Die Bedeutung von Mykosen hat wegen der wachsenden Zahl immunsupprimierter Patienten in den letzten Jahren immer mehr zugenommen. Diese erkranken häufig an oberflächlichen sowie lebensbedrohlichen systemischen Infektionen mit dem opportunistisch humanpathogenen Hefepilz Candida albicans, da der Keim, der oftmals als harmloser Kommensale auf den Schleimhäuten im Gastrointestinaltrakt gesunder Menschen vorkommt, vom geschwächten Immunsystem nicht mehr in Schach gehalten werden kann. In dieser Arbeit sollten bestimmte Gene von C. albicans, die in anderen Organismen als essentiell für deren Lebensfähigkeit bzw. Virulenz beschrieben wurden, als potentielle Zielstrukturen für die Entwicklung neuer Antimykotika charakterisiert werden. Das CMP1-Gen kodiert für die katalytische Untereinheit der konservierten Calcium/Calmodulin-abhängigen Phosphatase Calcineurin, die in der Bäckerhefe Saccharomyces cerevisiae und in anderen Organismen verschiedene physiologische Prozesse reguliert und essentiell für die Virulenz des pathogenen Hefepilzes Cryptococcus neoformans ist. Um die Bedeutung von Calcineurin für das Überleben und die Virulenz von C. albicans zu untersuchen, wurden homozygote cmp1 knock-out-Mutanten sowohl in einem auxotrophen C. albicans-Laborstamm als auch, mit Hilfe eines neuen dominanten Selektionsmarkers, in einem prototrophen Wildstamm hergestellt. Die Mutanten erwiesen sich als hypersensitiv gegenüber Natrium, Calcium, Mangan und Lithium sowie gegenüber alkalischem pH-Wert. Darüber hinaus konnten die mutierten Zellen Membranstreß, der durch SDS- oder Fluconazol-Zugabe verursacht wurde, nicht tolerieren und waren unter diesen Bedingungen stark in ihrem Wachstum gehemmt. Andere wichtige Virulenzeigenschaften wie die Toleranz gegenüber Wirts-Körpertemperatur und die Fähigkeit zur Hyphenbildung zeigten sich durch die CMP1-Deletion in vitro nicht beeinträchtigt. Dennoch machte die Anwendung eines murinen Modells einer systemischen Candidose in vivo deutlich, daß die Mutanten sehr stark in ihrer Virulenz attenuiert waren. Der Virulenzdefekt war vermutlich zumindest zum Teil dadurch bedingt, daß die Calcineurin-defizienten Zellen im Gegensatz zum Wildtyp in humanem Serum nicht wachsen konnten und deshalb möglicherweise schlechter über die Blutbahn disseminieren konnten. Außer Calcineurin wurden in Kooperation mit einem Industriepartner drei weitere Gene, YML127, YPR143, und YML93, die in S. cerevisiae als essentiell beschrieben wurden und die keine signifikanten Homologien zu Vertebraten-Genen aufwiesen, in der C. albicans-Genomsequenz identifiziert und auf ihre Eignung als potentielle Targets hin untersucht. Die Funktion dieser Gene war zu Beginn dieser Arbeit unbekannt; vor kurzem wurde jedoch gezeigt, daß sie in S. cerevisiae eine Rolle beim Chromatin-Remodeling bzw. bei der rRNA-Prozessierung haben. Nachdem sich alle Gene auch in C. albicans als essentiell herausgestellt hatten, wurden konditional letale Mutanten hergestellt, in denen die Gene durch induzierbare Deletion mit Hilfe der site-spezifischen Rekombinase FLP aus dem Genom entfernt wurden. Dadurch wurde eine Population von Nullmutanten erhalten, in denen der terminale Phänotyp der Gendeletion analysiert werden konnte. Die funktionelle Analyse des YML127 (RSC9) Gens wies darauf hin, daß es in C. albicans eine ähnliche Funktion hat wie in der Bäckerhefe, in der das Rsc9-Protein ein Bestandteil des RSC-Protein-Komplexes ist, der die Struktur des Chromatins in Abhängigkeit von Zellzyklus und Umweltbedingungen umorganisiert und damit die Aktivität von Genen steuert. Mit Hilfe eines HA-Epitop markierten YML127-Gens konnte das Genprodukt im Zellkern von C. albicans lokalisiert werden. Die C. albicans yml127-Nullmutanten produzierten verlängerte, mehrfach knospende Zellen, was einen Verlust der Koordination zwischen Mitose und Zytokinese vermuten ließ. Die beiden Gene YPR143 und YML93 (UTP14) scheinen wie ihre homologen Vertreter in S. cerevisiae an der Prozessierung der ribosomalen RNA beteiligt zu sein. Heterozygote Mutanten wiesen eine Haploinsuffizienz auf, die sich in einer erhöhten Suszeptibilität gegenüber Hemmstoffen der rRNA-Synthese und der Ribosomenaktivität zeigte, und in den induzierten Nullmutanten akkumulierten Vorstufen der reifen rRNAs. In beiden Fällen führte die Gendeletion zu Anomalien im Zellzyklus; die ypr143-Mutanten wiesen eine vergrößerte unförmige Zellmorphologie auf, und die yml93-Mutanten bildeten große, rundliche Zellen. Die Ergebnisse dieser Arbeit erlauben nicht nur wichtige Einblicke in die Funktion der untersuchten Gene in essentiellen zellulären Prozessen, sondern zeigen auch deren Bedeutung für die Virulenz bzw. für das Überleben des humanpathogenen Hefepilzes C. albicans. Die entsprechenden Genprodukte sollten sich deshalb prinzipiell als Angriffspunkte für die Entwicklung neuer antimykotischer Medikamente eignen. / The importance of fungal infections has steadily increased during the past decades due to the growing number of immunocompromised patients. These patients often suffer from superficial as well as life-threatening systemic infections with the opportunistic human pathogenic yeast Candida albicans, which is a harmless commensal on mucosal surfaces in many healthy people but cannot be controlled any more by a weakened immune system. On the other hand, virulence traits of the fungus also contribute to its pathogenicity, because they enable adaptation to different host niches. The success of medical treatment is limited by the emergence of resistance and by toxic side effects of antifungal drugs. Therefore, there is an urgent need to develop novel antimycotic agents. In this work selected C. albicans genes, which were known to be essential for viability or virulence in other organisms, were characterized as potential targets for the development of new antifungal drugs. The CMP1 gene encodes the catalytic subunit of the conserved calcium/calmodulin-dependent phosphatase calcineurin, which regulates a variety of physiological processes in the model yeast Saccharomyces cerevisiae and other organisms and is essential for virulence of the pathogenic yeast Cryptococcus neoformans. To investigate the importance of calcineurin for survival and virulence of C. albicans, homozygous cmp1 knock-out mutants were constructed in an auxotrophic C. albicans laboratory strain as well as, using a new dominant selection marker, in a prototrophic