Orientador: Cristiane Yumi Koga-Ito / Resumo: A crescente incidência da resistência às drogas antifúngicas tornou-se um grande desafio para áreas médica e odontológica, fazendo com que a busca por métodos de tratamento alternativos ou em combinação com as já utilizadas sejam urgentemente necessárias. O objetivo deste projeto foi avaliar os efeitos da associação entre o plasma frio em pressão atmosférica e antifúngicos poliênicos convencionais utilizados no tratamento tópico da candidose bucal. Para tanto, foram realizados: a) Determinação das concentrações inibitórias dos antifúngicos poliênicos nistatina e anfotericina B e do plasma frio em pressão atmosférica sobre biofilmes de C. albicans; b) Avaliação do efeito dos tratamentos isolados e associados em condições/concentrações sub-inibitórias para fins de comparação e c) Avaliação dos diferentes protocolos de aplicação dos tratamentos, visando a obtenção da condição experimental mais eficaz para o controle do biofilme fúngico. Os dados de unidades formadoras de colônia foram comparados estatisticamente entre os grupos por One-way ANOVA e post hoc Tukey, com nível de significância de 5%. A partir dos resultados obtidos, pode-se concluir que o jato de plasma apresentou efeito antifúngico similar quando aplicado isoladamente ou em associação com nistatina e anfotericina B. Ainda, foi possível concluir que o jato de plasma frio apresenta efeito antifúngico mais eficaz do que os tratamentos dos biofilmes utilizando nistatina e anfotericina B no tempo de 5 minutos. / Abstract: The increasing incidence of antifungal resistance represents a great challenge in the medical areas and Dentistry. For this reason, the search for alternative methods or combination with the conventional ones is urgently necessary. The aim of this project is to evaluate, the effects of association between atmospheric pressure cold plasma and conventional polyene antifungals used for the treatment of oral candidiasis will be studied. With this purpose, the following methodologies were be used: a) To determine the inhibitory concentrations of the polyene antifungals nystatin and amphotericin B and cold plasma on C. albicans biofilms, b) To evaluate the combination of treatments in subinhibitory conditions, including isolated treatments for comparison purposes and c) To evaluate different protocols of treatment application, aiming to obtain the most effective protocol against fungal biofilm. Data was compared by One-way ANOVA and post hoc Tukey, with a significance level of 5%. Considering the results, it could be concluded that cold plasma showed similar effects when applied alone or in association to nystatin and amphotericin B. Cold plasma showed more effective antifungal effect on biofilms when compared to nystatin and amphotericin B, after 5 min exposure. / Mestre

Orientador: Juliana Campos Junqueira / Banca: Antonio Olavo Cardoso Jorge / Banca: Graziella Nuernberg Back Brito / Resumo: Estudos realizados in vitro tem demonstrado que Streptococcus mutans podem produzir metabólitos capazes de inibir Candida albicans, tornando interessante a identificação e desenvolvimento de novas substâncias para o tratamento da candidose bucal. Assim, o objetivo desse estudo foi extrair, fracionar e identificar as substâncias produzidas por S. mutans e avaliar seus efeitos sobre a patogenicidade de C. albicans e na resposta imunológica em modelos de estudo in vivo. As substâncias do sobrenadante da cultura de S. mutans foram extraídas com acetato de etila e posteriormente fracionadas em coluna de sílica derivatizada C-18 (150 g, Φ = 3,5 cm) utilizando diferentes soluções de MeOH:H2O (36:64, 49:51, 60:40, 76:24, 100:0) como eluente, obtendo cinco diferentes frações (SM-F1, SM-F2, SMF3, SM-F4 e SM-F5). A identificação das substâncias contidas no extrato bruto e frações foi realizada por cromatografia gasosa acoplada a espectrometria de massas. Foram testados os efeitos do extrato bruto e frações do sobrenadante da cultura de S. mutans sobre a candidose experimental induzida em modelo invertebrado de Galleria mellonella e em camundongos imunossuprimidos. Para a escolha da concentração a ser testada nos modelos in vivo foi realizada a determinação da Concentração Inibitória Mínima do extrato bruto e frações sobre C. albicans. No modelo de infecção experimental com G. mellonella, os efeitos do extrato e frações foram analisados pelos testes de curva de sobrevivência, quantificaç... (Resumo completo, clicar acesso eletrônico abaixo) / In vitro studies have shown that Streptococcus mutans can produce metabolites capable of inhibiting Candida albicans, becoming interesting the identification and development of new substances for the treatment of oral candidiasis. Thus, the objective of this study was to extract, fractionate and identify the substances produced by S. mutans and evaluate their effects on the pathogenicity of C. albicans and on the immune response in in vivo study models. Substances from the S. mutans culture supernatant were extracted with ethyl acetate and subsequently fractionated on a C-18 derivatized silica