Orientador: Juliana Campos Junqueira / Banca: Antonio Olavo Cardoso Jorge / Banca: Ewerton Garcia de Oliveira Mima / Resumo: O objetivo deste estudo foi avaliar in vitro e in vivo as interações entre as diferentes espécies de Candida por meio da formação de biofilme em placas de 96 poços, indução de candidose experimental em modelo de Galleria mellonella e de camundongos imunossuprimidos. Foram estudadas as cepas padrão das seguintes espécies: C. albicans (ATCC 18804), C. krusei (ATCC 6258) e C. glabrata (ATCC 90030). A partir de cada espécie, foram formados biofilmes monotípicos e heterotípicos no fundo da placa de 96 poços por 48 h. A seguir, a quantidade de biofilme formado foi analisada pela determinação do número de unidades formadoras de colônias (UFC/mL). G. mellonella foi inoculada com suspensões homotípicas e heterotípicas de Candida (105 células/mL) e incubadas a 37°C. Durante 5 dias, o número de lagartas mortas foi avaliado diariamente para análise da curva de sobrevivência. Em outro experimento, após 0, 2, 4, 8, 12 e 24 h da infecção por Candida, a hemolinfa das lagartas foi extraída para contagem das células fúngicas (UFC/mL). Para a indução da candidose bucal em camundongos, os animais foram inoculados com suspensões microbianas homotípicas ou heterotípicas contendo 108 células/mL. Após 48 h da última inoculação, amostras do dorso da língua foram coletadas e semeadas em ágar cromogênico HiCrome para contagem de UFC/mL recuperadas da cavidade bucal. Em seguida, os animais foram eutanasiados e as línguas retiradas para análise macroscópica e microscópica. A análise dos dados de UFC/mL dos biofilmes in vitro, de Candida na hemolinfa de G. mellonella e da recuperação dos camundongos foi feita por Análise de Variância, Teste de Tukey ou t de Student. A análise da curva de sobrevivência foi realizada utilizando o teste Log-rank (Mantel-Cox). Para avaliação dos escores obtidos na análise macroscópica e histológica foram aplicados os testes de Krusk-Wallis ou Mann-Whitney ... / Abstract: The aim of this study was to evaluate in vitro and in vivo interactions between different Candida species through the formation of biofilms in 96-well plates, induction of experimental candidiasis in Galleria mellonella model and immunosuppressed mice. We studied the standard strains of the following species: C. albicans (ATCC 18804), C. krusei (ATCC 6258) and C. glabrata (ATCC 90030). Monotypic biofilms of each species and heterotypic biofilms were performed on the bottom of 96-well plate for 48 h. Then, the amount of biofilm was analyzed by determining the number of colony forming units (CFU/mL). The larvae of G. mellonella were inoculated with homotypic and heterotypic suspensions of Candida (105 cells/mL) and incubated at 37°C. For 5 days, the number of dead larvae was assessed daily for survival curve analysis. In another experiment, after 0, 2, 4, 8, 12 and 24 h of infection with Candida, hemolymph of worms was extracted for counting the fungal cells (CFU/mL). For induction of oral candidiasis in mice, the animals were inoculated with homotypic or heterotypic microbial suspensions containing 108 cells/mL. After 48 h of the last inoculation, samples of the tongue were collected and seed in HiCrome chromogenic agar for counting of CFU/mL recovered from the oral cavity. Then, the animals were euthanized and their tongues removed for macroscopic and microscopic analysis. The analysis of Candida biofilms in vitro CFU/mL, the hemolymph of G. mellonella and recovery of mice were made by ANOVA, Tukey test or Student's t test. The analysis of the survival curve was performed using GraphPad Prism using the Log-rank test (Mantel - Cox). Kruskal-Wallis or Mann-Whitney were applied for evaluation scores of macroscopic and histological analysis (p≤0,05). The results of biofilms in vitro have demonstrated a lower number of CFU/mL of C. albicans biofilms in heterotypic biofilm compared ... / Mestre

Glucans are (1→3)-β-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.

