD e peróxidos alcalinos não diferiram entre si (p>0,05), para C. glabrata os tratamentos com peróxidos alcalinos apresentaram valores similares e menores (p>0,05),quando comparados com o tratamento com AD (p<0,05). Na comparação entre as espécies de Candida observou-se que C. glabrata apresentou os maiores níveis de células viáveis quando os dados foram avaliados na situação de imediatamente após os tratamentos com os peróxidos alcalinos e foi diferente de C albicans (p<0,05). Entretanto não houve diferença para a recolonização (p>0,05). Os resultados sugerem que os limpadores a base de peróxidos alcalinos não foram efetivos na remoção total dos micro-organismos e também não impediram a recolonização por Candida spp
Abstract: The denture liners exhibits surface changes in oral environment by constant loss of its constituent elements, which facilitate microorganisms adherence that leads to biofilm formation. Denture liners surface can be cleaned by brushing or using denture cleaners, which are recommended, in order to avoid mechanic injuries to denture liners by brushing it. Therefore, the aim of this study was to evaluate the long term efficacy of denture cleansers on Candida spp. biofilm recolonization on liner surface. Specimens of poly (methylmethacrilate) were lined according to manufacturer instructions (10 mm diameter X 3.0 mm thickness). Surface roughness was measured at baseline and after the treatments. Next, biofilms of C. albicans ATCC 90028 and C. glabrata ATCC 2001 were allowed to develop on liner surface for 48 h. Subsequently, the specimens were randomly assigned for the cleaning treatments (n=16): distilled water (DW - control), 15 min; Polident 3 minutes (POL) - 3min; Efferdent (EFF)-15 min; sodium hypochlorite (HYP) - 10 min. After the treatments, specimens (n=8) were sonicated for biofilm disruption and the viable cells were counted (cell/mL). To determine the long term effectiveness of the cleaning process, a set of cleaned specimens (n=8) were submitted to new biofilm growth conditions. After 48 h, biofilm were disrupted by sonication and cell number estimated. Scanning electron microscopy was performed to analyze the specimen topography after denture cleanser treatment. Data were analyzed by ANOVA and Tukey's HSD test was used as post-ANOVA employing a significance level fixed at 5%. The liner surface was rougher after the treatments (P<0.05). Results showed significant differences in cleanliness among the treatments (p<0.05), however for Candida species (p<0.05) no significant difference was observed in the recolonization condition (p>0.05). Alkaline denture cleansers showed similar cleaning performance and both showed lower cells counts compared with the control (p<0.05). Hypochlorite was the only effective treatment as no viable cells were detected even after the recolonization test. Within the limits of this study, it can be concluded that alkaline denture cleansers were not effective on biofilm removal, once denture liner surface by Candida spp biofilm recolonization was not prevented
Mestrado
Protese Dental
Mestre em Clínica Odontológica

Orientador: Altair Antoninha Del Bel Cury
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O biofilme de Candida spp. formado na superfície de próteses removíveis, é o principal fator etiológico da candidose, sendo a C. albicans e a C. glabrata as espécies mais prevalentes nesta condição. O antifúngico fluconazol (FLZ) é frequentemente utilizado no tratamento da candidose, porém o sucesso tem sido limitado devido a resistência desenvolvida pela Candida a esse medicamento. Considerando a importância da estrutura e morfologia do biofilme de Candida na candidose, o objetivo neste estudo foi avaliar o efeito de FLZ na bioatividade, bioestrutura e morfologia celular de biofilmes de Candida spp. desenvolvidos na presença deste antifúngico. Espécimes (10 mm x 2 mm) foram confeccionados utilizando resina de poli(metilmetacrilato) (PMMA), polimerizada por banho de água quente. Películas de saliva foram formadas na superfície da PMMA, e biofilmes de um isolado referência e dois isolados clínicos de C. albicans (ATCC 90028, P01, P34) e C. glabrata (ATCC 2001, P11, P31) foram desenvolvidos por 48h. Dois grupos foram formados: controle e experimental. FLZ a 2,56 µg/mL, concentração biodisponivel na saliva, foi adicionado ao meio de cultura do grupo experimental. Os meios de cultura do grupo controle e experimental foram trocados a cada 24 h. As bioatividades dos biofilmes foram avaliadas utilizando análise colorimétrica de redução por XTT. A bioestrutura analisada através do Microscópio Confocal à Laser e a morfologia celular avaliada utilizando o Microscópio Eletrônico de Transmissão. Os dados foram analisados pelo Test t de Student com nível de significância de 5%. A presença do FLZ reduziu a bioatividade de todos os biofilmes de C. albicans (p<0.001), porém não alterou a estrutura e morfologia da C. albicans P34. Quanto à bioatividade e bioestrutura dos biofilmes de C. glabrata, não foram encontradas diferenças estatisticamente significantes entre os grupos controle e experimental. Pode-se concluir que as alterações da bioatividade, bioestrutura e morfologia celular, como resposta ao tratamento com FLZ, na concentração biodisponível na saliva, depende da cepa de Candida spp. avaliada.
Abstract: Candida spp. biofilm formed on removable denture surfaces is considered the main etiologic factor of candidosis, being the C. albicans and C.glabrata the species most frequently found in this condition. The antifungic fluconazol (FLZ) is commonly used in the treatment of candidosis, however its success is limited due to the resistance developed by Candida to this medicament. Considering the importance of the structure and morphology of Candida biofilms in the candidosis, the aim of this study was to evaluate the effect of FLZ on the bioactivity, biostructure and morphology of Candida spp. biofilms formed in the presence of this antifungal agent. Specimens (10 mm x 2 mm) were fabricated using water bath poly(methylmethacrylate) resin (PMMA). Salivary pellicles were formed on the PMMA surface, and biofilms of a reference strain and two clinical isolates of C. albicans (ATCC 90028, P01, P34) and C. glabrata (ATCC 2001, P11, P31) were developed for a period of 48h. Two groups were formed: control and experimental. FLZ at 2.56 µg/mL, concentration bioavailable in saliva, was added to the medium of the experimental group. The culture mediums of the control and experimental groups were changed at 24 hours. The bioactivities of the biofilms were evaluated with XTT reduction colorimetric assay. The biostructure was analyzed by the Confocal Scanning Laser Microscopy and the cell morphology analyzed by the Transmission Electron Microscopy. The data were analyzed by Student's t-test, with significance level set at 5%. The presence of FLZ decreased the bioactivity of all C. albicans biofilms (p<0.001), it did not change the structure and morphology of P34. As regards C. glabrata biofilms bioactivity and biostructure, no statistically significant differences were found between control and experimental groups for biofilms of all strains. It could be concluded that the alterations in bioactivity, biostructure and cell morphology in response to the treatment with fluconazole, bioavailable concentration present in saliva, depends on the Candida spp. strain
Doutorado
Protese Dental
Doutor em Clínica Odontológica

This thesis describes the cloning the characterisation of three C. albicans genes: CaTBP1 encoding the TATA-binding protein, CaRAD6 encoding a ubiquitin-conjugating (E2) enzyme, the CaALS7 encoding an agglutinin-like cell-surface protein. Finally, the ALS7 promoter has been discussed. The CaTBP1 open reading frame is 716 bp long and encodes a functional TATA Binding Protein (TBP) of 27 kDa. Comparison of the predicted amino acid sequences of TBPs from a range of organisms reveals at least 80% amino acid sequence identity in the C-terminal domain. CaTbp1p binds specifically to a TATA box in vitro, substitutes for the human TBP to activate basal transcription in vitro, and suppress the lethal Δspt15 (tbp1) mutation in S. cerevisiae. CaRAD6 contains a 573-nucleotide ORF with the potential to encode a 179 amino acid polypeptide with a molecular mass of 19.7 kDa. The CaRAD6 open reading frame is interrupted by two introns which both contain consensus splicing signal sequences. A comparison of the predicted amino acid sequences of Rad6 proteins from a range of organisms reveals strong conservation within their N-terminal domains (70% amino acid sequence identity). The CaRAD6 gene complements the UV sensitivity and defective UV mutagenesis of a S. cerevisiae rad6Δ mutant. RAD6 expression decreases during hyphal development in C. albicans. Elevated RAD6 expression levels inhibit hyphal development, and Rad6p depletion enhances hyphal growth. These effects are dependent on the Efg1p morphogenetic signalling pathway. Therefore, RAD6 is a negative regulator of hyphal development, revealing for the first time, a mechanistic link between ubiquitination and fungal morphogenesis. CaALS7 is a member of the ALS gene family encoding agglutinin-like cell surface proteins in C. albicans. The ALS7sequence has a 3141-nucleotide open reading frame with the potential to encode a 1047 amino acid polypeptide.