wild-type strain. The mutants showed hypersensitivity to increased concentrations of ions and to alkaline pH. In addition, the mutated cells could not tolerate membrane stress resulting from SDS or fluconazole treatment and their growth was strongly inhibited under these conditions. Other characteristics that are important for virulence, like tolerance to the host body temperature and the ability to switch to a hyphal growth form, were not affected by the CMP1 deletion. Nevertheless, the mutants were avirulent in a murine model of systemic candidiasis. The virulence defect could be explained at least in part by the fact that, in contrast to the wild-type, the cmp1 mutants were unable to grow in human serum and therefore might have a reduced capacity to disseminate via the bloodstream. In addition to CMP1, three other genes, YML127, YPR143, and YML93, were selected in cooperation with an industrial partner from the available C. albicans genome sequence and evaluated as potential targets. These genes had been reported to be essential in S. cervisiae and they did not exhibit significant homology to mammalian genes. At the beginning of the present work the function of the three genes was unknown, but recently it was demonstrated that their counterparts in S. cerevisiae have roles in chromatin remodeling or rRNA processing. It was demonstrated that all three genes are also essential in C. albicans. Therefore, conditional lethal mutants were constructed in which the genes could be excised from the genome by inducible deletion using the site-specific FLP recombinase. In this way, populations of null mutants were obtained in which the terminal phenotype of the gene deletion could be analyzed. The functional analysis of the YML127 (RSC9) gene showed that it has a similar function in C. albicans as in S. cerevisiae, where the Rsc9 protein is a component of the RSC complex that remodels the structure of chromatin in a cell cycle dependent manner and in response to environmental conditions and thereby controls gene activity. Using an HA-epitope-tagged YML127 gene the Yml127 protein could be localized in the nucleus in C. albicans. The C. albicans yml127 null mutants produced elongated, multi-budded cells, pointing to a loss of coordination of mitosis and cytokinesis. The genes YPR143 and YML93 (UTP15) seem to be involved in the processing of the ribosomal RNA, like their counterparts in S. cerevisiae. Heterozygous mutants exhibited a haploinsufficient phenotype, which was evident from their hypersusceptibility to inhibitors of rRNA synthesis and ribosome activity, and the induced null mutants accumulated precursors of the mature rRNAs. In both cases the gene deletion resulted in cell cycle defects; the ypr143 null mutants produced enlarged, misshapen cells, and the yml93 mutants formed large, round cells. The results of this work not only provide valuable clues about the function of the investigated genes in essential cellular processes, but also demonstrate their importance for virulence and viability of the human pathogenic fungus C. albicans. In principle, the corresponding gene products should therefore be suitable targets for the development of novel antifungal drugs.

In den letzten Jahren haben Pilzinfektionen zugenommen und bakterielle Infektionen nahezu überholt, wofür vor allem der massive Einsatz von Medikamenten sowie operative Eingriffe verantwortlich sind. Einer der gefährlichsten Auslöser schwerer Pilzinfektionen, die innere Organe schädigen und sehr schwer zu behandeln sind, ohne dabei den Wirtsorganismus zu schädigen, ist der opportunistische Hefepilz Candida albicans. Da aufgrund der immer größer werdenden Zahl von Resistenzen von Candida albicans nur ein relativ kleines Repertoire für die Therapie zur Verfügung steht, war das Ziel der vorliegenden Arbeit die Synthese einer Reihe peptidischer Inhibitoren mit elektrophilen Bausteinen als potentielle irreversible Inhibitoren der sekretorischen Aspartat-Proteasen (SAPs) des Hefepilzes Candida albicans und deren Testung an dem am stärksten exprimierten SAP-Isoenzym SAP2 sowie anderen Proteasen. Dabei sollte geklärt werden, ob neben der HIV-1-Protease auch andere Aspartat-Proteasen durch cis-konfigurierte Epoxide irreversibel hemmbar sind, ob andere elektrophile Ringe sowie elektronenarme Michael-Systeme in der Lage sind, als irreversible Aspartat-Protease-Inhibitoren zu fungieren, und ob die Z-Konfiguration der Olefine für die Hemmung von Aspartat-Proteasen ebenso wichtig ist wie die cis-Konfiguration bei Epoxiden. Die Aziridin-2-carboxylat-Bausteine wurden als Racemate über Cromwell-Synthese gewonnen und die Aziridin-2,3-dicarboxylat-Bausteine stereoselektiv aus Tartraten dargestellt. Die Oxiran-2-carboxylat-Bausteine wurden enantioselektiv ausgehend von Threonin bzw. als Racemate über Darzens-Glycidester-Synthese dargestellt. Die Synthese der Oxiran-2,3-dicarboxylat-Bausteine gelang mittels tertButylhydroperoxid / BuLi aus den Maleaten. Die Z-Olefinbausteine wurden durch Kupplung von Alkoholen bzw. AS an Maleinsäureanhydrid erhalten oder über Wittig- bzw. Horner-Wadsworth-Emmons-Reaktion dargestellt. Die Kupplung von AS bzw. Peptiden an die elektrophilen Bausteine erfolgte mit gängigen AS- / Peptidkupplungsmethoden. Die als irreversible Inhibitoren der SAP2 konzipierten Verbindungen wurden in einem neu entwickelten fluorimetrischen FRET-Assay auf ihre SAP2-Hemmung getestet. Dazu wurde ein Verdünnungsassay nach Kitz und Wilson durchgeführt und die zunehmende Fluoreszenz durch das Spaltprodukt der enzymatischen Hydrolyse des Substrats bei 540 nm detektiert (Anregung 355 nm). Als Substrat diente das Undecapeptid Dabcyl-Arg-Lys-Pro-Ala-Leu-Phe / Phe-Arg-Leu-Glu(EDANS)-ArgOH (/ markiert die Spaltstelle). Von den Inhibitoren wurden IC50-, k2nd- und, falls möglich, ki- und Ki-Werte ermittelt. Von den 41 an der SAP2 getesteten AS- / Peptid-verknüpften Verbindungen stellen die beiden Aziridine A-07 und A-08 mit k2nd-Werten im mittleren fünfstelligen Bereich [M-1min-1] die besten Inhibitoren dar. Bis auf zwei Verbindungen zeigen alle aktiven Verbindungen an der SAP2 sinkende IC50-Werte bei längerer Inkubationszeit und somit eine zeitabhängige und irreversible Hemmung. Zur Untersuchung der Selektivität wurden die Verbindungen mittels kontinuierlicher Assays an den Cystein-Proteasen Cathepsin B (human), Cathepsin L (Paramecium tetraurelia) und Rhodesain (Trypanosoma brucei