column (150 g, Φ = 3.5 cm) using different solutions of MeOH:H2O (36:64, 49:51, 60:40, 76:24, 100:0) as eluent, obtaining five different fractions (SM-F1, SM-F2, SM-F3, SM-F4 and SM-F5). The identification of the substances contained in the crude extract and fractions was performed by gas chromatography coupled to mass spectrometry (GC-MS). The crude extract products and the fractions of the supernatant of the S. mutans culture were assessed on experimental candidiasis induced in invertebrate model of Galleria mellonella and in immunosuppressed mice. For a choice of the concentration to be tested in the in vivo models, a determination of the Minimum Inhibitory Concentration (MIC) of the crude extract and fractions on C. albicans was performed. In the model of experimental infection with G. mellonella, the effects of extract and fractions were analyzed by the survival curve test, (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Candida albicans, a commensal of gastrointestinal and uro-vaginal tract can cause superficial as well as life threatening disseminated infections under conditions of lowered immunity of the host such as HIV infection, drug induced immune suppression [given during organ transplantation to prevent rejection] and radiation therapy [head and neck cancer patients] (Odds, 1988; Fidel and Sobel, 1996). Candida albicans shows a range of morphologies, it can switch from budding yeast morphology to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa) (Mitchell, 1998). The various factors that contribute to its virulence include its ability to undergo yeast to hyphal transition, formation of biofilms, adhesion and secretion of aspartyl proteinases. Hyphae are considered to be involved in invasive growth as they are frequently identified in infected tissues and strains defective in morphological transition (yeast to hyphal) are avirulent (Leberer et al., 1996; Lo et al., 1997; Stoldt et al., 1997). Morphological switching is not only necessary for successful establishment of infection but important for evading components host defense system like macrophages or dendritic cells. A network of signaling pathways that operate in C. albicans continuously assess the nutrient availability, cell density and other environmental conditions. The integrated output of these pathways determine the response of C. albicans under given set of environmental/media conditions and eventually determines the gene expression and morphogenic transition (Liu., 2001). C. albicans utilizes at least two major signaling pathways besides others for regulating the morphological transition. One of these two pathways uses Cph1 as transcription factor and is the homolog of Ste12 in S. cerevisiae which is shown to be involved in Pseudohyphal growth and mating. The other pathway includes Efg1 (homolog of Phd1 in S. cerevisiae) as transcription factor. Biofilm formation by Candida species is an important virulence factor and has gained considerable interest recently as these specialized survival structures are found in implanted devices such as indwelling catheters and prosthetic heart valves (Hawser and Douglas, 1994; Douglas, 2003). These biofilms lead to the failure of implants besides providing multiple drug resistance (Baillie and Douglas, 1999). A better understanding of the C. albicans interaction with the host at the site of infection and with the components of immune system will help in identifying new potential drug targets. (a) Genome wide expression profile of Candida albicans from patient samples and characterization of CaRPB4/7: To get a better insight in C. albicans response at the site of infection we were interested in mapping the expression profile of Candida albicans in active state of human infections. Patients suffering from head and neck cancer undergoing radiation therapy have high risk of C. albicans infection. We identified five such patients with heavy oral thrush infections and C. albicans samples were collected from them. Candida albicans was confirmed in these samples by various microbiological tests following which the samples were used for RNA isolation. The whole genome expression analysis leads to the identification of 188 up regulated and 88 down regulated genes in patient samples. Our data analysis revealed that Protein Kinase A pathway and many downstream genes of the same were differentially expressed. Analysis of saliva (saliva is known for antifungal and antibacterial activity) from these patients showed that unlike healthy individuals, the patient saliva favours yeast to hyphal transition of C. albicans cells. This might be a reason for high risk of infection. A major class of upregulated genes is found to be functionally involved in transcription which includes some RNA polymeraseII and III subunits. CaRPB4, the forth largest subunit of RNA polymeraseII, was found to be upregulated in patient samples. RPB4 has been shown to form sub complex with RPB7, the seventh largest subunit of RNA polymeraseII, and both subunits are known to play a role in a variety of stress conditions and pseudohyphal