The term denture stomatitis describes an inflammation of the oral mucosa in contact with the fitting surface (usually maxillary) of the denture. Although considered to have a multi-factorial aetiology, the incidence of denture stomatitis is strongly associated with poor denture hygiene and the presence of Candida albicans, in denture plaque. Dentures provided an ideal abiotic substratum for microbial attachment, retention and growth, providing hard, non-shedding of variable topography. Although a wide range of denture hygiene products and procedures are available for use, targeted activity towards Candida is not typically reported. In addition, denture cleansing protocols often use abrasive pastes and/or brushes which can alter denture topography and potentially increase the susceptibility to plaque accumulation and reduce cleanability. The aim of this work was to investigate interactions between denture surface topography and C.albicans, with a view to identifying factors that would enhance denture hygiene. Two sets of denture acrylic test surfaces were abraded in a linear direction; one using emery papers of increasing grit size, and the other by abrasion using toothbrush and dentifrices. All test surfaces were characterised using white light profilometry, enabling derivation of roughness parameters (Ra/Sa) and measurement of feature dimensions (width, depth). A method was developed that allowed biofilm growth from adherent blastospores or hyphae on these surfaces. Initial retention on the abraded surfaces increased with increasing roughness values, but there was no effect on topography on the resultant biofilm. Biofilms developed from hyphae had significantly higher biomass than those from blastospores, and also presented a more open network structure. After biofilm removal, surface roughness significantly affected retention of remaining cells: rougher surfaces retained more cells, and retention was increased by the presence of hyphae. The orientation of hyphae on the abraded surfaces did not seem to be effected by topographic features, nor by the proximity of other cells. This latter observation was confirmed using an optical tweezer method, which enabled the specific placement of individual cells, with subsequent monitoring of germ tube formation. The production of quorum sensing (QS) molecules was investigated as a factor influencing biofilm development. Using gas chromatography and mass spectrometry, volatile compounds were collected from planktonic and biofilms of C.albicans over time. Ethanol and the QS molecule farnesol were amongst the molecules identified, being produced at 4hour and 10hours respectively during the 24 hour incubation period. Farnesol was also shown to inhibit hyphal production by attached cells, when applied to the surfaces as a conditioning film, as well as in vapour form. A denture cleanser with improved anti-Candida activity was effective against blastospore and hyphal biofilms during extended 1 and 16 hour soak times. Using confocal scanning laser microscopy (CSLM) live-dead staining of Candida biofilm indicated that the denture cleanser inactivated cells throughout the biofilm, with increased effect and penetration over time, but no difference between hyphal and blastospore biofilms was observed. When mixed biofilms were generated with C.albicans and Streptococcus oralis, the presence of the bacteria appeared to increase the effect of the cleanser on Candida biofilm. It is proposed that the inactivation of the bacteria may have disrupted the biofilm and enhanced its susceptibility. However this requires further investigation. In order to reduce the accumulation of denture plaque and C.albicans, there is a need to limit substrate abrasion, reduce retention on the surface, prevent hyphal growth and ideally kill and remove denture plaque. Strategies towards these aims include targeted but gentle cleaning and the potential use of quorum sensing molecules.

La maladie de Crohn (MC) est avec la rectocolite hémorragique (RCH) une des formes de Maladies Inflammatoires Chroniques de l'Intestin (MICI). L'étiologie de la MC reste encore inconnue. Cependant, il est établi que les mécanismes physiopathologiques de la MC reposent sur des facteurs de susceptibilité génétique, des facteurs environnementaux, associés à une dérégulation de la réponse immunitaire intestinale, notamment vis-à-vis de la flore endogène. Parallèlement au processus inflammatoire responsable des lésions, les patients développent une réponse humorale dirigée contre différents antigènes microbiens. Parmi eux, des anti-levures sont fréquemment rapportés chez les patients atteints de MC. Il s'agit des anticorps anti-Saccharomyces cerevisiae. Le laboratoire est à l'origine du test immuno-enzymatique de détection de ces anticorps, qui utilise le mannane d'une souche de S. Cerevisiae (SU1) et qui ont été dénommés ASCA. Les premières études séro-épidemiologiques issues du laboratoire ont montré que les ASCA sont présents chez 60% des patients atteints de MC et 10% des patients atteints de RCH. Ils sont également présents chez 20 à 25% des parents sains du premier degré de patients atteints de MC, contre seulement 7% dans la population témoin. Les ASCA sont maintenant largement utilisés par les gastroentérologues pour le diagnostic et la stratification phénotypique des patients MC. Malgré de nombreuses publications sur les ASCA comme marqueur de la MC, l'immunogène et les mécanismes à l'origine de leur production sont toujours inconnus. La première étape a consisté à étudier l'influence de la génétique sur les ASCA, par l'étude de leur prévalence chez des jumeaux monozygotes et dizygotes atteints de MICI. Nos résultats suggèrent que les ASCA serait le marqueur d'une réponse à un antigène environnemental, dont le niveau de réponse serait génétiquement déterminé. L'étape suivante a concerné la recherche d'un immunogène pour les ASCA. Nous avons exploré si, contrairement à la levure exogène S. Cerevisiae, Candida albicans, une levure commensale du tube digestif humain, mais aussi un pathogène fongique opportuniste majeur, pouvaient exprimer les épitopes majeurs reconnus par les ASCA. Un programme en plusieurs étapes, basé sur des observations cliniques et des études expérimentales sur les relations structure/immunogénicité des oligomannosides de levures dans la MC et dans les candidoses nous a permis de démonter que C. Albicans peut être un immunogène à l'origine des ASCA. Ces conclusions nous ont conduits à étudier les relations entre la colonisation intestinale par C. Albicans, les réponses sérologiques anti-levures, et le génotype des patients dans des familles affectées par la MC. Nous avons confirmé que la colonisation par C. Albicans influence à la fois la production des anticorps anti-C. Albicans et des ASCA chez les parents sains des patients MC. Chez les patients atteints, les ASCA étaient indépendants de la colonisation par C. Albicans. Cependant nous avons pu observer une corrélation entre la colonisation par C. Albicans et la seropositivité ASCA chez les patients MC positifs en anticorps anti-C. Albicans. Ces résultats renforcent notre conclusion que C. Albicans est un immunogène pour les ASCA chez l'homme. L'ensemble de ces travaux, qui correspondent à une première étape dans la description des mécanismes à l'origine de la synthèse des ASCA, nous a permis de faire progresser nos connaissances dans le domaine de la physiopathologie de la MC.