rhodesiense) getestet. Als Substrat wurde dabei Cbz-Phe-Arg-AMC verwendet. Erfreulicherweise waren bis auf das E-konfigurierte Olefin E-Ol-04 alle Verbindungen an den Cystein-Proteasen inaktiv. Die Ergebnisse zeigen, dass neben den HIV-Proteasen auch die sekretorische Aspartat-Protease SAP2 durch cis-konfigurierte Epoxide irreversibel hemmbar ist. Desweiteren zeigt sich, dass mit Aziridinen auch andere elektrophile Ringe als irreversible Aspartat-Protease-Inhibitoren fungieren können. An der SAP2 zeigen sich die Aziridine sogar aktiver. Auch elektronenarme Michael-Systeme sind in der Lage Aspartat-Proteasen zu hemmen, auch wenn ihre Hemmung deutlich schwächer ist als die der Aziridine. Die Ergebnisse zeigen jedoch, dass nicht, wie angenommen, die Z-Konfiguration der Olefine entscheidend ist, sondern dass E-Olefine sogar bessere Hemmungen aufweisen. In Kooperation mit der Arbeitsgruppe von Prof. Dr. Joachim Morschhäuser und Dr. Peter Staib vom Institut für Molekulare Infektionsbiologie der Universität Würzburg, konnte gezeigt werden, dass die Aziridine A-07 und A-08 neben dem isolierten Enzym auch die SAP2-Produktion in Candida albicans-Zellkulturen hemmen ohne auf die Pilzzellen toxisch zu wirken. Neben der Hemmung der SAP2 wirken die Aziridine A-07 und A-08 auch antiplasmodial. Bei Testungen am Malaria-Erreger Plasmodium falciparum zeigten beide Aziridine einen IC50-Wert im unteren mikromolaren Bereich. Der Grund der Hemmung des Parasiten ist jedoch noch unklar, da A-07 und A-08 weder an den isolierten Cystein-Proteasen des Malaria-Erregers Falcipain 2 und 3 aktiv sind, noch dessen Aspartat-Protease Plasmepsin II hemmen. / Over the last years fungal infections have increased dramatically and now nearly exceed the number of bacterial infections. Reasons are the massive use of antibiotics and the increasing number of surgeries. One of the most serious pathogens that causes superficial as well as severe systemic infections, which are difficult to treat without affecting the host organism, is the opportunistic fungal pathogen Candida albicans. Due to an increase of resistances of Candida species towards antifungal drugs only a limited repertoire of drugs is available for systemic therapy. The goal of the present work was the synthesis of series of peptide inhibitors containing electrophilic building blocks as potential irreversible inhibitors of the secreted aspartic proteases (SAPs) of Candida albicans. The synthesized compounds should be tested against SAP2, which is the mostly expressed SAP-isoenzyme, and other proteases. This work should elucidate whether cis-configured epoxides can be used to irreversibly block other aspartic proteases than the HIV-1-protease and whether other small electrophilic building blocks like aziridines and electron poor Michael acceptor systems can react as irreversible inhibitors of aspartic proteases as well. Additionally, the role of the configuration of the Michael systems (Z / E) for inhibition potency should be investigated. The aziridine-2-carboxylates were obtained as racemates by Cromwell synthesis and the aziridine-2,3-dicarboxylates were synthesized stereoselectively by a chiral pool synthesis starting from tartrates. The oxirane-2-carboxylates were synthesized enantioselectively starting from threonine or were obtained as racemates by Darzens glycide ester synthesis. The oxirane-2,3-dicarboxylates were obtained by Weitz-Schäffer epoxidation of maleates with tertbutylhydroperoxide / butyllithium. The Z-configured olefinic building blocks were synthesized by reactions of alcohols or amino acids with maleinic anhydride or by Wittig and Horner-Wadsworth-Emmons reactions. The electrophilic building blocks obtained by these pathways were coupled with amino acids and peptides using known methods of peptide chemistry. The compounds which were designed as irreversible aspartic protease inhibitors were tested for SAP2 inhibition using a newly-developed FRET assay. The inhibition constants (IC50-, k2nd-, ki- and Ki-values) were determined in dilution assays measuring the increase of fluorescence at 540 nm. The undecapeptide Dabcyl-Arg-Lys-Pro-Ala-Leu-Phe / Phe-Arg-Leu-Glu(EDANS)-ArgOH (/ designates the cleavage site) was used as substrate. Within the series of 41 synthesized compounds the aziridines A-07 and A-08 exhibiting k2nd-values of about 50000 M-1min-1 were found to be the most active inhibitors. With the exception of two compounds all inhibitors showed time-dependent inhibition indicating irreversible inactivation of the target enzyme. In order to elucidate the selectivity the compounds were tested against the cysteine proteases cathepsin B (human), cathepsin L (Paramecium tetraurelia) und rhodesain (Trypanosoma brucei rhodesiense) using a continuous fluorometric microplate assay. In all cases, the substrate Cbz-Phe-Arg-AMC was used. With the exception of the E-configured olefin E-Ol-04 all compounds were found to be inactive against cysteine proteases. In summary, the results prove that besides the HIV-proteases other aspartic proteases like SAP2 can also be inhibited irreversibly by cis-configured epoxides. Furthermore, it is shown that cis-configured aziridines can also be used as building blocks for irreversible inhibitors of aspartic proteases, being even more active against SAP2 than corresponding epoxides. Electron poor Michael acceptor systems can also be used, but they are obviously weaker than the three-membered heterocycles. The results obtained with the olefins show that the E-configured compounds are superior to Z-configured ones. In collaboration with the group of Prof. Dr. Joachim Morschhäuser and Dr. Peter Staib (Department of Molecular Infection Biology, University of Würzburg) it was proven that the aziridines A-07 and A-08, which are the most active inhibitors of the target enzyme, also inhibit SAP2 in Candida albicans cell cultures leading to growth inhibition without being cytotoxic against the fungi. These aziridines (A-07 and A-08) display antiplasmodial activity as well. Tests against the malaria parasite Plasmodium falciparum revealed for both aziridines IC50-values in the low micromolar range. The reasons for the antiplasmodial activity are uncertain at the moment: A-07 and A-08 are only weakly active against the plasmodial cysteine proteases falcipain 2 and falcipain 3 and, furthermore, they do not inhibit the parasitic aspartic protease plasmepsin II.