development in Saccharomyces cerevisiae. We characterized the CaRPB4 and CaRPB7 (homolog in Candida albicans) for their ability to complement their S. cerevisiae counterparts. CaRPB4 and CaRPB7 were able to complement majority of the phenotypes associated with these subunits in S. cerevisiae. Overexpression of CaRPB7 in S. cerevisiae enhances pseudohyphal growth. Considering the high degree of conservation of signaling pathways between S. cerevisiae and C. albicans it can be speculated that CaRPB7 might be involved in pseudohyphal development in C. albicans. We found that over expression of CaRPB4 in Candida albicans shows enhanced agar invasive growth which can be thought analogous to tissue invasion in host and hence might contribute for establishment of infection. This suggests that both the RNA polII subunits have a role to play in the virulence of C. albicans. (b) Characterization of UDP-Galactose 4-Epimerase (GAL10) from Candida albicans and their role in virulence. Enzyme UDP-Galactose-4-Epimerase [GAL10] is responsible for conversion of UDP-galactose to UDP-glucose which then gets metabolized by the cells through glycolysis and TCA cycle. The enzyme catalyzes a reversible reaction and can convert glucose to galactose in the absence of galactose as shown in Trypanosoma brucei and also involved in its virulence. In this study, we have identified the functional homolog of GAL10 in Candida albicans. S. cerevisiae and C. albicans GAL10 homologs are similar in their domainal organization as the proteins have a mutarotase and an epimerase domain. The former is responsible for conversion of ﾟ-D-galactose to a-D-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. The synteny of galactose metabolizing structural genes is conserved among some fungi. To study the importance of CaGAL10 we generated deletion mutant of the gene in C. albicans. Our studies show that CaGAL10 [C. albicans GAL10] is involved in cell wall organization and in oxidative stress response. The mutant strain of GAL10 is hyperfilamentous in Lee’s and spider medium and the biofilm formed is morphologically different from the wild type strain. These set of results suggests that CaGAL10 plays an important role in organization/integrity of cell wall in C. albicans and speculate that it might be involved in virulence. (c) Study of Candida albicans-macrophage interaction and identification of transcriptional regulator of genes encoding proteins of translation machinery: Macrophages serve as the effector cells of cell mediated immunity in the control of infections. They are considered to be important for resistance to muco-cutaneous and systemic candidiasis. Our studies were aimed at understanding the response of Candida albicans cells to the presence of macrophages for extended period of time. The response was monitored using microarrays. Specifically genes involved in galactose, protein and lipid metabolism and stress response undergo concerted changes in their transcript levels. We analyzed the promoters of coregulated genes to identify common DNA elements present in them which might be involved in their transcriptional regulation. Promoter analysis of differentially expressed genes revealed presence of CPH1 and EFG1 transcription factor binding sites. Besides identifying CPH1 and EFG1 Binding sites, we identified two novel DNA elements in promoters of coregulated gene. A conserved motif TGAAAAGGAAG was identified in the promoters of genes involved in energy generation. Another 18 mer consensus palindromic sequence TAGGGCTNTAGCCCTAAT was identified in the promoters of about 48 genes. Majority of these genes encode ribosomal proteins. With the help of techniques like EMSA (Electophoretic Mobility Shift Assay) and south-western we had shown the presence of a protein of ~66 KDa molecular weight binding to the sequence with high specificity.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Marcela Vaisová Supervisor: Lucie Křivčíková Title of diploma thesis: Effect of tyrosol on minimal inhibitory concentration of antimycotics Previous studies concerning quorum sensing in Candida albicans discovered two quorum sensing molecules, farnesol and tyrosol. Tyrosol has been formerly confirmed as a substance which shortens the lag phase of Candida albicans cell cycle and stimulates its conversion to the hyphal form, farnesol stimulates blastospores. This work covers the effect of tyrosol combined with two antimycotics, fungistatic fluconazole and fungicidal amphotericine B. Candida albicans strains used in the experiment were mostly isolated from samples taken from patients suffering from variously located Candida albicans infections and four laboratory standard strains were also used. The method used for the experiment was a modified broth dilution method using combinations of both tyrosol and antimycotics various concentrations, which were prepared by two-fold dilution. Using amphotericine B, 28 strains did not show any difference in MIC compared with the control sample, 6 strains showed mildly higher MIC and 3 strains shower mildly lower MIC. Using fluconazole, 24...