Thesis (MSc)--Stellenbosch University, 2011.
ENGLISH ABSTRACT: The ascomycetous yeast, Candida albicans, has been almost exclusively studied in a clinical context, due to the medical risk and costs associated with the yeast. Most environmental research into the external survival of this opportunistic pathogen has been concerned with short-term, severe pollution challenges. However, a study of literature indicates that the habitat characteristics of the oxygenlimited zones in wetlands and riverbanks are comparable to those of the gastrointestinal source of sewage-borne C. albicans. Interestingly, these are the external, environmental regions to which sewage-borne C. albicans is often exposed. In addition, oxygen-limitation is the predominant parameter in stimulating conjugation of C. albicans. Based on these observations, this study aimed to assess polluted river bank and wetland environments in the Western Cape of South Africa as potential habitats to accommodate a niche for C. albicans, particularly comparing the presence of this yeast in oxygen-limited, plant debris-rich zones and aerobic, clear, flowing zones. The second objective was to employ in vitro microcosm studies to investigate the survival and growth of C. albicans in various microhabitats similar to those comprising the oxygen-limited zones of wetlands. These included the rhizosphere of wetland flora, various soil and mud types and decomposing plant debris. The final objective was to establish the presence of sufficient nutrient and energy sources within this environment for the growth of C. albicans. In particular, cellulosic substrates and mono- and disaccharides released by the natural degradation of wetland plant debris were investigated as potential energy sources for this human commensal in the wetland environment. These study objectives combined to demonstrate the potential of such an oxygen-limited, plant debris-rich environment as a niche for C. albicans external to its human host. Both semi-quantitative culturing techniques and quantitative Real-Time PCR demonstrated the improved survival of C. albicans in oxygen-limited, plant debris-rich zones in wetland and river bank environments, in comparison to aerobic, clear subsurface water zones in the same environments. These zones were compared in the Plankenburg and Diep Rivers, situated in the Western Cape of South Africa. Correlations between coliform concentrations and total yeast concentrations were demonstrated in each of the different river zones, with higher pollution levels characteristic of the dry season. Candida albicans numbers in flowing water (zone W), rock-filtered (zone R) and plant-filtered water (zone P) were compared during the progress of the rainy and dry seasons. No C. albicans was observed in clear, flowing water throughout the analysis. Early in the rainy season, both rock-filtered (aerobic, poor in plant debris) and plant-filtered (oxygen-limited, rich in plant debris) water demonstrated C. albicans numbers at approximately equivalent levels of 10²-10³ cells/100 mL. However, as the rainy season progressed and total yeast and coliform numbers in all zones of the rivers dropped to negligible levels, C. albicans could no longer be detected in aerobic, rock-filtered zones; but its numbers remained at constant levels in oxygen-limited, plant-filtered zones. This suggests that oxygen-limited wetland and river bank zones rich in plant matter, analogous to the human gastrointestinal tract, may provide an ideal habitat in which C. albicans could establish a niche external to its host. The survival of this yeast in the various microhabitats that comprise this anaerobic, reducing wetland environment was evaluated with in vitro microcosms. The rhizosphere of wetland plants had no influence on C. albicans growth and survival in comparison to bulk soil away from the plant, and wetland mud microbiota was demonstrated to be inhibitory to its survival. However, decaying plant debris was shown to increase the survival of the yeast in this inhibitory mud environment. Candida albicans was shown to compete well saprophytically in anaerobic plant debris microcosms. In addition, the tendency of C. albicans to associate with plant matter in an aquatic environment was demonstrated by inoculating the yeast in water containing Hydrilla, a submerged macrophyte found in South African aquatic environments. Plate and liquid analyses, as well as an ANKOM NDF analysis, indicated unequivocally that the C. albicans strains evaluated in this work were unable to utilise the complex carbohydrates of the wetland habitat, including cellulose and fibre. However, HPLC, along with GCMS, demonstrated the anaerobic assimilation by C. albicans of monosaccharides released by natural lignocellulose degradation of wetland plant matter. An analysis of total nitrogen by digestion in a nitrogen analyser, as well as evaluation of ammonium, nitrate and nitrite in a KCL extract, also showed that C. albicans assimilates nitrogenous compounds released by the decomposition of wetland plant matter. This decay process occurs constantly in wetland and river bank habitats. It may therefore provide energy and nutrients for C. albicans, particularly in the anaerobic zones where conjugation may possibly occur and a niche may be established, as indicated by the results obtained for the Plankenburg and Diep Rivers.
AFRIKAANSE OPSOMMING: Die askomisete gis Candida albicans is feitlik eksklusief in ‘n kliniese konteks bestudeer weens die mediese risiko en koste daaraan verbonde. Die meeste omgewingsnavorsing op die eksterne oorlewing van hierdie opportunistiese patogeen was toegespits op die uitdagings van ernstige korttermyn besoedeling. ‘n Literatuurstudie toon egter dat die habitat-eienskappe van die suurstof-beperkte sones in vleilande en rivieroewers vergelykbaar is met dié van die gastroïntestinale bron van C. albicans wat in riool gevind word. Interessant genoeg is dit juis hierdie eksterne omgewingsgebiede waaraan C. albicans vanuit riool dikwels blootgestel word. Hierby is suurstof-beperking die vernaamste parameter in die stimulering van konjugasie in C. albicans. Op grond van hierdie waarnemings poog dié studie om besoedelde vleilande en rivieroewers in die Wes-Kaap Provinsie van Suid-Afrika te evalueer as potensiële habitatte wat ‘n nis van C. albicans kan akkommodeer, en veral om die teenwoordigheid van hierdie gis in suurstof-beperkte sones ryk aan plantafval te vergelyk met aerobe, helder, vloeiende sones. Die tweede doelwit was om in vitro mikrokosmos studies te gebruik om die oorlewing en groei van C. albicans in verskillende mikrohabitatte soortgelyk aan suurstof-beperkte sones in vleilande te ondersoek. Dit sluit die risosfeer van vleilandflora in, asook verskillende grond- en moddertipes en ontbindende plantafval. Die laaste doelwit was om die teenwoordigheid van genoegsame voedings- en energiebronne in dié omgewing te bepaal vir die groei van C. albicans. In besonder is sellulose substrate, asook die mono- en di-sakkariede, wat deur die natuurlike afbraak van vleiland plantafval vrygestel word, as potensiële energiebronne van hierdie mens-kommensaal in die vleiland-omgewing ondersoek. Hierdie studiedoelwitte het gesamentlik die potensiaal van so ‘n suurstofbeperkte, plantafvalryke omgewing as ‘n nis vir C. albicans buite die menslike gasheer aangetoon. Beide semi-kwantitatiewe kweektegnieke en kwantitatiewe in-tyd PKR het die verbeterde oorlewing van C. albicans in suurstofbeperkte, plantafvalryke sones in vleiland en rivieroeweromgewings gedemonstreer, in teenstelling met aerobe, helder oppervlakwatersones in dieselfde omgewings. Hierdie sones in die Plankenburg- and Dieprivier in die Wes-Kaap Provinsie, Suid-Afrika, is met mekaar vergelyk. Korrelasies tussen coliform konsentrasies en totale giskonsentrasies is in elk van die verskillende sones in dié riviere gedemonstreer, met hoër vlakke van besoedeling kenmerkend aan die droër seisoen. Candida albicans getalle in vloeiende water (sone W), rots-gefiltreerde (sone R) en plant-gefiltreerde water (sone P) is deur die verloop van die reën- en droë seisoene met mekaar vergelyk. Geen C. albicans is deur die loop van die analises in helder, vloeiende water bespeur nie. Vroeg in die reënseisoen het beide rots-gefiltreerde (aerobe, min plantafval) en plant-gefiltreerde (suurstofbeperk, ryk in plantafval) water vergelykbare vlakke van C. albicans getoon, naamlik 10²-10³ selle/100 mL. Soos die reënseisoen egter verloop het en die totale gis- en coliforme getalle in al die sones van die riviere tot weglaatbare vlakke gedaal het, kon C. albicans egter nie meer in die aerobe, rots-gefiltreerde sones bespeur word nie, hoewel die getalle in suurstofbeperkte, plant-gefiltreerde sones konstant gebly het. Dit dui daarop dat suurstof-beperkte vleiland en rivieroewer sones ryk in plantmateriaal, analoog tot die menslike gastroïntestinale kanaal, die idealke habitat mag bied waarin C. albicans ‘n nis mag vind buite sy gasheer. Die oorlewing van hierdie gis in die verskillende mikrohabitatte wat uit hierdie anaerobe, reduserende vleilandomgewing bestaan, is met in vitro mikrokosmosse geëvalueer. Die risosfeer van vleilandplante het in vergelyking met die grond weg van die plant geen effek op die groei en oorlewing van C. albicans gehad nie, en vleiland modder-mikrobiota is gevind om die oorlewing daarvan te inhibeer. Verrottende plantafval het egter die oorlewingsvlakke van giste in hierdie inhiberende modderomgewing verbeter. Candida albicans kan egter saprofities goed kompeteer in anaerobe plantafval mikrokosmosse. Hierby is die geneigdheid van C. albicans om met plantmateriaal in waterige omgewings te assosieer gedemonstreer deur die gis te innokuleer in water wat Hydrilla, ‘n onderwater makrofiet wat in Suid-Afrikaanse akwatiese omgewings aangetref word, bevat. Plaat en vloeibare analises, asook ‘n ANKOM NDF data-analise, het onteenseglik getoon dat die C. albicans stamme wat in dié werk gebruik is, nie in staat was om die komplekse koolhidrate, insluitende sellulose en vesel, van die vleiland habitat te benut nie. HPLC, saam met GC-MS, toon egter C. albicans se anaerobe assimilasie van monosakkariede wat deur natuurlike lignosellulose afbraak van vleiland plantmateriaal vrygestel is. ’n Totale stikstof analise deur vertering in ’n stikstof analiseerder, en ’n evalueering van ammonium, nitraat en nitriet in ‘n KCl ekstrak, het ook getoon dat C. albicans stikstofverbindings assimileer wat deur die afbraak van vleiland plantmatriaal vrygestel word. Hierdie afbraakproses kom deurlopend in vleiland en rivieroewer habitatte voor en verskaf potensieel energie en voedingstowwe aan C. albicans, spesifiek in die anaerobe sones waar konjugasie moontlik kan plaasvind, en ‘n nis gevestig kan word, soos aangedui deur die Plankenburg- and Dieprivier.

Orientador: Denise Madalena Palomari Spolidorio
Banca: Cristiane Duque
Banca: Janaina de Cássia Orlandi Sardi
Resumo: O gênero Candida pode ser encontrado em até 50% dos indivíduos saudáveis não causando danos aparentes, porém, sob condições predisponentes como doenças sistêmicas ou condições fisiológicas, pode tornar-se patogênico causando inflamação e destruição tecidual. Candida spp. na forma de biofilmes são importantes no desenvolvimento de infecções, pois estão associados a altos níveis de resistência a agentes antimicrobianos. Terapias alternativas com extratos naturais abrem novas perspectivas para prevenção e controle das doenças bucais na busca de efeitos terapêuticos favoráveis. O Terpinen-4-ol é um monoterpeno que atua na indução da perda da membrana e apresenta amplo espectro de atividade antimicrobiana e atividade anti-inflamatória. O objetivo deste estudo foi avaliar o efeito antifúngico, sinérgico/aditivo, inibição da adesão em células orais e alteração dos fatores de virulência do Terpinen-4-ol sobre Candida albicans e Candida tropicalis. Assim, foi realizada a identificação da Concentração Inibitória Mínima (CIM) e Concentração Fungicida Mínima (CFM) do Terpinen-4-ol sobre cepas padrão de C. albicans (ATCC 90028) e C. tropicalis (ATCC4563), empregando-se o método de microdiluição em caldo. Biofilmes mono e dual-espécies foram preparados usando o modelo de placa de microtitulação estática e quantificados por unidades formadoras de colônias (UFC/mL). O efeito do Terpinen-4-ol na adesão de C.albicans e C. tropicalis foi realizado em co-cultura com células orais NOK Si como tam... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The genus Candida can be found in up to 50% of healthy individuals without causing apparent damage, however, under predisposing conditions such as systemic diseases or physiological conditions, can become pathogenic causing inflammation and tissue destruction. Candida spp. in the form of biofilms are important in the development of infections because they are associated with high levels of resistance to antimicrobial agents. Alternative therapies with natural extracts open new perspectives for prevention and control of oral diseases in search of favorable therapeutic effects. The Terpinen-4-ol is a monoterpene engaged in membrane loss of induction and presents broad spectrum of antimicrobial and anti-inflammatory activity. The aim of this study was to evaluate the antifungal effect, synergistic / additive inhibition of adhesion on oral cells and modification of virulence factors of Terpinen-4-ol of Candida albicans and Candida tropicalis. Thus, the identification of Minimum Inhibitory Concentration was performed (MIC) and Minimum Fungicidal Concentration (MFC) of Terpinen-4-ol on standard strains C. albicans (ATCC 90028) and C. tropicalis (ATCC4563), using the method microdilution broth. Biofilms mono and dual-species were prepared using the microtiter plate static model and quantitated by colony forming units (CFU / mL). The effect of Terpinen-4-ol in the adhesion on C. tropicalis and C. albicans was carried out in co-culture with oral cells NOK Si as well as virulence facto... (Complete abstract click electronic access below)
Mestre

During the infection process Candida albicans has to respond to various stresses imposed by host environment including oxidative and osmolarity stress generated by phagocytic cells such as macrophages and neutrophils. Exposure to caspofungin and other antifungal antibiotics also imposes stress on the C. albicans cell wall. These various stress responses are orchestrated through the activation of multiple stress pathways including the cAMP-PKA, several MAPK pathways and the Ca2+-calcineurin pathway which influence cell wall shape and composition. Such changes were predicted to influence recognition of C. albicans by innate immune cells. During my Ph.D. studies I primarily investigated the effect of the activation or inhibition of these pathways on the interaction with the innate immune cells by examining phagocytosis, the cytokine profile induced by mononuclear and polynuclear cells of the innate immune system. I found that the activation and inhibition of these pathways plays an important role in remodeling of cell wall and hence the immunological profile. Inactivation of cAMP, Calcium signaling pathway by the deletion of TPK1 and CNA1 resulted in marked reduction in pro-inflammatory cytokine production. Inactivation of MAPK pathway by deletion of HOG1 altered major pro-inflammatory cytokine secretion. Cytokine production was also affected by exposure of C. albicans signaling mutants to Calcofluor White, caspofungin, oxidative and osmotic inducing stresses. Cytokine stimulation was also affected by deletion of URA3, exposure of C. albicans to rifmapicin and antimycin A. These results suggest that stress signaling pathways act to regulate collateral changes in the cell wall, which in turn affects the immune reactivity. Pro and anti-inflammatory cytokine and antifungal profiles of Candida haemulonii was also found to be highly variable. Thus regulation and exposure to different microenvironments significantly modifies immunological signature of fungal cells, suggesting that responses to local stresses make the fungal cell surface a moving target for immunological surveillance.