Der Hefepilz Candida albicans ist Teil der natürlichen Mikroflora auf den Schleimhäuten des Verdauungs- und Urogenitaltrakts der meisten gesunden Menschen. Allerdings kann C. albicans vor allem in immunsupprimierten Patienten auch schwerwiegende Infektionen verursachen. Diese reichen von oberflächlichen Mykosen bis hin zu lebensbedrohlichen systemischen Infektionen. C. albicans besitzt eine Reihe von Eigenschaften, die es diesem opportunistisch humanpathogenen Pilz ermöglichen unterschiedliche Wirtsgewebe zu kolonisieren und zu infizieren. Ein wichtiger Virulenzfaktor sind sekretorische Aspartylproteasen (SAPs), die von einer großen Genfamilie von zehn SAP-Genen codiert werden. Die SAPs werden während der Infektion differentiell exprimiert und übernehmen unterschiedliche Rollen im Infektionsverlauf. So tragen sie zur Adhärenz bei, können Wirtsbarrieren und Moleküle der Wirtsimmunabwehr zerstören oder liefern Nährstoffe, indem sie Proteine abbauen. Unter den zehn SAP-Genen ist SAP2 für ein Wachstum von C. albicans auf Proteinen als alleiniger Stickstoffquelle verantwortlich. Allerdings ist wenig über die Regulation der SAP2-Expression und über die Aufnahme der proteolytischen Abbauprodukte in die Zelle bekannt. In dieser Arbeit wurde eine Familie von Oligopeptidtransportern von C. albicans funktionell analysiert. Da aus früheren Arbeiten bekannt war, dass SAP2 durch Peptide mit mindestens acht Aminosäuren induziert werden kann, könnten einzelne Mitglieder dieser Familie neben der Transportfunktion auch eine Sensorfunktion für Peptide übernehmen und somit über einen Signalweg SAP2 induzieren. In der Genomsequenz von C. albicans wurden neben dem bereits beschriebenen OPT1-Gen sieben weitere Gene identifiziert, deren Genprodukte signifikante Homologie zu Opt1p aufwiesen und die deshalb als OPT2-OPT8 bezeichnet wurden. Um die Rolle dieser putativen Oligopeptidtransporter bei der SAP2-Induktion und beim Transport der durch Sap2p-Aktivität bereitgestellten proteolytischen Abbauprodukte zu untersuchen, wurden Mutanten hergestellt, in denen die OPT-Gene spezifisch deletiert waren. Zu diesem Zweck wurde eine Methode zur gezielten Geninaktivierung etabliert, die auf einem neuen, recycelbaren dominanten Selektionsmarker (caSAT1) beruht, der Resistenz gegen Nourseothricin verleiht. Die “SAT1-Flipping“-Strategie kann direkt in C. albicans Wildstämmen angewendet werden und umgeht somit alle Probleme, die mit der Verwendung von auxotrophen Ausgangsstämmen verbunden sind. Alle Mutanten, in denen jeweils eines der OPT-Gene inaktiviert war, verhielten sich wie der Wildtyp und zeigten keinen Wachstumsdefekt auf bovinem Serumalbumin (BSA) als alleiniger Stickstoffquelle, während eine sap2-Nullmutante unter diesen Bedingungen nicht wachsen kann. Somit ist kein einzelnes OPT-Gen für C. albicans notwendig, um auf BSA als alleiniger Stickstoffquelle zu wachsen. Dagegen zeigten opt123-Triplemutanten ähnlich wie die sap2-Mutante einen starken Wachstumsdefekt auf BSA als alleiniger Stickstoffquelle, der durch Reintegration einer intakten Kopie von OPT1, OPT2 oder OPT3 wieder aufgehoben werden konnte. Der Wachstumsdefekt der opt123-Triplemutanten war nicht auf eine fehlende Induktion von SAP2 zurückzuführen, sondern auf das Unvermögen dieser Mutanten, die durch den proteolytischen Abbau von BSA entstandenen Peptide zu transportieren. Mit Hilfe von Reportergenen konnte gezeigt werden, dass die einzelnen OPT-Gene differentiell exprimiert werden. Während keines der Gene in einem Vollmedium (YPD) exprimiert wurde, wurde eine starke Induktion von OPT1 und OPT3 in Gegenwart von BSA als alleiniger Stickstoffquelle beobachtet. Nach Expression von OPT4 und OPT5 unter Kontrolle des konstitutiven ADH1-Promotors in den opt123-Triplemutanten konnte deren Wachstumsdefekt auf BSA als alleiniger Stickstoffquelle ebenfalls kompensiert werden, während die zusätzliche Deletion dieser Gene in den dabei entstandenen opt1234-Quadruple- und opt12345-Quintuplemutanten den Wachstumsdefekt noch verstärkte. Diese Ergebnisse bestätigten, dass die Gene OPT1-OPT5 für funktionelle Oligopeptidtransporter codieren. Weitere Experimente zeigten, dass die Oligopeptidtransporter unterschiedliche Substratpräferenzen haben. Während das Tetrapeptid LWMR für Stämme, die spezifisch OPT3, OPT4, oder OPT5 exprimierten, ein besseres Substrat war als das Tetrapeptid LSKL, konnten Stämme, die spezifisch OPT2 exprimierten, das LSKL-Peptid verwerten, nicht aber das LWMR-Peptid. Experimente mit Peptiden definierter Länge und Zusammensetzung wiesen außerdem darauf hin, dass die Oligopeptidtransporter in der Lage sind, auch längere Peptide mit bis zu mindestens acht Aminosäuren zu transportieren. Die Evolution einer Genfamilie, die für Oligopeptidtransporter mit unterschiedlicher Substratpräferenz codieren, hat deshalb vermutlich dazu beigetragen, dass C. albicans Proteine sehr effizient als Stickstoffquelle verwerten und sich an die Nahrungsbedingungen in verschiedenen Wirtsnischen optimal anpassen kann. / The yeast Candida albicans is a member of the microflora on mucosal surfaces of the gastrointestinal and urogenitary tract in most healthy people. However, in immunocompromised patients C. albicans can cause superficial as well as life-threatening systemic infections. C. albicans exhibits a variety of characteristics that enable this opportunistic human fungal pathogen to colonize and infect different host tissues. Among these virulence factors are secreted aspartic proteinases (SAPs), which are encoded by a family of ten SAP genes. The SAPs are differentially expressed during infection and play different roles in disease progression by contributing to adherence, by degrading tissue barriers and host defence molecules or by providing nutrients through the digestion of proteins. Of the ten SAP genes, SAP2 enables C. albicans to grow on proteins as a sole source of nitrogen. However, little is known about how SAP2 expression is regulated and how proteolytic products are taken up into the cell. In this work a family of oligopeptide transporters of C. albicans was functionally characterized. Since earlier studies had demonstrated that SAP2 expression can