Background: Candida albicans is an opportunistic fungal pathogen that induces strong proinflammatory responses, such as IL-1β production. Much less is known about the induction of immune modulatory cytokines, such as the IL-1 receptor antagonist (IL-1Ra) that is the main natural antagonist of IL-1, by C. albicans. Methods: Peripheral blood mononuclear cells (PBMC) of healthy individuals were stimulated with C. albicans and different components of the fungal cell wall. The role of pathogen recognition receptors (PRRs) for the induction of IL-1β and IL-1Ra was investigated by using specific blockers or in PBMC from Dectin-1 deficient patients. Results: C. albicans induced a strong IL-1Ra response, and this induction was primarily induced by the cell-wall component β-glucan. Blocking IL-1Ra significantly increased C. albicans β-glucan hyphae induced IL-1β and IL-6 production. Surprisingly, blocking the β-glucan receptor Dectin-1 or the downstream Syk or Raf-1 pathways only marginally reduced C. albicans-induced IL-1Ra production, while blocking of the complement receptor 3 (CR3), TLR2 or TLR4 had no effect. In line with this, blocking MAP kinases had little effect on Candida-induced IL-1Ra production. PBMC isolated from Dectin-1 deficient patients produced normal IL-1Ra amounts in response to C. albicans stimulation. Interestingly, the IL-1Ra synthesis induced by β-glucan was blocked by inhibitors of the Akt/PI3. K pathway. Conclusions: β-glucan of C. albicans induces a strong IL-1Ra response, which is independent of the β-glucan receptors dectin-1 and CR3. These data strongly argue for the existence of an unknown β-glucan receptor that specifically induces an Akt/PI3. K-dependent anti-inflammatory IL-1Ra response upon recognition of C. albicans.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. / Introduction Despite the availability of several antifungal drugs, Candida infections remain a major health threat worldwide. The Candida infections problem has been amplified by the emergence of multidrug resistant Candida species towards the conventional antifungal drugs. In addition, activation of antioxidant defense system by Candida species has been known to be forefront mechanism to escape drug toxicity.This indicates an urgent need for the development of new therapeutic strategies and antifungal drugs. Natural products have served for centuries for the treatment of infectious diseases and are among the major sources for finding new antifungal drugs. Berberine (BER), an isoquinoline alkaloid found in a variety of plant species, has been shown to possess multiple biological and pharmacological properties including antimicrobial activity against C. albicans and other Candida species. However, the mechanism of action exerted by BER and its effect on Candida cells is not yet fully elucidated. Therefore, this study was conducted to evaluate the role of antioxidant enzymes in the susceptibility to fluconazole (FLC) in C. albicans. Another aspect was to determine the effect of BER on growth, antioxidant enzymes and their gene expression in C. albicans. Materials and methods Candida albicans clinical isolates (10 FLC susceptible and 10 FLC resistant) and one ATCC strain were obtained from the Department of Clinical Microbiology and Infectious Diseases, University of the Witwatersrand. Species identification was confirmed using API 20C AUX. Antifungal susceptibility was determined following CLSI M27-A3 guidelines. Gene expression of SOD1, SOD2, GPx2, GLR1, GTT11, and CAT1 in untreated and BER treated C. albicans cells was measured by RT-qPCR. The activity level of the corresponding enzymes in the presence of BER was determined using a spectrophotometer. Results Gene expression analysis showed an increase in mRNA expression level of SOD1, SOD2, GPx2, GLR1 and GTT11 genes in FLC resistant isolates than in the susceptible group. The most significantly expressed gene was SOD1 with 50.69-fold increase. The other genes showed moderate increase in the expression with fold change ranging from 1.2 to 4.2. The susceptibility test showed MICs ranging from 125 to 500 μg/ml with a significant difference in the activity of BER between FLC susceptible and resistant C. albicans. BER treatment induced upregulation in the mRNA expression and enzymatic activities of major antioxidants. In FLC resistant C. albicans, treatment with ½ MIC value of BER caused downregulation of the targeted antioxidant genes indicating that BER at this concentration induced an intense oxidative stress, therefore, surpassing the antioxidant capacity of the cells. Conclusion The findings in this study showed that drug resistance is not only caused by mutations in a particular gene but could also arise from proteomic modulations. The study . The study also demonstrated that C. albicans activates several antioxidant enzymes that form an integral component of the cell’s response against oxidative stress. Candida albicans showed efficient antioxidant response at lower concentrations of BER. However, BER at ½ MIC value induced robust oxidative stress, especially in FLC resistant C. albicans, surpassing the antioxidant capacity of the cells. This demonstrates that BER at sub-inhibitory concentrations is able to render C. albicans avirulent by suppressing its antioxidant defense response without compromising cell viability of the fungi. Therefore, BER has a potential to be developed into BER has a potential to be developed into a therapeutic agent a therapeutic agent for for the the treatment oftreatment of C. albicansC. albicans infections and other pathogenic fungi to infections and other pathogenic fungi to overcome drug resistance. overcome drug resistance. / E.K. 2019