Orientador: Cristiane Yumi Koga-Ito
Co-orientador: Marcos José Salvador
Banca: Janete Dias Almeida
Banca: Sônia Khouri
Banca: Fernanda Lourenção Brighenti
Banca: Antônio Carlos Victor Canettieri
Resumo: O objetivo deste estudo foi avaliar a efetividade frente a Candida albicans da fração enriquecida em fenólicos (FE) das folhas de Buchenavia tomentosa além das substâncias ácido gálico (AG), corilagina, kaempferol e vitexina, substâncias fenólicas que foram previamente detectadas no extrato aquoso de B. tomentosa livres ou encapsuladas em ciclodextrinas. Para tal, foi realizado teste de microdiluição com cepas padrão e isolados clínicos e análise química da FE por espectrometria de massa (ESI-MS). Detectou-se que o extrato acetônico foi a FE e AG foi a substância fenólica mais eficiente contra C. albicans. O efeito de FE e AG contra os fatores de virulência de C. albicans foram analisados. FE e AG foram encapsulados em 2-hidroxipropil-beta-ciclodextrina e tiveram sua análise química realizada. As CIMs e CFMs dos encapsulados foram determinadas, porém apenas o AG encapsulado teve sua ação antibiofilme e in vivo verificadas. A citotoxicidade de AG e FE livres e encapsulados foi determinada. As CIMs variaram de 5,0 e 0,625 mg/ml para o ácido gálico e 2,5 e 0,019 mg/ml para FE. AG e as outras moléculas foram encontradas na FE. Não foram encontrados CFMs. Os fenólicos estudados também foram encontrados em FE por ESI/MS. Tanto FE quanto AG tiveram efeito direto nos fatores de virulência de C. albicans, exceto sobre a secreção de exoenzimas. Não houve diferença na CIM entre as substâncias livres e encapsuladas. AG encapsulado teve melhor ação anti-biofilme do sua forma livre. Foi verificada melhora clínica de lesões eritematosas no palato de ratos, porém não foi possível. A citotoxicidade das substâncias livres ou encapsuladas variou de moderava a leve para FE e foi moderada para AG. Após as análises, observou-se o efeito anti-C. albicans de FE e AG. AG encapsulado apresentou promissor efeito anti-biofilme e aparente melhora clínica nas lesões sugestivas de candidose eritematosa na mucosa palatar dos ratos
Abstract: The aim of this study was to evaluate the effectiveness against Candida albicans of fraction enriched in phenolic (FE) of Buchenavia leaves tomentosa beyond substances gallic acid (GA), corilagin, kaempferol and vitexin, phenolic substances previously detected in the aqueous extract of B. tomentosa, free or encapsulated in cyclodextrins. Microdilution test with standard strains and clinical isolates besides the chemical analysis of FE by mass spectrometry (ESI-MS) were carried out. The acetone extract was the FE and AG was the most efficient phenolic substance against C. albicans. The effect of FE and AG against the virulence factors of C. albicans was also analyzed. FE and AG were encapsulated into 2- hydroxypropyl-beta-cyclodextrin (HP-β-CD) and had their chemical analyses made. MICs and MFCs of encapsulated have been determined. Solely the GA encapsulated had its anti-biofilm and in vivo action verified. Cytotoxicity of free and encapsulated GA and FE were determined. The MIC ranged from 5,0 to 0,625 mg/ml for GA and 2,5 and 0,019 mg/ml for FE. MFCs values were not found. All phenolics molecules were found in FE by ESI/MS. AG and FE had a direct effect on virulence factors of C. albicans, except on the secretion of exoenzymes. There was no difference in the CIM between free and encapsulated substances. The anti-biofilm effect was better in GA encapsuladed than its free form. A clinical improvement of sugestives erythematous lesions on the palate of rats was observed, although the hyphaes were not found in the palatar mucosa. The cytotoxicity for all substances was moderated. After the analysis, we observed the anti-C. albicans effect of GA and FE. AG encapsulated showed promising anti-biofilm effect and apparent clinical improvement in lesions suggestive of erythematous candidiasis in palatar mucosa of rats
Doutor

ABSTRACT We followed the localization of GFP-tagged myosin I (Myo5), septin (Cdc12) and rhodamin-stained actin during bud and shmoo formation in opaque-phase cells of Candida albicans, and monitored the mating-associated processes of cell fusion, zygote budding, septum formation, daughter cell development and the dynamics of nuclear migration and division. The localization of Myo5p, Cdc12p and actin during budding in opaque and white cells is similar. In pheromone-stimulated cells, these proteins localize in shmoos in patterns consistent with hyphal formation in white-phase cells. MTLa cells generate shmoos 5-7 hours earlier than MTLα cells in mixed populations. In the daughter cell generated after mating, Cdc12p, Myo5p and actin localize as they do under vegetative budding conditions. Intriguingly, isogenicity for the mating type locus is involved in the positioning of the nuclear division; in MTLa cells the nucleus divides within the mother cell up to 70% of the time, rather than across the mother-bud neck.
RÉSUMÉ Nous avons déterminé la localisation de l'actine ainsi que de la myosine I (Myo5) et de la septine (Cdc12) modifiées avec la protéine fluorescente verte (GFP) chez Candida albicans en phase opaque (reproductive) et blanche (végétative). Plus particulièrement, nous avons porté notre attention sur les processus associé à la reproduction tel le bourgeonnement et l'expansion cellulaire (shmoo), la conjugaison et la fusion cellulaire, le bourgeonnement et le développement des zygotes (cellules -filles issues de la conjugaison). Nous avons aussi observé la configuration subcellulaire de Myo5p, Cdc12p et de l’actine lors de la migration et de la division nucléaire en phase blanche. Que ce soit en phase opaque ou en phase blanche, la localisation de Myo5p, Cdc12p et de l'actine reste similaire lors du bourgeonnement. Lors d’une stimulation à la phéromone, en phase opaque, ces trois protéines ont le même patron d’organisation cellulaire que lors de la formation d'hyphes en phase blanche. Les cellules de type MTLa produisent des shmoo entre 5 et 7 heures plus tôt que les cellules de type MTLα dans une population mixte. Dans les zygotes, Cdc12p, Myo5p et l'actine ont la même localisation que celle observée dans les cellule-filles issues du bourgeonnement en mode végétatif. Étonnamment, l'isogénicité du locus génétique déterminant le type sexuel de la cellule influence la position du noyau lors de la division; Ainsi, dans 70% des cas, le noyau des cellules de type MTLa se divise à l'intérieur de la cellule-mère plutôt qu'au travers du col entre la cellule-mère et le bourgeon.

Candida albicans, one of the most prevalent fungal pathogens in humans, is diploid, appears to lack a natural sexual cycle and possesses a different genetic code from most other species. These features combine to make the study of its biology by traditional methods extremely difficult, particularly with respect to gene isolation and expression. A cosmid library was constructed from C. albicans genomic DNA which represents an 11-fold coverage of its genome. Assessments made of the breadth of library coverage and clonal stability gave favourable results and the library has already proved to be of use within the Candida research community. Fluorescently-labelled restriction fingerprints of the cosmid clones were used to construct a physical map consisting of 364 contigs. Screening of the library with 22 STS markers reduced the number of contigs to 353 and allowed redundancy among the fingerprinted contigs to be estimated. The fingerprinted contigs therefore represent a first generation physical map covering 86% of the C. albicans genome, which when complete will greatly facilitate further study of the organism and its pathology. The results obtained from a pilot sequencing project, involving several of the library clones are discussed and included in an evaluation of restriction fingerprinting as a general approach to physical mapping. Finally, as the Human genome Mapping Project reaches its conclusion, the transition to a post-genomic era and its relevance to future work with C. albicans are discussed.

The study of the regulation of chitin synthesis in the pathogen Candida albicans is a challenging field. It not only offers a possibility of a better understanding of the dimorphic transition but also may help in the development of an effective antifungal. The suggestion that the regulation of cell wall synthesis may be closely coupled to the turgor pressure of the fungus has been investigated. The synthesis of chitin by the enzyme chitin synthase was studied under conditions of osmotic stress. Mixed membrane fractions from protoplasts of C.albicans incubated in medium of low osmolality exhibited up to four-fold greater native enzyme activity as compared to protoplasts incubated at high osmolality. This was also the case for preparations from whole cells of C.albicans, Coprinus cinereus and Saccharomyces cerevisiae and also from protoplasts from S.cerevisiae. Trypsin-treated enzyme preparations did not show this regulation to the same degree. The addition of nikkomycin Z, a differential chitin synthase inhibitor, partially restored this regulation. The synthesis of chitin, assessed by following the incorporation of (¹⁴C)-GlcNAc into chitin in the cell wall, was also greater in C.albicans cells incubated in medium of low osmolality. However, the incorporation of (¹⁴C)-GlcNAc into the cell wall of regenerating protoplasts exhibited the opposite effect. This was substantiated further by measuring the fluorescence of regenerating protoplasts following the addition of Calcofluor white. Following the attempted cloning of the C.albicans CHS3 (CSD2) gene, a detailed northern analysis of three chitin synthase genes during growth and dimorphism of C.albicans was performed. CHS1 was expressed during both the yeast and hyphal phases of growth while CHS2 and CHS3 were preferentially expressed in the hyphal form. There was no difference in expression of the chitin synthase genes in invasive and non-invasive clinical isolates of C.albicans and all three genes showed highest levels of mRNA when grown in medium of neutral pH.