be induced by peptides of at least eight amino acids in length, oligopeptide transporters, in addition to transporting peptides, might also serve as sensors which in the presence of peptides activate a signalling pathway that induces SAP2 expression. Beside the already described OPT1 gene, seven additional genes were identified in the C. albicans genome sequence whose encoded products exhibit significant similarity to Opt1p and hence were designated as OPT2-OPT8. To elucidate the role of these putative oligopeptide transporters in SAP2 induction and in the uptake of proteolytic products provided by Sap2p activity, a series of mutants lacking specific OPT genes was constructed. For this purpose, a method for targeted gene inactivation was established that relies on the use of a new recyclable, dominant selection marker, caSAT1, which confers resistance to nourseothricin upon C. albicans transformants. The SAT1 flipping strategy can be used directly in C. albicans wild-type strains and, therefore, circumvents all problems related to the use of auxotrophic host strains. All knockout mutants lacking single OPT genes behaved like the wild-type parental strain and did not show a growth defect in a medium containing bovine serum albumin (BSA) as the sole nitrogen source, conditions in which a sap2 null mutant can not grow. Therefore, no single OPT gene is required for growth of C. albicans on BSA as the sole source of nitrogen. In contrast, opt123 triple mutants, similar to a sap2 mutant, had a severe growth defect on BSA as the sole nitrogen source, which could be rescued by reintroduction of an intact copy of either OPT1, OPT2 or OPT3. The poor growth of the opt123 triple mutants was not caused by failure to induce SAP2 expression but by the inability of these mutants to efficiently transport the peptides produced by proteolytic degradation of BSA into the cell. By using reporter genes it could be demonstrated that individual members of the OPT gene family are differentially expressed. While none of the OPT genes was detectably expressed in rich YPD medium, a strong induction of OPT1 and OPT3 was observed in the presence of BSA as the sole nitrogen source. Forced expression of OPT4 and OPT5 under control of the constitutive ADH1 promoter in the opt123 triple mutants also complemented their growth defect on BSA as a sole nitrogen source, whereas the additional deletion of these genes in the resulting opt1234 quadruple and opt12345 quintuple mutants exacerbated the growth defect. These results confirmed that at least OPT1-OPT5 encode functional oligopeptide transporters. Additional experiments showed that the individual oligopeptide transporters differ in their substrate preferences. While the tetrapeptide LWMR was a better substrate than the tetrapeptide LSKL for strains that specifically expressed OPT3, OPT4 or OPT5, strains specifically expressing OPT2 grew on the LSKL peptide, but not on the LWMR peptide. Furthermore, experiments with peptides of defined length and sequence suggested that the oligopeptide transporters are also able to transport longer peptides up to at least eight amino acids in length. Therefore, the evolution of a gene family encoding oligopeptide transporters with different substrate preferences probably contributed to the ability of C. albicans to efficiently utilize proteins as a nitrogen source and adapt to the nutritional conditions in different host niches.

Orientador: Maria Valnice Boldrin Zanoni / Co-orientador: Thaís Tasso Guaraldo / Banca: Raquel Fernandes Pupo Nogueira / Banca: Regina Helena Pires Gonçalves / Resumo: O acesso à água de qualidade, livre de microrganismos patogênicos e de subprodutos potencialmente nocivos ao homem é um desafio constante do ser humano. Dentre os diversos microrganismos patogênicos, a presença daqueles pertencentes ao Reino Fungi tem chamado a atenção, principalmente em águas de hemodiálise, devido às consequências que a presença destes e de suas respectivas micotoxinas podem ocasionar a um paciente imunocomprometido. O presente trabalho teve como objetivo investigar a potencialidade de fotoânodos do tipo W/WO3 em sistemas de desinfecção fotoeletrocatalítica de águas contaminadas por Candida parapsilosis. Para isto, foram sintetizados eletrodos de trióxido de tungstênio usando anodização oxidativa através do crescimento eletroquímico de filmes de WO3 sobre W como substrato. O eletrodo proposto mostrou a formação de um filme homogêneo com nanoporos ao redor de 100nm, conforme a imagem de FEGSEM e apresentou um band gap em torno de 2,5 eV calculado a partir do gráfico de Tauc pelo método Kubelka-Munk. O material construído é fotoativo e A curva de Iph vs E sob radiação UV em NaCl 0,1 molL-1 revelou uma fotocorrente igual a 19 mA. Estudos da degradação de C. parapsilosis em NaCl 0,1 molL-1, pH 7, potencial igual a 1,5V e irradiação UV-Vis e Vis, condições previamente otimizadas, mostraram a morte das leveduras em 30 segundos de tratamento. Não foi verificada a formação significativa de cloro ativo nas condições empregadas assim como não há formação de íons clorato e perclorato durante a degradação fotoeletrocatalítica. Para as medidas fotoeletrocatalíticas realizadas no dialisato, verificou-se a morte microbiana em torno de 5 minutos e remoção de carbono orgânico total em torno de 73% e 45% para as soluções ácidas e básicas respectivamente. / Abstract: The access to quality water, free of pathogenic microorganisms and potentially harmful to the community is a constant challenge of being human. Among the various pathogenic microorganisms, the presence of those belonging to the kingdom Fungi has drawn attention, especially in hemodialysis water, due to the consequences that the presence of these and their respective mycotoxins causes to an immunocompromised patient. The aim of this study was to investigate the capability of W/WO3 as photoanode in disinfection systems using photoelectrocatalysis in water contaminated by Candida parapsilosis. For this, tungsten trioxide was synthesized using electrodes via electrochemical anodization by the growth of WO3 films in tungsten foil as a substrate. The prepared electrode was