Orientador: Daniela Cardoso Umbelino Cavallini / Coorientador: Carla Raquel Fontana / Banca: Juliana Cabrini Carmello / Banca: Marlus Chorilli / Resumo: Os microrganismos probióticos associados a produtos alimentícios já são amplamente utilizados para a manutenção da saúde e redução do risco de certas doenças. Nesse contexto, diversos estudos mostram que cepas probióticas específicas têm grande potencial de manutenção e melhora da saúde oral, apresentando atividade anticariogênica, redução da halitose e prevenção de infecções oportunistas, como a candidose, frequentemente presente em indivíduos imunocomprometidos. Essas condições patogênicas apresentadas estão relacionadas à formação e estabelecimento de biofilmes na cavidade oral. As infecções provenientes de biofilmes são duradouras, altamente resistentes à farmacoterapia e de difícil eliminação. Dessa forma, o objetivo do presente estudo foi obter filmes orodispersíveis (ODF) contendo cepa probiótica Enterococcus faecium CRL 183, avaliando sua capacidade de inibir a formação e/ou maturação de biofilme de Candida albicans in vitro. Essa cepa foi selecionada devido as suas características de cultivo, informações disponíveis sobre efeito probiótico, segurança e caráter inovador, pois não há na literatura informações sobre a atividade antifúngica dessa cepa na cavidade oral. A segurança da cepa probiótica foi avaliada através do teste susceptibilidade a antimicrobianos. Para testar a capacidade de inibição de biofilmes de C. albicans in vitro foram formados biofilmes polimicrobianos (C. albicans + E. faecium), em diferentes proporções de fungo: probiótico (1:1; 1:10; 1:... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Probiotic microorganisms associated with food are already widely used to maintain health and reduce risk of certain diseases. In this context, several studies show that certain probiotic strains have great potential for maintenance and improvement of oral health, presenting anticariogenic activity, reduction of halitosis and prevention of opportunistic infections, such as candidiasis, often present in immunocompromised individuals. These pathogenic conditions are related to formation and establishment of biofilms in the oral cavity. Infections caused by biofilms are long-lasting, highly resistant to pharmacotherapy and difficult to eliminate. Thus, the objective of the present study is to obtain orodispersible films (ODF) containing Enterococcus faecium CRL 183, evaluating its capacity of inhibiting the formation and / or maturation of Candida albicans biofilms in vitro. This strain was selected due to its cultivation characteristics, available information on probiotic effect and safety and innovative character, as there is no information on the antifungal activity of this strain in the oral cavity. The safety of the probiotic strain was evaluated by antimicrobial susceptibility test. In order to test the ability to inhibit the formation of C. albicans biofilms in vitro, combined polymicrobial biofilms (C. albicans + E. faecium) with different proportions of opportunistic pathogens: probiotic were formed (1: 1, 1:10, 1: 100), during the formation (24 hours) and maturation (48... (Complete abstract click electronic access below) / Mestre

The work deals with the synthesis and characterization of cerulenin analogues as inhibitors of efflux pump mediated resistance of Candida albicans isolates and as inhibitors of the fatty acid synthesis enzyme KasA of Mycobacterium tuberculosis. Cerulenin was chosen as the lead structure, being a substrate of the efflux pumps in Candida albicans on one hand and therefore variations on the structure could lead to a blocking of the efflux pumps as in the case of tetracycline and inhibitor 13-CPTC of the TetB efflux pump. On the other hand, cerulenin is a known inhibitor of the FAS system but inhibition is unselective in type I and II FAS. Therefore, analogues could result in increased selectivity towards the type II FAS system in M. tuberculosis. The first cerulenin derivatives were prepared by coupling 2,3-dihydrofuran to the before synthesized 1-octaniodide, followed by ring opening and oxidation in one step by chromic acid and transfer of the resulting 4-keto acid to amides to give analogues 4a-d, 4e was prepared in analogy. To include the epoxide function especially with regard to the mechanism of action of cerulenin in the FAS system (considering known crystal structures of cerulenin and the KasA analogue of E. coli) tetrahydro- and dihydrocerulenin analogues were synthesized. Starting from the corresponding aldehyde, lactone 5 (tetrahydrocerulenin analogues) was obtained via two different routes A and B. Route A included the coupling of the aldehyde 1-nonanal to propiolic acid via a Grignard reaction with subsequent hydrogenation with the Lindlar catalyst under hydrogen pressure to give 5. Via Route B 1-nonanal was coupled to methyl propiolate by n-BuLi with subsequent hydrogenation under reflux with the catalytic system Lindlar cat./NH4HCO2 to yield 5. These hydrogenations were also executed in a microwave oven resulting in better yields and/or reaction times. The lactone 5 was then epoxidized, the ring opened by amidation and the remaining alcohol was oxidized via Collins oxidation to result in tetrahydrocerulenin analogues 8a-e. The same procedure was used for dihydrocerulenin analogues 10a-c except that to obtain the corresponding lactone 9a only route A was used and a further step had to be executed for ring closure. To obtain analogues with all structural features of cerulenin including