The research described in this thesis was aimed at the characterization of the Candida adhesin, and elucidation of the nature of the human epithelial cell receptor with which it combines. Previous data have shown that the fibrillar mannoprotein layer, produced when Candida albicans is grown in high concentrations of galactose, contains the proteinaceous adhesin. Different Candida species from leukoplakia patients were assayed for their ability to adhere to buccal cells. The results supported previous conclusions that there is a relationship between the ability of different Candida albicans strains to adhere to epithelial cells and their capacity for cell surface modification. Extracellular polymeric material (EPM) was isolated from culture supernates of C. albicans after growth in medium containing 500 mM galactose. When used to pretreat buccal epithelial cells, EPM inhibited adherence, which suggested that it contains an adhesin that binds to, and blocks epithelial cell receptors. Fractionation of EPM by affinity chromatography was performed. An index, the adhesion inhibition index (AII) was used to compare the various "lectin-like" components isolated from crude EPM. These studies indicated that use of different buccal cell donors gave different results, with the same C. albicans strain. Attempts to resolve EPM by SDS-polyacrylamide gel electrophonesis had previously proved unsuccessful. However here, success was achieved using the silver-stain technique. Chemical and enzymatic digestion of the EPM indicated that the protein portion , of the glycoprotein was more important than the carbohydrate at inhibiting adherence. N-Glycanase, papain, mild alkali treatment of EPM, followed by Synsorb-H-2 affinity adsorption chromatography, resulted in a purification of the yeast adhesin of more than 220 fold, relative to the crude EPM (on a protein weight basis). The nature of the epithelial cell receptor for C. albicans was investigated with potential receptor analogues such as sugars, lectins, monoclonal antibodies and saliva. The adhesion of C. albicans to the buccal cells of blood groups A and O, was found to be significant with respect to secretor status but not blood group. Caution should be shown in the interpretation of sugar inhibition tests. Nevertheless, N-acetyl-D- galactosamine was the most effective single sugar as an inhibitor of adhesion for buccal cell donors of blood group A; N-acetyl-D-galactosamine is the immunodominant blood group sugar for group A cells and the possibility exists that the blood group oligosaccharide on the buccal cell surface functions as the receptor for yeast adhesion. Further work would be needed to establish whether the purified adhesin had any therapeutic value in treating Candida infections.

Cells that grow by apical extension, such as neurons, pollen tubes, root hairs and fungal hyphae, orient their growth in response to tip contact with physical cues in the environment (thigmotropism). I use Candida albicans, an opportunistic human fungal pathogen, as a model to assess tip re-orientation growth responses after contact. Thigmotropism is associated with virulence (Brand et al., 2008), therefore this thesis aims to characterise the responses that C. albicans displays after contact events in an enclosed chamber featuring various obstacles and shapes. It was found that hyphae grow along surfaces in a nose-down manner, presumably to identify and exploit gaps in the substrate. Further, hyphae preferentially grow nose-down on softer surfaces when given the option of two contrasting surfaces, implying novel substrate sensing mechanisms. Contact-dependent hyphal responses are outlined, where perpendicular contact with an obstacle induced various growth responses after re-orientation. Further, important cytoskeletal regulators of thigmotropism were identified, which subsequently regulate substrate indentations. The applied force generated by hyphal tips was quantified, which was enough to penetrate mammalian membranes without the need of hydrolytic enzymes, and this was modulated by a change in environmental carbon source. This thesis describes several new exploratory behaviours in C. albicans, which may apply to hyphae in general, since behaviours described here have also been observed in other filamentous fungi. Further, the role of septins as regulators of directional growth is discussed. The first chapter describes contact-dependent behaviours that support the ability of hyphae to be opportunistic and exploit their topographical environment to invade surfaces. The second chapter presents a detailed description of how the fungus responds to perpendicular contact events. Finally, the third chapter identifies cytoskeletal regulators important for thigmotropism. Together, this thesis brings together multiple aspects of cell biology and biophysics that apply during polarised tip growth, which adds knowledge to the narrative of why hyphae are such successful space invaders.

Candida albicans is a fungus that colonizes oral cavity surfaces and is carried by approximately 50% of humans. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans. The objective of this study was to better understand how streptococci communicate with C. albicans in oral biofilms. Mannoproteins comprise a major component of the C. albicans cell wall. Initial aims of the work were to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hypha I functions associated with biofilm community development. A C. albicans mnt1- mnt2L1 mutant, with deleted a1,2-mannosyltransferase genes and thus defective in 0- mannosylation, was abrogated in biofilm formation under various growth conditions, and produced hypha I filaments that were not ·recognized by S. gordonii. Cell wall proteomes of hyphae-forming mutant cells showed reduction, compared to wild type, in a range of protein components including Als1, Als3, Rbt1, Scw1 and Sap9. Hyphal filaments formed by mnt1- mnt2L1 mutant cells, unlike wild type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins, or with S. gordonii Ssp8 partner adhesin. These observations implied that early stage O-mannosylation was critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii, and microbial community development. C. albicans was enhanced in hypha formation when incubated planktonically with S. gordonii. This was supported by transcriptome RNASeq analysis which identified C. albicans genes, including the filamentation and pathogenesis associated genes FRG42, ALS1, CA T1 and TEC1, which were up-regulated in the presence of S. gordonii. Taken collectively these results identify new inter-Kingdom communication mechanisms that provide better understanding of the ways that microbial communities develop, and of potential means to control C. albicans infections.

The copper-containing protein superoxide dismutase is required for the virulence of C. albicans in a mouse model. Previous work in our laboratory has shown that copper uptake and regulation in C. albicans has some similarities to Saccharomyces cerevisiae, including the activation of the copper transporter gene CaCTR1 in low copper conditions by the transcription factor CaMac1p. However, further analysis in this study has demonstrated that the actual mechanism of regulation by CaMac1p is different from that of S. cerevisiae Mac1p.;This thesis demonstrates for the first time that the CaMAC1 gene is transcriptionally autoregulated in a copper-dependent manner. This is in contrast to the S. cerevisiae MAC1 homologue, which is constitutively transcribed. The presence of one binding site for CaMac1p in the promoters of CaCTR1, CaMAC1 and the ferric/cupric reductase gene CaFRE7 is sufficient for copper-responsive regulation. In contrast, two promoter elements are essential for normal levels of copper-dependent activation by S. cerevisiae Mac1p. CaMac1p is also involved in the regulation of the iron-responsive transcriptional repressor gene SFU1 and the alternative oxidase gene AOX2. This work describes key features of the copper uptake system in the human pathogen C. albicans that distinguishes it from similar processes in the model yeast S. cerevisiae. Transcriptional autoregulation of the CaMAC1 gene could enable C. albicans to respond more precisely to environmental changes, conferring an adaptation to the human host that may be an advantage in the disease process.

Among the prosthetic and implant biomaterials in the oral cavity currently exciting tremendous interest, titanium and resin are the main components of implants and dentures respectively. From their introduction in the oral cavity, a highly complex heterogeneous biofilm coats these biomaterials. The present thesis analyzes the complex relationships that are formed between one single microorganism (Candida albicans), one defence system of the oral cavity (oral peroxidases) and the two aforementioned biomaterials (titanium and resin). These biomaterials are indeed in contact with peroxidases: myeloperoxidase from neutrophils and sialoperoxidase from salivary secretions. Oral peroxidases belong to the salivary non immune innate defence mechanisms that control the oral microbial flora. In the presence of hydrogen peroxide (H2O2), they catalyze in vivo the oxidation of thiocyanate (SCN-) into hypothiocyanite (OSCN-), in vitro the oxidation of iodide (I-) into hypoiodite (OI-). In the salivary compartment and in oral biofilms, H2O2 is mainly formed by bacteria. In our investigations, H2O2 was produced by an enzyme sequence glucose (G) / glucose-oxidase (GOD). The OSCN- and OI- are antibacterial, antiviral and antifungal oxidants. Few studies have considered their action on Candida biofilms.

Candida albicans is a commensal yeast of the oral cavity which can turn parasitic when the host immune defences are weakened. This fungus forms biofilms on biomaterials within the mouth, especially on dentures, the decontamination of these prostheses is therefore essential to avoid the risk of candidosis. (Candida et prothèses dentaires. Ahariz M. Loeb I. Courtois Ph. Rev. Stomatol. Chir. Maxillofac. 111: 74-78, 2010).

In vitro, our investigations aimed to analyze the relationships between peroxidase systems and Candida. The effect of peroxidase systems (G / GOD / KI or KSCN / peroxidase) on Candida suspensions, on biofilms already formed or in formation was evaluated with various inputs of hydrogen peroxide and was studied by incorporation of enzymatic sequences in the culture media used for Candida biofilms formation. The susceptibility of Candida albicans ATCC 10231 to OSCN- versus to OI-, produced by lactoperoxidase (LPO), was studied in three different experimental models: - in a liquid culture medium – on a solid medium (with agarose gel), - in a biofilm model developed in the context of this work. The latter consisted of titanium powder suspended in Sabouraud broth contaminated with Candida albicans. The growth of Candida in the supernatant (planktonic phase) was evaluated by turbidimetry and the biomass of yeasts adherent to biomaterials (attached phase) by the tetrazolium salt MTT method. Enzymatic studies have allowed the optimization of the concentrations and activities of peroxidase systems components and the illustration of the competition between thiocyanate and iodide for lactoperoxidase. Peroxidase systems G/GOD/I-/LPO and G/GOD/SCN-/LPO prevented or limited the growth of Candida in the planktonic and attached phases on titanium powder for at least 21 days. At a dose of GOD (0.2 U / ml), the system G/GOD/I-/LPO has limited the development of planktonic and attached phases for 4 days while the system G/ GOD/SCN-/LPO has shown an inhibitory effect only in the first 2 days of incubation. (Candida albicans susceptibility to lactoperoxidase-generated hypoiodite. Ahariz M. Courtois Ph. Clinical, cosmetic and investigational dentistry; 2: 69-78, 2010).