characterized morphologically and electrochemically and was subsequently applied in a photoelectrochemical system. The FEMSEM image showed a formation of a homogeneous film in the surface foil with nanopores around 100 nm. There was obtained a band gap of 2.5 eV calculated from Tauc's plot of the Kubelka-Munk method and photocurrent observed in NaCl 0.1 molL-1 was equal to 19 mA. Studies of C. parapsilosis degradation in NaCl 0.1 molL-1, pH 7, the potential equal to 1.5V and irradiating UV-Vis and Vis, previously optimized conditions, showed the death of yeast at 30 seconds of treatment. There was no significant formation of active chlorine under the conditions employed just as there is formation of chlorate and perchlorate ions during photoelectrocatalytic degradation. For photoelectrocatalytics measurements in the dialysate, there was microbial death in about 5 minutes and removal of total organic carbon at around 73% and 45% for acidic and basic solutions, respectively. / Mestre

Orientador: Juliana Campos Junqueira / Banca: Aguinaldo Silva Garcez Segundo / Banca: Antonio Olavo Cardoso Jorge / Resumo: A terapia fotodinâmica (TFD) tem demonstrado ação antimicrobiana sobre as leveduras do gênero Candida, sendo considerada uma técnica promissora para o tratamento de candidose. Recentemente foi relatado que a aplicação de TFD também pode resultar em ativação do sistema imunológico, contribuindo para a melhora da infecção. Assim, o objetivo desse estudo foi avaliar a ação da TFD e da terapia laser sobre a resposta imunológica à candidose experimental utilizando Galleria mellonella como modelo hospedeiro de infecção. Larvas de G. mellonella foram infectadas com diferentes cepas de Candida albicans e, após 30 min, foram tratadas com TFD mediada por azul de metileno e laser diodo emitido em 660 nm. A seguir, as larvas foram incubadas a 37°C por sete dias e analisadas diariamente para determinação da curva de sobrevivência. Para o estudo da resposta imunológica, após os tempos de 3, 6, 18 h da TFD foram realizados testes de determinação da densidade de hemócitos na hemolinfa de G. mellonella. Os dados obtidos na curva de sobrevivência foram avaliados pelo teste de Log-rank (Mantel Cox) e os resultados da análise imunológica por análise de variância ANOVA e teste de Tukey, com significância de 5%. Os resultados obtidos demonstraram que tanto para a cepa ATCC 18804 como para a cepa clínica 17 de C. albicans, a TFD prolongou a sobrevivência das larvas de G. mellonella infectadas por uma dose fúngica letal. Entretanto, houve diferença estatisticamente significante e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Photodynamic therapy (PDT) has demonstrated antimicrobial activity on the yeast of the genus Candida and is considered a promising technique for the treatment of candidiasis. Recently it was reported that the application of PDT may also result in activation of the immune system, contributing to the improvement of the infection. The objective of this study is to evaluate the action of PDT and laser therapy on the immune response to experimental candidiasis using Galleria mellonella as host of the infection. G. mellonella larvae were infected with different Candida albicans strains and, after 30 min were treated with methylene blue-mediated PDT and laser diode emitted at 660 nm. Then, the larvae were incubated at 37° C for seven days and analyzed daily in order to determine the survival curve. For the study of the immunological response, after intervals of 3, 6, 18 h of the PDT, tests were performed to determine the density of hemocytes in the hemolymph of G. mellonella. The data obtained in the survival curve were evaluated by the Log-rank test (Mantel Cox) and the results of the immunological analysis by analysis of variance ANOVA and Tukey test, with significance of 5%. The results demonstrated that for both the ATCC 18804 strain and the C. albicans clinical strain 17, PDT prolonged the survival of the infected G. mellonella larvae by a lethal fungal dose. However, there was a statistically significant difference between the PDT and the control groups only... (Complete abstract click electronic access below) / Mestre

O uso de antissépticos complementa a higienização bucal reduzindo a microbiota e, consequentemente minimizando a colonização, a formação de biofilme e, assim promovendo a saúde bucal. Diante das opções de antissépticos bucais e produtos naturais, faz-se necessária a análise microbiológica da eficácia desses produtos e suas implicações no controle do biofilme. Neste sentido, têm-se como objetivos: determinar a diluição inibitória máxima (DIMax) de antissépticos bucais (Listerine®, Colgate Plax® Tea Fresh, Periogard®) e o óleo de melaleuca sobre as cepas de Candida albicans e Candida glabrata, clínicas e padrão; quantificar as unidades formadoras de colônias por tubo traqueal (UFC/TT) das leveduras em TTs revestidos com os respectivos produtos e, analisar a formação de biofilme em fragmentos de tubos revestidos ou não com antissépticos e óleo de melaleuca por C. glabrata. Trata-se de um estudo de natureza laboratorial, in vitro, realizado com cepas clinicas e padrão e subsidiado em métodos clássicos da microbiologia para o processamento das avaliações propostas. Para determinar a DIMax, realizou-se a diluição dupla seriada (1/4 a 1/4096) dos antissépticos e óleo de melaleuca respectivamente, sendo as placas incubadas a 37°C por 24 horas. Considerou-se DIMax a maior diluição capaz de inibir o crescimento de todas as cepas avaliadas. Na formação de biofilme foram empregadas duas técnicas: determinação das UFC/TT e microscopia eletrônica de varredura (MEV). Na aleatorização das cepas C. glabrata utilizou-se a análise estatística pelo modelo de regressão logístico multinomial. A partir da análise dos resultados observou-se que o Listerine® apresentou a menor ação inibitória na DIMax de 1/4, óleo de melaleuca (1/16), Colgate Plax® Tea Fresh (1/64) e o Periogard® (1/128). Em termos de formação de biofilme, o tubo revestido com Colgate Plax® Tea Fresh apresentou diferença no teste de comparações múltiplas (p=0,0031), com atividade antibiofilme em todas as cepas de