The fungal cell wall serves as the interface between the organism and its environment. Complex carbohydrates are a major component of the cell wall, , glucan, mannan and chitin. β-Glucan is a pathogen associated molecular pattern (PAMP) composed of β-(1 → 3,1 → 6)-linked glucopyranosyl repeat units. This PAMP plays a key role in fungal structural integrity and immune recognition. Glycogen is an α-(1 → 4,1 → 6)-linked glucan that is an intracellular energy storage carbohydrate. We observed that glycogen was co-extracted during the isolation of β-glucan from SC5314. We hypothesized that glucan and glycogen may form a macromolecular species that links intracellular glycogen with cell wall β-(1 → 3,1 → 6)-glucan. To test this hypothesis, we examined glucan-glycogen extracts by multi-dimensional NMR to ascertain if glycogen and β-glucan were interconnected. H NMR analyses confirmed the presence of glycogen and β-glucan in the macromolecule. Diffusion Ordered SpectroscopY (DOSY) confirmed that the β-glucan and glycogen co-diffuse, which indicates a linkage between the two polymers. We determined that the linkage is not via peptides and/or small proteins. Our data indicate that glycogen is covalently linked to β-(1 → 3,1 → 6) glucan via the β -(1 → 6)-linked side chain. We also found that the glucan-glycogen complex was present in , and , but was not present in or hyphal glucan. These data demonstrate that glucan and glycogen form a novel macromolecular complex in the cell wall of and other species This new and unique structure expands our understanding of the cell wall in species.

We have inestigated the expression of the genes hydratase-dehydrogenase-epimerase (HDE), acyl-CoA oxidase (AOX) and catalase (CATL) of the diploid yeast Candida tropicalis. These genes encode enzymes which are localized to the peroxisome. Expression of each gene was monitored by immunoblot analysis of yeast lysates using antibodies directed against each protein. We demonstrate that carbon sources influence expression of these genes, and do so in a coordinate fashion. We expressed C. tropicalis HDE in Saccharomyces cerevisiae and demonstrate that this trifunctional enzyme can be regulated by S. cerevisiae in a fashion that closely resembles that of C. tropicalis. Expression of constructs containing deletions in the upstream region of the HDE gene allowed us to localize regions responsible for regulating the expression of this gene. Regions were identified that are responsible for both repression by glucose and induction by oleic acid. A glucoseresponsive region lies between nucleotides -466 and -334. An oleic acid-responsive region lies between nucleotides -333 and -281. An additional region controlling derepression by nonfermentable carbon sources is located downstream of nucleotide -281. Comparison of the upstream nucleotide sequences of HDE, AOX and CATL both to each other, and to upstream regions of other oleic acid-responsive genes of C. tropicalis has identified possible consensus nucleotide sequences for glucose-and oleic acid-responsive upstream elements. Since the regulation of the HDE gene in S. cerevisiae closely resembles that of C. tropicalis, this implies that similar mechanisms of transcriptional control operate in both yeasts. / Thesis / Master of Science (MS)

Strains of pathogens are typically described as virulent or non-virulent. However, in the majority of pathogens, strains often vary continuously and quantitatively in their virulence and pathogencity. Biofilm formation is one of the recently recognized virulence factors in many human pathogens and little is known about the variation and evolution of biofilms among natural strains. In this study, I examined quantitative variation of biofilms among natural strains of the human pathogenic yeast Candida albicans. A total of 115 natural strains of C. albicans from three sources (vaginal, oral and environmental) were quantified by two mebods: (i) the XTT tetrazolium reduction assay, and (ii) optical density following staining by crystal violet dye. Mature biofilm was confirmed by observation using confocal laser scanning microscopy. My analyses indicated that strains from each of the three scurces varied widely in biofilm formation abilities and that biofilm formation ability was positively correlated to cell surface hydrophobicity (CSH). For each strain, multilocus genotypes were determined by PCR-RFLP, my comparative genotype and biofilm analyses denonstrated that natural clones and clonal lineages of C. albicans exhibited extensive quantitative variation for biofilm formation. I also examined potential interactions among strains within C. albicans and between different Candida species. My preliminary results suggest significant variation and complex patterns of strains or species interaction during Candida biofilm development. / Thesis / Master of Science (MS)