In other experiments, peroxidase was adsorbed on titanium sheets in order to modify their surface and give them the property of inhibiting biofilm formation of Candida after addition of the enzyme substrates. Enzymatic studies and X-ray photoelectron spectroscopy (XPS) showed the adsorption of lactoperoxidase to titanium. In vivo, peroxidases are adsorbed on titanium healing abutments. (Adsorption of peroxidase on titanium surfaces: A pilot study. Ahariz M. Mouhyi J. Louette P. Van Reck J. Malevez C. Courtois P. J. Biomed. Mater. Res. 52: 567-571, 2000).

The development of the Candida biofilm was followed on titanium (powder or sheets) as well as on resin. Planktonic and attached phases have been monitored for 21 days. The presence of an exopolysaccharide matrix secreted by the yeasts has been observed with light microscopy and confirmed with fluorescence using the calcofluor method. In a series of experiments on titanium and resin sheets, an attached phase was demonstrated by the same techniques. The efficiency of the peroxidase system using iodide as a substrate was demonstrated when the enzyme was in solution and when it was preadsorbed on titanium (Candida albicans biofilm on titanium: effect of peroxidase precoating. Ahariz M. Courtois Ph. Medical Devices: Evidence and Research; 3: 33-40, 2010).

The incorporation in oral gels of other molecules present in exocrine secretions is a research direction that was also discussed: the present studies have demonstrated the in vitro benefits of peroxidase systems (with thiocyanate, chloride or especially iodide as substrates) acting in synergy with colostrum, lactoferrin and lysozyme. But the formulation of specialities that contain these natural antimicrobials is difficult to transpose in vivo as the complexity of the oral environment is very large (Denture contamination by yeasts in the elderly. Vanden Abbeele A. de Meel H. Ahariz M. Perraudin J.-P. Beyer I. Courtois P. Gerodontology; 25: 222-228, 2008).

Investigations pursued in vivo in 155 patients allowed the determination of the wild strains of Candida sp present on the fitting surface of the removable dental appliance and on the corresponding palatal mucosa. The link between the presence of yeasts and a reduced salivary flow was confirmed. These wild strains were directly grown and identified on Petri dishes (ChromAgar™ medium) from the macroscopic morphology of colonies and from additional tests (germination test in human serum, formation of chlamydoconidies on RAT medium, API™ galleries identification system ). For a period of two weeks, 14 patients accepted the daily application of a gel on the fitting surface of their denture. It was a double-blind comparison of an active gel containing the thiocyanate - lysozyme - lactoferrin - colostrum complete system with a control gel inactivated by heating. Data analysis showed a reduction in the number of colonies on the palatal mucosa by Candida sp, but not on the denture itself. By contrast, decontamination ex vivo of dentures by immersion in a bath (at room temperature or 37° C) containing either G/GOD producing H2O2 or the complete peroxidase system G/GOD/KI/L producing OI- demonstrated the efficiency of hypoiodite.

Parmi les biomatériaux prothétiques et implantaires qui connaissent actuellement un essor considérable dans la sphère orale, le titane est le composant principal des implants et la résine celui des prothèses dentaires. Dès leur introduction dans la cavité orale, un biofilm hétérogène très complexe les recouvre. Cette thèse analyse la complexité des relations qui se nouent entre un seul micro-organisme (Candida albicans), un système de défense de la cavité orale (les peroxydases orales) et les 2 biomatériaux précités (titane, résine). Ces biomatériaux sont en effet, dans le milieu oral, au contact de peroxydases: la myéloperoxydase des neutrophiles et la sialoperoxydase des sécrétions salivaires. Les peroxydases orales appartiennent aux mécanismes salivaires de défense innée non immunitaires qui contrôlent la flore microbienne orale. En présence de peroxyde d’hydrogène (H2O2), elles catalysent in vivo l’oxydation du thiocyanate (SCN-) en hypothiocyanite (OSCN-) et in vitro l’oxydation d’iodure (I-) en hypoiodite (OI-). Dans le compartiment salivaire et dans les biofilms oraux, l’H2O2 provient essentiellement des bactéries. Dans nos investigations, l’H2O2 était produit par une séquence enzymatique glucose (G) / glucose-oxydase (GOD). L’OSCN- et l’OI- sont des oxydants antibactériens, antiviraux et antifongiques. Peu d’études envisagent leur action sur les biofilms à Candida.

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Doctorat en Sciences dentaires
info:eu-repo/semantics/nonPublished

This thesis describes attempts to investigate the regulation of the Candida albicans hyphal-specific gene HYR1 by a functional dissection of the HYR1 promoter, protein localisation studies and analysis of HYR1 expression in C. albicans morphological mutants. Sequencing of the HYR1 promoter revealed several putative cis-acting elements within 700 bp of the determined HYR1 transcriptional start site. The possibility of using the LAC4 gene from Kluyveromyces lactis as a reporter for dissection of the HYR1 promoter in C. albicans was investigated. Expression of LAC4 in S. cerevisiae and C. albicans was driven by the C. albicans ADH1 promoter. LAC4 expression was carbon-source-dependent in Saccharomyces cerevisiae as shown by a plate assay and -galactosidase assay, and was confirmed by northern analyses which showed high levels of LAC4 mRNA. However, -galactosidase activity was not detectable in C. albicans transformants using the plate assay or the enzyme assay, and this lack of LAC4 expression was confirmed by northern analysis of the LAC4 mRNA. Preliminary Southern analysis revealed that the LAC4 sequences in S. cerevisae and C. albicans are maintained at approximately equal copy numbers between transformants. Hence LAC4 was not sufficiently sensitive to act as reporter of HYR1 expression and therefore the recently developed yEGFP gene was used for a preliminary HYR1 promoter dissection. However, the HYR1 promoter-yEGFP fusions failed to confirm a role for these elements in the regulation of HYR1 expression . Nevertheless, the hyphal-specific nature of HYR1 expression was confirmed by analysis of the HYR1-yEGFP mRNA by northern analysis. The yEGFP reporter also proved to be too insensitive for use as a reporter of HYR1 expression in C. albicans. To investigate the proposed localisation of the Hyr1p, an in-frame Hyr1-yEGFP fusion was created and expressed in C. albicans and S. cerevisiae. However, this work was inconclusive and the status of Hyr1p as a component of the hyphal cell wall remains to be confirmed.

This thesis had the purpose to understand pathogenicity in Candida albicans. The study focused on the previously known virulence factors. In addition, new genes related to virulence were investigated. To achieve this objective, two strains were initially compared; SC5314 (virulent strain) and RV4688 (attenuated strain) by transcript profiling and phenotypic analysis. Transcript profiling revealed that genes related to oxidative stress were down-regulated in the attenuated strain as compared to the virulent strain. RV4688 also formed hypha less readily and adhered less to buccal epithelia than SC5314. It was also determined that the virulence status of these two strains could not be changed. As suggested by microarrays, genes related to oxidative stress might be related to virulence in C. albicans. Therefore, the role of three different genes involved in stress response was analysed: SOD1, SOD2 and TTR1. The D sod1/sodl and D sod2/sod2 mutants were kindly provided by Dr. Sa-Ouk Kang. TTR1 was disrupted and subsequently reintegrated. To investigate the role of two genes in oxidative stress and virulence, a double mutant for SOD1 and TTRI was also generated and each gene was individually reintegrated. The oxidative stress mutants had delayed evagination to form germ tubes. The SOD1 and TTR1 genes seem to have specific roles to respond to the oxidative stress inducers menadione and diamide. However, both of them seem important for virulence in C. albicans, as the D sod1/sod1 and D ttr1/ttr1 mutants were attenuated for virulence in mice and quantitatively more killed by human neutrophils than the wild-type control. When different clinical isolates were characterized, it was demonstrated that SC5314 does not reflect what might happen with all the wild-type virulent strains because other clinical isolates showed clearly different phenotypes. Therefore, C. albicans virulence properties expression might be different in each strain.

It was of interest to clone key genes involved in O-glycosylation with a view to using reverse genetics to establish their function. The Candida homolog of the S. cerevisiae MNT1 gene (Hausler and Robbins, 1992) was cloned by heterologous probing of a genomic DNA library. The CaMNT1 gene was found to be regulated differentially in response to the environment and exhibited a transitory increase in the level of transcription during early germ tube formation. Low stringency Southern analysis of C. albicans genomic DNA identified several CaMNT1 homologs suggesting CaMNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases. In order to demonstrate that specific glycosyl residues were actively involved in the host-fungus interaction, the CaMNT1 gene was disrupted in two strains using the ura-blaster technique. Disruption at the CaMNT1 locus led to a 90% reduction in -1,2-mannosyltransferase activity when -methyl mannoside was used as an acceptor, but had no obvious influence on viability, growth rate, germ tube formation or proteinase production. CaMnt1 appears to be involved in O-glycosylation since the Camnt1 null mutant strain accumulated intracellularly the O-glycosylated enzyme chitinase. Mannosyltransferase-deficient Camnt1 mutants were significantly reduced in their ability to adhere to human buccal epithelial cells in vitro and were attenuated in virulence in systemic models of candidosis. O-linked mannan may therefore be important for direct interactions with epithelial surfaces or for the stabilization and function of cell surface adhesins. The low virulence potential displayed by Camnt1 mutants clearly demonstrates the important role glycosylation plays in the virulence of C. albicans. Given that O-glycosylation differs significantly between yeast and man, this protein modification may constitute a novel target for antifungal agents.