two double bonds and the epoxide function, a third pathway was chosen. To obtain the future side chain, aldehyde 12 was synthesized by coupling protected 4-pentyn-1-ol to either crotyl bromide or crotyl chloride, which then was deprotected, hydrogenated with Lindlar catalyst under hydrogen pressure and oxidized via a Swern oxidation. The following synthesis sequence starting from 12 was executed similar to that of dihydrocerulenins via the corresponding lactone (51) with the major exception of the oxidation procedure in the last step via TPAP/NMO to result in (4Z,7E)-cerulenin analogues 15a-b. A fourth class of cerulenin analogues was synthesized with the aromatic analogues 17a-e. This synthesis pathway started with the formation of the benzoyl acrylamides 16a-e from benzoylacrylic acid via a mixed anhydride which was prepared with isobutylchloroformate followed by the addition of the corresponding amine. Subsequent epoxidation with H2O2 in basic EtOH gave the aromatic cerulenin analogues 17a-e. Pharmacological testings for the synthesized substances were executed on efflux pump-resistant and -sensitive Candida albicans isolates, on the fatty acid synthesis enzyme KasA of Mycobacterium tuberculosis and on other organisms such as Leishmania major, Trypanosoma brucei brucei, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa within the Sonderforschungsbereich 630. / Die Arbeit befasst sich mit der Synthese und Charakterisierung von Ceruleninanaloga als Inhibitoren effluxpumpenresistenter Candida-albicans-Isolate und als Inhibitoren des Fettsäuresyntheseenzyms KasA von Mycobacterium tuberculosis. Der Naturstoff Cerulenin wurde als Leitstruktur gewählt, da er einerseits ein Substrat der bekannten Effluxpumpen von Candida albicans ist und deshalb Strukturvariationen zu einer Blockierung der Effluxpumpen, wie im Fall von Tetrazyklin und dem Inhibitor 13-CPTC der TetB Effluxpumpe, führen können. Andererseits ist Cerulenin ein bekannter Inhibitor der Fettsäuresynthese, allerdings unselektiv für Typ I und II. Ceruleninanaloga als Fettsäureinhibitoren könnten daher eine erhöhte Selektivität bezüglich des Typ II Fettsäuresystems in M. tuberculosis aufweisen. Die in dieser Arbeit zuerst synthetisierten Ceruleninderivate wurden durch Kupplung von 2,3-Dihydrofuran und dem zuvor dargestellten 1-Octaniodid gefolgt von Ringöffnung und Oxidation mittels Chromsäure zur 4-Ketosäure, und Umsetzung zum entsprechenden Amid und somit den Ceruleninanaloga 4a-d hergestellt. Substanz 4e wurde entsprechend synthetisiert. Um die Epoxidfunktion der Leitstruktur zu integrieren, die besonders hinsichtlich des Wirkmechanismus von Cerulenin im FAS-System wichtig zu sein scheint (wenn man die bekannten Kristallstrukturen von Cerulenin und dem KasA-Analogon in E. coli berücksichtigt), wurden Tetrahydro- und Dihydroceruleninanaloga synthetisiert. Ausgehend von dem entsprechendem Aldehyd wurde (im Fall der Tetrahydrocerulenine) Lacton 5 auf zwei verschiedene Arten dargestellt: mittels Route A und B. Route A beinhaltete die Kupplung des Aldehyds 1-Nonanal mit Propiolsäure durch eine entsprechende Grignardreaktion mit anschließender Hydrierung über Lindlar-Katalysator unter Wasserstoffdruck. Bei Route B wurden 1-Nonanal und Methylpropiolat mittels n-BuLi gekuppelt und anschließend hydriert durch Refluxieren mit Lindlar-Katalysator und NH4HCO2. Die Hydrierungen von Route A und B wurden auch in der Mikrowelle durchgeführt, wodurch bessere Ausbeuten und/oder Reaktionszeiten erzielt werden konnten. Das so dargestellte Lacton 5 wurde dann epoxidiert, der Lactonring durch den Angriff eines Amins geöffnet und der so entstandene Alkohol mittels Collins Oxidierung zu den Tetrahydroceruleninanaloga 8a-e oxidiert. Dihydroceruleninanaloga 10a-c wurden auf analogem Syntheseweg hergestellt mit dem Unterschied, dass die entsprechende Lactonzwischenstufe 9a nur durch Route A synthetisiert und ein weiterer Zwischenschritt zum Ringschluss benötigt wurde. Um Ceruleninanaloga mit allen strukturellen Komponenten des Cerulenins inklusive zweier Doppelbindungen und Epoxidfunktion zu erhalten, wurde ein dritter Syntheseweg gewählt. Zur Integration der späteren Seitenkette wurde zuerst Aldehyd 12 durch Kupplung von geschütztem 4-Pentyn-1-ol mit entweder Crotylbromid oder Crotylchlorid, anschließendem Entschützen und Hydrierung über Lindlar-Katalysator und unter Wasserstoffdruck und nachfolgender Swern Oxidation synthetisiert. Die anschließende Synthesesequenz startete von Substanz 12 und wurde in Anlehnung der Synthese an die Dihydroceruleninderivate über Lacton 51 durchgeführt. Die größte Abweichung stellte dabei die Oxidation im letzten Schritt dar, die mittels TPAP/NMO durchgeführt wurde und in den (4Z,7E)-Ceruleninanaloga 15a-b resultierte. Eine vierte Klasse von Ceruleninanaloga wurde mit den aromatischen Derivaten 17a-e synthetisiert. Diese Route startete mit der Synthese der Benzoylacrylamide 16a-e aus Benzoylacrylsäure über das gemischte Anhydrid, das mit Isobutylchloroformiat hergestellt wurde, gefolgt von der Zugabe des entsprechenden Amins. Die nachfolgende Epoxidierung mit H2O2 in basischem EtOH ergab die aromatischen Ceruleninanaloga 17a-e. Pharmakologische Testungen der synthetisierten Substanzen wurden an Efflux-pumpen-resistenten und -sensitiven Candida albicans Isolaten, am Fettsäuresyntheseenzym KasA von Mycobacterium tuberculosis und an anderen Mikroorganismen wie Leishmania major, Trypanosoma brucei brucei, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli und Pseudomonas aeruginosa im Sonderforschungsbereich 630 durchgeführt.