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Gandida albicans strain 105 from a normal human and strain 582 (from the American type Culture Collection) were used for studying the effect in vitro of pH, various amounts of carbon dioxide, nitrogen, and composition of media on filamentation in this yeast-like organism. The yeast phase of the organism was maintained on a glucose, glycine, yeast extract (GGY) medium (1%; 1%; 0.5%) at 37°C. The experiments were conducted on both solid and liquid media. All cultures were incubated at 37°C. for 48 hours. The two strains of c. albicans, although similar to one another in their yeast forms, behaved differently toward the environmental conditions used; strain 582 responded more readily to the factors inducing filament formation than did strain 105. Increasing the pH above 6.5 to 7.0, 7.5 and 8.0 induced maximum filamentation in strain 582, whereas no filaments were produced by strain 105. All the aerobic cultures on solid GGY medium showed alkalinity and were positive for ammonia at the end of the incubation period. In liquid media, no alkalinity was observed at any pH values. Presence of 75% carbon dioxide in the atmosphere increased filamentation in strain 582 to a maximum degree, and induced mycelial formation in strain 105. With 94% or 95% carbon dioxide, growth and filamentation decreased in both strains. None of the CO2 cultures showed alkalinity at the end of the incubation period. Moreover, all the CO2 cultures were negative for ammonia. Growth under nitrogen (9J%) was less than that of the aerobic cultures. However, colonies appeared larger in size. Nitrogen stimulated filamentation in strain 105 only at a pH of 8.0, whereas strain 582 formed a maximum amount of filaments at pH values of 7.0 to 8.0. All the solid cultures under nitrogen showed alkalinity, while the liquid cultures were acid at all pH values. The occurrence of deamination in a medium without glucose in both strains of C. albicans showed that this organism was able to use glycine its source of both nitrogen and carbon. However, only a sparse growth was obtained in a medium lacking glucose. Strain 105 did not form filaments in such a medium, while strain 582 did so. Since more filaments were produced by the latter strain when a fresh subculture on a GGY medium was transferred to a medium without glucose, it was concluded that possibly glucose is required for both growth and filamentation. Comparative studies of the effect of a medium containing mannose with a glucose medium showed the two sugars behaved similarly with regard to fermentation and filament induction in both strains or c. albicans. Under conditions where glucose induced filamentation (e.g., with C02 or N2), mannose also induced filamentation. The decreased growth in the presence of oleic or stearic acid in a concentration of 40 micrograms per liter was attributed to the toxic effect of the fatty acids. Moreover, it was noted that the two acids had different effects on filamentation in the two strains. Oleic acid in a solid GGY medium induced hyphal formation in strain 105 only under nitrogen; without glucose, oleic acid did not bring about filamentation under any of the atmospheric conditions tested. In liquid media, oleic acid induced filamentation for strain 105 only when glucose was omitted. With strain 562, oleic acid promoted filamentation in both liquid and solid media with or without glucose, except for solid cultures incubated under nitrogen in the absence or glucose. Stearic acid did not stimulate filamentation in strain 105 under any conditions, but did increase hypha! formation in strain 582. In the presence of stearic acid, maximum filamentation occurred in aerobic cultures wnen glucose was absent. Although maximum filamentation occurred with an increase in the pH of the medium under aerobic conditions, in the presence of 75% C02, under nitrogen or in the presence of stearic acid in a medium without glucose, yeast cells were also present, indicating that this Y to f transformation was not complete. / 2999-01-01

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Durant les 20 dernières années, les infections fongiques ont augmenté de façon considérable et causent un défi dans le domaine médical. C. albicans est responsable de 20 % à 75 % de la majorité des candidoses. La candidose buccale se produit à plus de 80% chez les patients infectés par le VIH, et C. albicans a pu être identifié chez 78-100 % des porteurs de prothèses dentaires souffrant de stomatite prothétique. Lors de traitement, les antifongiques ont une efficacité limitée. Dans le but de trouver un nouvel antifongique qui pourrait résoudre les problèmes de résistance de C. albicans aux antifongiques disponibles actuellement, nous avons évalué in vitro l'effet du famesol sur la croissance et la transformation de C. albicans ainsi que son innocuité sur les cellules gingivales humaines. Nous avons d'abord étudié l'effet du famesol à différentes concentrations (0 à 300 uM) sur la croissance et la transformation de C. albicans. Pour tester l'innocuité du famesol, nous avons utilisé des cellules épithéliales et des fibroblastes isolés de biopsies de tissus palatins. L'effet du famesol sur la croissance et la transformation de C. albicans a été analysé par numération cellulaire. L'innocuité a été étudiée par des techniques de microscopie optique et par numération cellulaire. Avec les différentes cultures cellulaires qu'on a réalisées, nous avons montré que le famesol réduit la croissance de C. albicans et inhibe sa transformation de la forme blastospore à la forme hyphe. Les études de l'innocuité du famesol sur les cellules gingivales humaines ont montré que le famesol était nocif pour les fibroblastes (70 uM) mais les cellules épithéliales étaient plus résistantes (150 uM). Nos travaux montrent aussi que le famesol et les cellules gingivales humaines ont des effets synergiques contre la transformation de C. albicans. Ces études suggèrent que le famesol pourrait être utilisé comme un agent antifongique.