ALS3 encodes a hypha-specific cell wall protein of the Agglutinin-Like Sequence family of adhesins. The first aim of this project was to define the region of the ALS3 promoter required for its hypha-specific activation. Previous work in our laboratory defined a region of 190 bp between -496 and -306 that is important for ALS3 activation. Furthermore, two sequences that bind the transcriptional repressor Nrgl (NREs), were shown to be required for repression under yeast inducing conditions. In this study, the importance of these NREs for ALS3 repression was confirmed. Between the two studies, eight 13-68 bp overlapping sequences of the ALS3 promoter were placed upstream of the basal -306 to +4 region of ALS3 promoter (previously shown to cause no activation). None of these induced expression of the reporter. Further deletions downstream from -471, or upstream from -321 disrupted the hypha-specific regulation of ALS3. These results suggest that no short region or known response element, in isolation, is sufficient for hypha-specific activation of ALS3. During this study an efficient Streptococcus thermophilus lacZ reporter was developed for C. albicans. This was adopted to enable a high throughput mutagenesis screen on the ALS3 promoter. Another aim of this study was to contribute to the annotation of the C. albicans genome sequence with the EU Galar Fungail Consortium. This was achieved by annotating 6.5% (401 ORFs) of the predicted open reading frames annotated by the Consortium as a whole. These data were used by Christophe d'Enfert to design whole genome C. albicans microassays and to generate the database CandidaDB (http://genolist.pasteur.fr/CandidaDB/). Protocols for the use of these microarrays and for transcript profiling data analysis were established. Validation of the microarrays was carried out by the transcript profiling of cells which had undergone a temperature shift from 25°C to 37°C. This showed the up-regulation of some heat shock and stress response genes, as expected.

The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Previous research on this switch has focused on genes involved in the morphological switch from yeast to hyphal growth. However, very few genes have been found that directly control hyphal morphogenesis, suggesting primary control at the post‐translational level. The Cyclin Dependent Kinases (CDK), Cdk1, has been previously implicated in this process; therefore it was sought to identify phospho­‐regulatory targets of Cdk1 involved in C. albicans hyphal morphogenesis. Fkh2 was a likely CDK target that shows differential phosphorylation between yeast and hyphal growth; being phosphorylated in conjunction with cell cycle progression during yeast growth, but not in hyphae, where phosphorylation occurs for a short period on hyphal induction. Further investigations have found that Fkh2 is phosphorylated at C-­‐terminal CDK consensus sites on hyphal induction, but is not a regulatory target of Cdk1, or any other CDK related protein present in C. albicans. Although the kinase responsible remains elusive, the physiological consequences of the phosphorylation have been found to be quite profound, with phosphorylation of Fkh2 being a requirement for the maintenance of invasive filamentous growth. Loss of Fkh2 phosphorylation specifically prevents the expression of genes required for: filamentous growth, pathogenesis, host interaction and biofilm formation, which are required for the transition from commensalism to pathogenesis. Thus phosphorylation of Fkh2 on hyphal induction provides a cell cycle­‐independent function that contributes to pathogenesis.

El propósito del presente fue de identificar las especies de Candida implicadas en estomatitis subprotésica en pacientes del Departamento de Odontoestomatología del Centro Médico Naval “CMST” – 2007 Se analizaron los 30 primeros pacientes con diagnóstico de estomatitis subprotésica que acudían al Departamento, a los cuales se les realizó cuatro frotises, dos para el examen directo microscópico (con coloración Gram) para confirmar la presencia de levaduras, y dos para el cultivo en Agar Sabouraud+Cloranfenicol, del crecimiento en este agar, se hizo la prueba del tubo germinal para determinar la presencia de Candida albicans, de salir negativo esta prueba, se llevaba a cabo la identificación de la especie mediante el sistema Api Candida. Se obtuvieron entre otros resultados que Candida albicans fue la especie más implicada en la estomatitis subprotésica con un 96.66% seguido de Candida tropicalis con un 3.33%.
-- The purpose of this was to identify the species of Candida involved in stomatitis subprotésica in patients of the Department of Dental Naval Medical Center "CMST" - 2007 We analyzed the first 30 patients diagnosed with stomatitis subprotésica who went to the Department, to which were conducted four frotises, two for the direct microscopic examination (colouring Gram) to confirm the presence of yeast, and two to be planted in Agar Sabouraud + Chloramphenicol, growth in the agar, it was the germ tube test to determine the presence of Candida albicans, to leave this negative test was carried out to identify the species by the API system Candida. Among other results were obtained that Candida albicans was the most involved in stomatitis subprotésica with a 96.66% followed by Candida tropicalis with a 3.33%.
Tesis

Candida spp. se tornaram, nas últimas décadas, importantes agentes causadores de infecções invasivas, responsáveis por altos índices de morbidade e mortalidade. Acredita-se que a aspartato protease secretada (Sap) seja um fator diretamente associado aos processos infecciosos exercendo papel determinante na patogenicidade de Candida spp. e que a exposição a concentrações subinibitórias de antifúngicos, pode aumentar a produção de Sap e selecionar isolados resistentes. Analisaram-se isolados de Candida albicans e Candida não-albicans oriundos de casos de infecção hospitalar. Avaliou-se o perfil proteico destes isolados por eletroforese SDS-PAGE. Avaliou-se, também, o perfil de sensibilidade dos isolados frente a antifúngicos de uso convencional em esquemas terapêuticos (fluconazol e anfotericina B) e a antifúngicos mais novos que ainda não fazem parte das rotinas hospitalares (voriconazol e caspofungina). Determinou-se a atividade proteolítica qualitativa e quantitativa de Sap e atividade metabólica de C. albicans e Candida não-albicans cultivados na presença e ausência de concentrações subinibitórias de fluconazol e anfotericina B. Estabeleceu-se a correlação entre os testes e por fim, avaliou-se a expressão do gene SAP2 em C. albicans ATCC 64548 e C. krusei ATCC 6258. 100% dos isolados foram sensíveis a anfotericina B, voriconazol e caspofungina e 89,9% a fluconazol. Dois isolados (7,4%) apresentaram sensibilidade dependente da dose e um (3,7%) apresentou resistência ao fluconazol. Na análise qualitativa da atividade proteolítica, 77,7% dos isolados apresentaram atividade. A maioria dos isolados de C. albicans (50%) e Candida não-albicans (32%) apresentou atividade proteolítica moderada. A adição de fluconazol ao cultivo não promoveu alterações significativas na atividade proteolítica dos isolados padrões, enquanto anfotericina B inibiu o crescimento de C. glabrata ATCC 90030 e C. krusei ATCC 6258. Na análise quantitativa, todos os isolados se apresentaram ativos, sendo que a maior atividade foi observada em Candida complexo “psilosis” 210 (100%) e a menor em C. albicans 257 (2,44%). A maioria dos isolados de C. albicans (50%) foi classificada como fracamente proteolítica, enquanto Candida não-albicans (53%) como moderadamente proteolítica. A presença de antifúngicos no cultivo alterou significativamente o percentual de degradação da maioria dos isolados. A maior diferença de percentual foi observada em C. lusitaniae 286, que na presença de ¼ da IC90 de anfotericina B aumentou 13,7x em relação à ausência do fármaco. O método quantitativo de determinação da atividade proteolítica apresentou maior sensibilidade. 63% dos isolados de C. albicans apresentaram alta atividade metabólica e 68% de Candida não-albicans, atividade moderada. A maior atividade metabólica foi observada em C. albicans 120 (61,72%) e a menor em C krusei ATCC 6258 (2,35%). A atividade metabólica da maioria dos isolados foi significativamente alterada. A maior diferença de percentual foi observada em Candida complexo “psilosis” 210, que na presença de ½ da IC50 de fluconazol apresentou redução de 8x na atividade metabólica em relação à ausência do fármaco. Houve expressão de SAP2 apenas em C. albicans ATCC 64548, que foi reduzida significativamente na presença de ¼ da IC90 de anfotericina B. A atividade de Sap pode ser considerada um fator potencial associado à virulência, uma vez que o isolado que apresentou a maior atividade apresentou sensibilidade antifúngica reduzida.
Candida spp. has become in recent decades, major causative agents of invasive infections, responsible for high rates of mortality and morbidity. It is believed that the secreted aspartate protease (Sap) is a factor directly associated with infection exerting decisive role in the pathogenicity of Candida spp. and that the exposure to subinibitory concentrations of antifungals may increase the production of Sap and to select resistant isolates. We analyzed isolates of Candida albicans and Candida non-albicans from cases of hospital infection. We evaluated the protein profile of the isolates of Candida spp. by SDS-PAGE. We evaluated also the susceptibility profile of the isolates against antifungals of use conventional in regimens therapeutics (fluconazole and amphotericin B) and newer antifungals that are not yet part of the hospital routine (voriconazole and caspofungin). It was determined the proteolytic activity qualitative and quantitative of Sap and metabolic activity of isolates of C. albicans and Candida non-albicans grown in the presence and absence of subinhibitory concentrations of fluconazole and amphotericin B. Was established the correlation between the tests and, finally, the expression of the SAP2 gene in C. albicans ATCC 64548 and C. krusei ATCC 6258 was evaluated. 100% of the isolates were susceptible to amphotericin B, voriconazole and caspofungin and 89.9% to fluconazole. Two isolates (7.4%) showed susceptibility dose dependent and one (3.7%) showed resistance to fluconazole. In the qualitative analysis of proteolytic activity, 77.7% of the isolates showed activity. The most isolates of C. albicans (50%) and Candida non-albicans (32%) had moderate proteolytic activity. The addition of fluconazole to culture did not promote significant changes in the proteolytic activity of the isolates patterns, while amphotericin B inhibited the growth of C. glabrata ATCC 90030 and C. krusei ATCC 6258. In quantitative analysis, all isolates were active, being that the highest activity was observed in Candida complex “psilosis” 210 (100%) and the lowest in C. albicans 257 (2.44%). The most of the C. albicans isolates (50%) were classified as weakly proteolytic, while Candida non-albicans (53%) as moderately proteolytic. The presence of antifungals in the culture significantly changed the percentage of substrate degradation of the most of isolates. The greatest difference of percentage was observed in C. lusitaniae 286, which in the presence of ¼ IC90 of amphotericin B increased 13.7x regarding to absence of the drug. The quantitative method of determining the proteolytic activity presented greater sensitivity. 63% of the isolates of Candida albicans showed high metabolic activity and 68% of Candida non-albicans had moderate activity. The higher metabolic activity was observed in C. albicans 120 (61.72%) and lowest in C krusei ATCC 6258 (2.35%). The metabolic activity of the most of the isolates was significantly altered. The major difference of percentages was observed in Candida complex “psilosis” 210, which in the presence of ½ IC50 of fluconazole showed reduction of 8x in the metabolic activity regarding to absence of the drug. There was expression of SAP2 only in C. albicans ATCC 64548, which was significantly reduced in the presence of ¼ IC90 of amphotericin B. The Sap activity may be considered a potential factor associated to virulence, once that the isolate that showed the greatest activity showed susceptibility reduced.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A técnica da espectroscopia infravermelha por transformada de Fourier (FT-IR) vem sendo largamente empregada como uma abordagem rápida e simples para a identificação de microrganismos, incluindo a do gênero Candida. A proposta deste estudo foi avaliar o emprego da microespectroscopia FT-IR na identificação e discriminação de 5 cepas clínicas de Candida albicans e 3 de Candida glabrata, as quais foram identificadas previamente por meio de métodos convencionais, e mais duas cepas ATCC de cada espécie. As amostras foram analisadas em triplicata, a partir de culturas independentes, por meio de filmes finos obtidos da suspensão em solução salina estéril a 0,85% da biomassa da colônia que ficou incubada por 24 horas em placas com ágar Saboraud Dextrose. Dez espectros com 32 varreduras foram obtidos na forma de ponto em 10 regiões selecionadas aleatoriamente nas amostras. A média foi obtida dos dez espectros de cada amostra para a análise estatística multivariada, dada pela análise de cluster. Além disso os espectros foram transformados em primeira derivada e três janelas espectrais foram determinadas (900-1250 cm-1, 1300-1600 cm-1, 2800-3000 cm-1). A representação dos resultados foi dada pela construção de um dendograma. Nesse, foi possível separar em dois grupos distintos as duas espécies de Candida estudadas. Dessa forma, pode-se concluir que a microespectroscopia FT-IR foi capaz de identificar e discriminar cepas clínicas de Candida albicans e de Candida glabrata, sendo um método promissor para identificação de leveduras
The technique of infrared spectroscopy Fourier transform (FT-IR) has been widely used as a new approach for rapid identification and simple micro-organisms, including the genus Candida. The purpose of this study was to evaluate the use of FT-IR microspectroscopy for the identification and discrimination of 5 clinical strains of Candida albicans and Candida glabrata 3, which were previously identified by conventional methods, and two-standard strains of each species. The samples were analyzed in triplicate from independent cultures by means of thin films obtained from a suspension in sterile saline and 0.85% of the biomass of the colony that was incubated for 24 hours in Sabouraud dextrose agar plates. Ten spectra with 32 scans were obtained in 10 randomly selected regions in the samples.The average of ten spectra was obtained from each sample for the multivariate analysis, given by cluster analysis. In addition, three windows were determined spectral (900-1250 cm-1, 1300-1600 cm-1, 2800-3000 cm-1) and the spectra were transformed into first derivative. The representation of the results was given by the construction of a dendrogram. In this, we separated into two groups of two Candida species studied. Thus, one can conclude that the FT-IR microspectroscopy was able to identify and distinguish clinical isolates of Candida albicans and Candida glabrata is an important method for identification of yeasts