Die sekretorischen Aspartatproteasen (Saps) des Hefepilzes Candida albicans gelten als wichtiger Virulenzfaktor dieses opportunistischen Krankheitserregers. Die zehn Sap-Isoenzyme werden von einer Genfamilie codiert, deren Vertreter (SAP1-SAP10) in der Vergangenheit bereits intensiv untersucht wurden. SAP-Expressionsanalysen und die Charakterisierung von sap-Deletionmutanten, die in einem auxotrophen Laborstamm hergestellt wurden, führten aber zu teilweise widersprüchlichen Ergebnissen, weshalb die Rolle der einzelnen Proteine bis heute nicht zweifelsfrei aufgeklärt ist. Eine differentielle Expression der SAP-Gene in unterschiedlichen Stadien einer Infektion wurde jedoch in vielen unabhängigen Studien gezeigt. In der vorliegenden Arbeit wurde zunächst die Expression der Gene SAP1-SAP6 in einem etablierten in vitro-Modell einer Candida-Vaginitis untersucht, das auf der Infektion von rekonstituiertem humanem Epithel (RHE) basiert. Dazu wurden Reporterstämme verwendet, die bereits früher für Expressionsanalysen in verschiedenen in vivo-Infektionsmodellen in Mäusen eingesetzt worden waren. Dies erlaubte es einerseits unterschiedliche Nachweismethoden zur SAP-Genexpression in einem standardisierten Modell zu vergleichen und andererseits das Expressionsmuster der SAP-Gene in einem in vitro-Infektionsmodell mit den Ergebnissen von in vivo-Infektionsexperimenten zu korrelieren. Es zeigte sich in Übereinstimmung mit Ergebnissen früherer in vivo-Experimente in Mäusen, dass bei der Infektion des vaginalen Gewebes vor allem das SAP5-Gen induziert wurde. Allerdings weicht dieses Ergebnis deutlich von Ergebnissen anderer Studien ab, die mit Hilfe anderer Methoden ein ungleiches Muster der SAP-Expression detektierten. Um die Rolle der Gene SAP1-SAP6 bei der Infektion genauer zu untersuchen, wurden deshalb Mutanten hergestellt, in denen einzelne oder mehrere SAP-Gene mit Hilfe einer neuartigen Mutagenesestrategie erstmals aus dem Genom eines wildtypischen C. albicans-Stammes deletiert wurden. Überraschenderweise konnte sowohl in Einzelmutanten der Gene SAP1-SAP6 als auch in sap1 sap2 sap3- und sap4 sap5 sap6-Triplemutanten keine verminderte Fähigkeit zur Invasion und Schädigung von humanem Gewebe in RHE festgestellt werden. Eine in früheren Arbeiten beschriebene abgeschwächte Virulenz solcher Mutanten konnte auch in einem murinen Modell für eine disseminierende Infektion nicht beobachtet werden. Die Sekretion von Aspartatproteasen ermöglicht es C. albicans Proteine als alleinige Stickstoffquelle zum Wachstum zu verwenden. Unter diesen Bedingungen wird spezifisch die Expression des SAP2-Gens induziert; jedoch ist über die Mechanismen dieser Regulation noch wenig bekannt. Aus diesem Grund wurden in dieser Arbeit Promotor-Deletionsanalysen des SAP2-Gens und des mit diesem co-regulierten Oligopeptidtransportergens OPT1 durchgeführt. Es zeigte sich, dass unterschiedliche Bereiche innerhalb der 3,5 kb großen regulatorischen Region von SAP2 gemeinsam eine Expression dieses Gens unter induzierenden Bedingungen ermöglichen. Für das OPT1-Gen konnte eine regulatorische Region eingegrenzt werden, die für die Expression dieses Gens essentiell ist. In den für die Expression von SAP2 und OPT1 wichtigen Regionen wurden ähnliche Sequenzen gefunden, die als Bindungsstellen für regulatorische Faktoren dienen könnten. In dieser Arbeit wurden neue Erkenntnisse zur Regulation und Bedeutung der sekretierten Aspartatproteasen von C. albicans erhalten. Um eine endgültige Bewertung der Rolle dieser Enzyme