Orientador: Rosilene Fernandes da Rocha
Banca: Maria Stella Amorim da Costa Zollner
Banca: Antonio Olavo Cardoso Jorge
Resumo: A Candidose bucal e um dos processos infecciosos micóticos mais comuns da cavidade oral e existe suscetibilidade aumentada para o mesmo durante o período neonatal principalmente devido à imaturidade dos mecanismos de defesa e a falta de uma microbiota bucal balanceada. Para verificar a presença de Candida spp. na cavidade bucal de lactentes foram examinados inicialmente cem bebês, nos quais foi feita coleta de material do dorso da língua com swab nas primeiras 24 horas de vida. Trinta e três recém-nascidos deste grupo foram acompanhados durante os primeiros quatro meses de vida sendo realizada mensalmente coleta de material da cavidade oral e avaliação das condições gerais de saúde, nutrição e higiene. A análise do material obtido nesse período mostrou positividade para Candida spp. em 64 (48,5%) das 132 amostras. A doença foi observada em 27,3% dos recém nascidos acompanhados. Candida albicans foi a espécie encontrada mais frequentemente (44,6%), e esteve presente em nove dos 11 casos de candidose bucal. Verificou-se com a ananmese que os possíveis fatores de risco para o grupo estudado foram o uso de chupeta e mamadeira, e, a introdução de outros tipos de alimento. Conclui-se que Candida albicans e a espécie prevalente nesta faixa etária
Abstract: Abstract: Oral candidosis is one of the most frequent mycotic infectious diseases of the oral cavity and there is increased susceptibility to this infection during the neonatal period due to immaturity of the defense mechanisms and the lack of a balanced buccal microbiota. To verify the presence of Candida spp. in the oral cavity of infants were examined initially a hundred babies from which were collected material of the lingual dorsum with swabs in the first 24 hours of life. Thirty-three newborn of this group were accompanied during the first 120 days of life and were submitted monthly to a material collection of the oral cavity and an evaluation of general conditions of health, nutrition and hygiene. The analysis of the material collected during this period showed positivity to Candida ssp. in 64 (48,5%) of the 132 samples. Disease was observed in 27,3% of those 33 newborns. Candida albicans was the most frequent species (44,6%) and it was present in nine of the 11 cases of oral candidosis. The anamnesis showed that the possible risk factors were pacifier use, bottle feeding and introduction of different food types. It was concluded that Candida albicans is the prevalent species in this age band
Mestre

Orientador: Graziella Nuernberg Back Brito
Banca: Adolfo José da Mota
Banca: Ricardo Sérgio Couto de Almeida
Banca: Renata Falchete do Prado
Banca: Samira Esteves Afonso Camargo
Resumo: Candida albicans é um fungo oportunista capaz de causar infecções superficiais e até sistêmicas. A maioria das infecções é mediada pela formação de biofilme que confere resistência aos agentes antifúngicos e ao sistema imune, porém os mecanismos de desenvolvimento do biofilme e patogenicidade ainda não foram completamente elucidados. No presente estudo foram selecionados 9 genes de C. albicans com função desconhecida, dentre 34 cepas mutantes que apresentaram fenótipo alterado para formação de biofilme. Os biofilmes foram formados em placas de 96 poços ou discos de poliestireno e avaliados em diferentes tempos de desenvolvimento. A seguir foram construídas 4 cepas complementadas que foram avaliadas quanto à susceptibilidade a agentes estressantes, crescimento sob limitação de nutrientes e testes de filamentação. A arquitetura dos biofilmes foi analisada por microscopia eletrônica de varredura (MEV). Os biofilmes também foram avaliados quanto à quantidade de β-1,3-glucana e quitina. Para os modelos de infecção, células epiteliais bucais (TR-146) foram utilizadas para análise de aderência, invasão e dano. A patogenicidade das cepas foi avaliada em ovos embrionados de galinha durante 7 dias, após a inoculação das cepas. Células planctônicas e biofilmes foram submetidos a testes antifúngicos com os agentes fluconazol, anfotericina B e caspofungina. A reação em cadeia da polimerase quantitativa foi realizada para verificar a expressão dos genes MRV8 e NDT80 em células fúngicas em interação com células epiteliais e MRV8, MRV1 e MRV6 em células crescidas em biofilme. Os resultados foram analisados por teste t de Student, ANOVA, Tukey e testes de Log-rank (Mantel-Cox) (p < 0,05). Foram construídas 4 cepas complementadas para os genes selecionados ORF19.823, ORF19.7170, ORF19.6847 e MRV8. A função de ORF19.823 ainda permanece desconhecida, pois nãofoi observado fenótipo msignificativo para a cepa....
Abstract: Candida albicans is an opportunistic fungi capable of causing superficial and systemic infections. Most of infections are mediated by biofilm formation which confers resistance to antifungal agents and immune system, but the mechanisms of biofilm development and pathogenicity were not thoroughly elucidated yet. In the presente study, 9 unknown function genes of C. albicans were selected among 34 mutant strains that presented altered phenotype for biofilm formation. The biofilms were formed on 96-well microtitle plates or on polystyrene disks and evaluated in different time intervals. Next, 4 complemented strains were constructed and evaluated for susceptibility to stressor agents, growth under nutrient limitation and filamentation tests. The biofilm architecture was analyzed by scanning electron microscopy (SEM). The biofilms were also assessed as the quantity of β-1,3-glucan and chitin. For the infection models, buccal epithelial cells (TR-146) were used for adherence, invasion and damage assays. The pathogenicity of the strains was evaluated in embrionated chicken eggs for 7 days, after inoculation of the strains. Planktonic cells and biofilms were submitted to antifungal tests with fluconazole, amphotericin B, and caspofungin. Quantitative polymerase chain reaction was performed to verify the expression of MRV8 and NDT80 genes in fungal cells in interaction with epithelial cells and MRV8, MRV1, and MRV6 genes in cells grown in biofilm. The results were submitted to the Student t test, ANOVA, Tukey's test, and Log-rank test (Mantel-Cox) (p < 0.05). Four complemented strains were constructed for the selected genes ORF19.823, ORF19.7170, ORF19.6847, and MRV8. The function of the ORF19.823 is still unknown, because the mutant strain did not show any significative phenotype for the tests performed. The mutant strain for the ORF19.7170 caused less damage on epithelial cells, but the result was not significant and the gene was dispensable...
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