in der Virulenz des Erregers zu ermöglichen, sind jedoch noch weitere detaillierte Studien unter Verwendung verschiedenster Infektionsmodelle nötig. / The secreted aspartic proteases (Saps) of the yeast Candida albicans are considered as an important virulence trait of this opportunistic pathogen. The ten Sap isoenzymes are encoded by a gene family, whose members (SAP1-SAP10) have been analysed intensively already in the past. However, the analysis of the expression pattern of the SAP genes and the characterisation of sap deletion mutants, which were generated from an auxotrophic laboratory strain, yielded partially contradictory results. Therefore, the role of the individual proteins is still not fully elucidated. A differential expression of the SAP genes in various stages of an infection has been demonstrated in many independent studies. In the present work the expression of the SAP1-SAP6 genes was analyzed in an established in vitro model of vaginal candidiasis that is based on the infection of reconstituted human epithelium (RHE). For this purpose, reporter strains were used which had already been utilized previously to study the SAP expression pattern in various in vivo infection models in mice. This allowed the comparison of different detection methods for SAP gene expression in a standardized model and also to correlate the SAP expression pattern in an in vitro infection model with the results of in vivo infection experiments. It was found that SAP5 was the predominantly expressed SAP gene during infection of the vaginal tissue, in agreement with the results of previous in vivo experiments in mice. However, this result contrasts with those of other studies that, using alternative methods, detected a different SAP expression pattern. To investigate the role of the SAP1-SAP6 genes during infection in more detail, a new set of mutants was generated, in which single or multiple SAP genes were deleted for the first time from the genome of a C. albicans wild-type strain with the help of a novel mutagenesis strategy. Surprisingly, neither single mutants lacking one the SAP1-SAP6 genes nor triple mutants lacking all of the SAP1-SAP3 or the SAP4-SAP6 genes exhibited a detectable defect in invasion and damage of reconstituted human epithelium. A previously reported attenuated virulence of sap mutants was also not observed in a murine model of disseminated candidiasis. The secretion of aspartic proteases enables C. albicans to utilize proteins as a sole source of nitrogen for growth. Under these conditions expression of the SAP2 gene is specifically induced, however, little is known about the mechanisms of this regulation. Therefore, promoter deletion analyses of the SAP2 gene and the co-regulated oligopeptide transporter gene OPT1 were performed in the present work. It was found that different regions within the approximately 3,5 kb large SAP2 promoter jointly allowed the expression of this gene under inducing conditions. For OPT1, a region could be delimited that is essential for the expression of this gene. The regions that were found to be important for SAP2 and OPT1 expression contain similar sequences, which may serve as binding sites for regulatory factors. This study provides new insights into the regulation and importance of the secreted aspartic proteases of C. albicans. For a definite evaluation of the role of these enzymes in the virulence of the pathogen, however, further detailed studies that employ a variety of different infection models are necessary.

Humane neutrophile Granulozyten spielen eine wichtige Rolle in der Immunabwehr invasiver Infektionen durch die humanpathogenen Pilze Candida albicans und Aspergillus fumigatus. Das Ziel der hier vorliegenden Arbeit bestand in einer Charakterisierung der Interaktion beider Pilzspezies mit neutrophilen Granulozyten, mit Fokussierung auf die unterschiedlichen Effektormechanismen dieser Zellen. C. albicans exprimiert eine Reihe von Aspartatproteasen, welche mit der Virulenz des Erregers assoziiert sind und zu Adhäsion, Gewebeinvasion und Immunevasion beitragen können. In dieser Arbeit wurde die Rolle der Aspartatproteasen Sap1-6, Sap9 und Sap10 in der Interaktion mit neutrophilen Granulozyten analysiert. Es konnte gezeigt werden, dass, im Gegensatz zu anderen Aspartatproteasen, das zelloberflächenassoziierte GPI-verankerte Enzym Sap9 einen maßgeblichen Einfluss auf die Erkennung von C. albicans durch neutrophile Granulozyten hat. SAP9-Expression ist erforderlich, um die gerichtete Motilität (Chemotaxis) neutrophiler Granulozyten zu C. albicans-Keimschläuchen hin zu induzieren. Dieser Prozess stellt eine Grundvoraussetzung zur effektiven Aktivierung neutrophiler Granulozyten darstellt. Die Chemotaxis neutrophiler Granulozyten kann durch autologe Sekretion des Zytokins IL-8 verstärkt werden. Es konnte jedoch kein Einfluss von SAP9 auf die IL-8 Sekretion beobachtet werden. Allerdings führte die Deletion von SAP9 zu reduzierter Freisetzung von reaktiven Sauerstoffspezies (engl. reactive oxygen species, ROS) in neutrophilen Granulozyten. Die mit der ROS-Generierung in Verbindung stehende und durch C. albicans induzierte Apoptose neutrophiler Granulozyten war ebenfalls vermindert. In Konfrontationsassays war die Abtötung einer SAP9-Deletionsmutante verglichen mit dem Wildtyp reduziert. Die Degranulation stellt neben der Produktion von ROS einen weiteren wichtigen Effektormechanismus zur Abtötung von Mikroben dar, jedoch verlief die Freisetzung von Elastase ebenso unabhängig von SAP9 wie die durch neutrophile Granulozyten ausgelöste Wachstumsinhibition von Keimschläuchen. Die hier präsentierten Daten verbinden die Aktivität der Protease Sap9, der zuvor bereits eine Rolle in der Immunevasion von C. albicans zugeschrieben wurde, mit der Initiation der protektiven angeborenen Immunität. Wie C. albicans stimuliert auch A. fumigatus die Aktivität der neutrophilen Granulozyten. Microarray- Analysen mit Fokus auf dem Zytokinprofil neutrophiler Granulozyten während der Interaktion mit A. fumigatus-Hyphen offenbarten, dass nur wenige Zytokine im Lauf der Infektion hochreguliert wurden. Zusammenfassend konnte gezeigt werden, dass die Sap-Granulozyten-Interaktion neue molekulare Mechanismen zur Aktivierung dieser Zellen birgt. Zudem brachten die Microarray Analysen die Erkenntnis, dass die de novo-Zytokinsynthese durch A. fumigatus nur geringfügig beeinflusst wird und eine schnelle Abtötung des Pilzes offenbar im Vordergrund steht. / Human polymorphonuclear neutrophils (PMNs) play a major role in the immune defence against invasive infections caused by the pathogenic fungi Candida albicans and Aspergillus fumigatus. The aim of this work was to further characterize the interaction of both fungi with PMNs with a focus on diverse PMN effector functions. C. albicans expresses a set of aspartic proteases which are associated with virulence and can contribute to adhesion, tissue invasion and immune evasion. Here, the role of the aspartic proteases Sap1-6, Sap9 and Sap10 in the interaction with human neutrophils was analysed. It could be demonstrated that, in contrast to other aspartic proteases, the cell surface associated GPI-anchored enzyme Sap9 has a major impact on recognition of C. albicans by PMNs. SAP9 expression is required for the effective induction of PMN chemotaxis towards C. albicans filaments, an essential prerequisite of effective PMN activation. Targeted motility can be enhanced by autologous secretion of IL-8, but no influence of SAP9 on the process of IL-8 secretion could be observed here. However, deletion of SAP9 led to a mitigated release of reactive oxygen intermediates (ROI) in human PMNs. Likewise, C. albicans induced apoptosis triggered by ROI formation was decreased. In confrontation assays, killing of a SAP9 deletion mutant was reduced in comparison to the wild-type. Beside ROI production, degranulation represents an additional important effector mechanism contributing to microbial killing, however, the release of elastase in response to C. albicans was found to be not dependent on SAP9, as well as the PMN-mediated growth inhibition of germ tubes. The data presented here clearly implicate Sap9 protease activity, which was already attributed to immune evasion before, with the initiation of protective innate immunity. Like C. albicans, A. fumigatus evokes PMN antifungal activity. Custom-made microarray analyses focusing on the cytokine profile of PMNs during interaction with A. fumigatus hyphae revealed that only few cytokines are upregulated during the course of infection. In summary it could be demonstrated that the interaction between Saps and PMNs is revealing novel molecular mechanisms which lead to an activation of these cells. Furthermore, microarray analyses lead to the awareness that de novo cytokine synthesis of PMNs is barely influenced by A. fumigatus, pointing out that instant killing of the fungus might be of higher importance.