C. glabrata, com exceção de um isolado clínico. As fotomicrografias revelaram reprodução por brotamento presente no TT revestido com óleo de melaleuca, lise celular na ação do Periogard® e, os TTs revestidos com Colgate Plax® Tea Fresh não apresentaram biofilme, exceto na cepa 33. A formação de biofilme por células leveduriformes foi significativa apresentando-se de forma diversificada nos diferentes tubos revestidos. Estudos adicionais sobre Candida spp. em tubos traqueais são recomendáveis em pacientes, com e sem pneumonia, submetidos à ventilação mecânica / Introduction: The use of oral antiseptics is increasingly common, as a complement to the regular oral hygiene by reducing the oral microbiota, biofilm formation and thereby promoting oral health. Facing of several oral antiseptics and natural products, it is necessary microbiological analysis of the effectiveness of these antiseptics and in its implications in the control of biofilm. Objective: The aims of the study were to determine the maximum inhibitory dilution (MID) oral antiseptics (Listerine®, Colgate® Plax, Fresh Tea and Periogard®) and tea tree oil of clinical and standard strains of Candida spp. and Candida glabrata; colony forming units assay (CFU/TT) of yeast in tracheostomy tubes (TT) coated with some products; and analyze the biofilm formed in fragments of tubes coated or not with oral antiseptics and tea tree oil for Candida glabrata. Methods: This is a laboratory investigation, in vitro study performed with clinical and standard strains and subsidized of classical microbiology methods for processing the proposals reviews. To determine the MID, serial dilution was carried out (1/4 a 1/2048) of oral antiseptics and tea tree oil respectively, and the plates were incubated at 37°C for 24h. The MID was considered the highest dilution capable of inhibiting the growth of all strains tested. In biofilm formation two techniques were employed: determination of colony forming units (CFU/TT) and scanning electron microscopy (SEM). The statistical analysis by multinomial logistic regression model was used to randomization strains of C. glabrata. Results: Listerine® showed the worst performance, MID (1/4), tea tree oil (1/16), Colgate Plax® Fresh Tea (1/64) and Periogard® (1/128). In terms of biofilm formation, the multiple comparisons test presented differences (p=0.0031) for tube coated Colgate Plax® Tea Fresh with antibiofilm activity in all strains of C. glabrata, except for one clinical isolate. The photomicrographs revealed reproduction by budding in the TT coated with tea tree oil, cell lysis in action of Periogard®, and the Colgate Plax® Fresh Tea coated tube produced no visible colony, except with strain 33. Conclusion: Biofilm formation by yeast was significant presenting diverse in different coated tubes. Additional studies of Candida spp. in tracheostomy tubes are recommended in patients with and without pneumonia, undergoing mechanical ventilation

Siu Yau Lung, Philip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 99-119). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.iv / Declaration --- p.v / Scientific publication --- p.vi / Abbreviations --- p.vii / Genetic symbols --- p.ix / Table of contents --- p.x / List of tables --- p.xiv / List of figures --- p.xv / Chapter Chapter One --- Introduction / Chapter 1.1 --- Thesis outline --- p.1 / Chapter 1.2 --- Candida albicans --- p.2 / Chapter 1.3 --- Physical characterization of C. albicans --- p.3 / Chapter 1.3.1 --- Strain identification --- p.3 / Chapter 1.3.2 --- Dimorphism --- p.5 / Chapter 1.3.3 --- Genome of C. albicans --- p.10 / Chapter 1.3.4 --- Karyotype --- p.11 / Chapter 1.4 --- Candidiasis --- p.12 / Chapter 1.4.1 --- Superficial candidiasis --- p.15 / Chapter 1.4.2 --- Systemic candidiasis --- p.16 / Chapter 1.4.3 --- Virulence --- p.16 / Chapter 1.4.4 --- Multi-drug resistance --- p.17 / Chapter 1.5 --- Parasexual genetics --- p.20 / Chapter 1.5.1 --- Mutant isolation --- p.20 / Chapter 1.5.2 --- Spheroplasts complementation --- p.21 / Chapter 1.5.3 --- Mitotic complementation --- p.22 / Chapter 1.6 --- Natural heterozygosity in C. albicans --- p.22 / Chapter 1.7 --- Adenine biosynthesis --- p.26 / Chapter 1.7.1 --- de novo pathway --- p.26 / Chapter 1.7.2 --- Salvage pathway --- p.29 / Chapter 1.7.3 --- Importance of C. albicans ADE1 and ADE2 genes --- p.29 / Chapter 1.8 --- Aim of study --- p.30 / Chapter Chapter Two --- Construction of disrupted C. albicans ADE1 gene / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.34 / Chapter 2.2.1 --- Strains --- p.34 / Chapter 2.2.2 --- Construction of plasmid pGEMTE-ADEl --- p.34 / Chapter 2.2.2.1 --- Isolation of Candida genomic DNA --- p.34 / Chapter 2.2.2.2 --- Isolation of C. albicans ADE1 gene from CAM --- p.36 / Chapter 2.2.2.2.1 --- Amplification of C. albicans ADE1 gene --- p.36 / Chapter 2.2.2.2.2 --- Purification of PCR product --- p.37 / Chapter 2.2.2.3 --- Cloning of ADEl gene into pGEMT-Easy vector --- p.38 / Chapter 2.2.2.3.1 --- Cloning vector pGEMT-Easy --- p.38 / Chapter 2.2.2.3.2 --- Ligation --- p.38 / Chapter 2.2.2.4 --- Transformation of E. coli DH5a cells --- p.39 / Chapter 2.2.2.4.1 --- Preparation of competent E. coli DH5a cells --- p.39 / Chapter 2.2.2.4.2 --- Plasmid DNA transformation --- p.40 / Chapter 2.2.2.4.3 --- Isolation ofplasmid DNA from E. coli --- p.40 / Chapter 2.2.3 --- Construction of pGEMTE-ADElA-URA3 --- p.41 / Chapter 2.2.3.1 --- Isolation of C. albicans URA3 gene from plasmid pCUB-6 --- p.41 / Chapter 2.2.3.2 --- Preparation of cloning vector pGEMTE-ADE 1Δ --- p.42 / Chapter 2.2.3.2.1 --- PCR amplification of vector pGEMTE-ADElΔ --- p.42 / Chapter 2.2.3.2.2 --- Modification of PCR vector pGEMTE-ADElΔ --- p.44 / Chapter 2.2.3.2.3 --- Dephosphorylation --- p.45 / Chapter 2.2.3.3 --- Cloning and isolation of plasmid pGEMTE-ADE1Δ-URA3 --- p.46 / Chapter 2.3 --- Results and Discussion --- p.47 / Chapter Chapter Three --- Gene disruption of C. albicans CAI4 by electroporation / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.2.1 --- Strains --- p.54 / Chapter 3.2.2 --- Transforming DNA --- p.54 / Chapter 3.2.3 --- Purification of PCR product --- p.55 / Chapter 3.2.4 --- DNA transformation --- p.55 / Chapter 3.2.5 --- Transformation efficiency --- p.56 / Chapter 3.2.5.1 --- Pulse length --- p.56 / Chapter 3.2.5.2 --- Amount of DNA --- p.57 / Chapter 3.2.6 --- Southern analysis of transformants --- p.57 / Chapter 3.2.6.1 --- Isolation of Candida genomic DNA --- p.57 / Chapter 3.2.6.2 --- Preparation of Candida genomic DNA for Southern analysis --- p.57 / Chapter 3.2.6.3 --- Southern hybridization --- p.58 / Chapter 3.2.6.4 --- Preparation of radioactive probe --- p.60 / Chapter 3.2.6.5 --- Radioactive labelling of the probe --- p.61 / Chapter 3.2.6.6 --- Hybridization of nylon membrane --- p.62 / Chapter 3.2.6.7 --- Stringency washes --- p.62 / Chapter 3.2.6.8 --- Auto-radiography --- p.62 / Chapter 3.3 --- Results and Discussion --- p.64 / Chapter Chapter Four --- UV mutagenesis of disrupted C. albicans / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Materials and Methods --- p.76 / Chapter 4.2.1 --- Strains --- p.76 / Chapter 4.2.2 --- Generation of recombinants by UV irradiation --- p.76 / Chapter 4.2.3 --- Analyses of twin-sectored colonies --- p.77 / Chapter 4.2.3.1 --- Replica analyses of twin-sectored colonies --- p.77 / Chapter 4.2.3.2 --- Southern analysis of segregants --- p.77 / Chapter 4.3 --- Results and Discussion --- p.78 / Chapter Chapter Five --- Concluding remarks and perspectives --- p.96 / Bibliography --- p.99

Candida albicans is an opportunistic fungal pathogen. It is the fourth leading cause of nosocomial infections and can endanger immunocompromised patients. Candida has the ability to form biofilms on plastic medical devices, such as catheters and central nervous system shunts. Two clinical isolate series were profiled using a number of phenotyping assays comprising in vivo, ex vivo, and in vitro tests. These tests shed light on host-pathogen relations as well as offer potential information useful in the treatment of these infections. Fluconazole, an antifungal, is the first line of treatment for fungal infections. The incidence of fluconazole-resistant infections is increasing annually, and there are not many other drugs available to treat infections. In 2013, Fazly et al. discovered the drug Filastatin, which prevents adhesion and filamentation of Candida albicans. In our study, two screens were performed to identify the target of Filastatin. Because there is no complete knockout library for Candida, an available, partial knockout library was screened. This library is enriched for transcription factors. We screened for regulators of biological pathways that may be important for adhesion and filamentation in Candida albicans, to identify potential Filastatin targets. The iron-uptake pathway was chosen as the focus for the remainder of this study.

As Candida spp. têm grande habilidade para colonizar e formar biofilmes em dispositivos médicos e biomateriais, provocando infecções nos hospedeiros humanos. O Polietileno de Alta Densidade (PEAD), Polihidroxibutirato (PHB) e o Ácido Polilático (PLA) apresentam propriedades interessantes para aplicações em dispositivos médicos. Os sais imidazólicos (SI) apresentam propriedades avançadas, inclusive antifúngicas, podendo ser utilizados como aditivos nestes polímeros. Neste estudo, foram preparados filmes poliméricos com diferentes teores dos SI cloreto de 1-n-hexadecil-3-metilimidazólico (C16MImCl) e metanossulfonato de 1-n-hexadecil-3- metilimidazólico (C16MImMeS), através dos processos de mistura no estado fundido (PEAD e PHB) e de Solvent Casting (PLA). Para avaliar se esses biomateriais apresentavam atividade antibiofilme foram realizados ensaios Antibiofilme, testados frente a isolados de Candida spp. Para determinar a porcentagem de impedimento da formação do biofilme foram realizados ensaios de Menor Concentração Antibiofilme, frente a isolados de Candida tropicalis. Análises de Microscopia Eletrônica de Varredura foram realizadas para verificar se ocorre o impedimento da formação do biofilme nos biomateriais. Foram avaliadas a citotoxicidade dos SI, a adesão celular e a biocompatibilidade celular de células-tronco mesenquimais humanas (CTMh) nos filmes de PEAD, PHB e PLA. Foram realizados ensaios histopatológicos dos biomateriais em pele de orelha de suínos para verificar se ocorre lesões microscópicas. Os filmes de PEAD, PHB e PLA foram caracterizados para avaliar a morfologia, a presença dos SI na superfície e as propriedades físicoquímicas dos mesmos. Os filmes de PEAD, PHB e PLA com diferentes teores dos SI C16MImCl e C16MImMeS apresentaram atividade antibiofilme frente a isolados de Candida tropicalis, C. parapsilosis e C. albicans. Com o ensaio MCA foi possível verificar que com o aumento do teor dos SI (C16MImCl e C16MImMeS) no PLA e PHB ocorre em geral um aumento na porcentagem de impedimento da formação do biofilme, sendo que isso em geral não ocorre com o PEAD. As micrografias de MEV mostraram o efetivo impedimento da formação do biofilme. Em geral os SI não apresentaram citotoxicidade em baixas concentrações. Os biomateriais mostraram uma boa adesão celular e são biocompatíveis com CTMh. Na avaliação histopatológica de pele da orelha suína com os biomateriais não foram observadas lesões microscópicas agudas. Em combinação com as boas propriedades morfológicas, físico-químicas e mecânicas, estes biomateriais são promissores para serem utilizados na confecção de dispositivos médicos. / Candida spp. have great ability to colonize and form biofilms on medical devices and biomaterials, causing infections in human hosts. High-density polyethylene (HDPE), Polyhydroxybutyrate (PHB) and Polylactic acid (PLA) exhibit interesting properties for applications in medical devices. Imidazolium salts (IS) have advanced properties, including antifungal, which could be explored as additives in these polymers. In this study, polymer films with different IS (1-n-hexadecyl-3- methylimidazolium chloride (C16MImCl) and 1-n-hexadecyl-3-methylimidazolium methanesulfonate (C16MImMeS)) contents were prepared, using either the melt blending (HDPE and PHB) or the solvent casting process (PLA). The antibiofilm assay with Candida spp. isolates was performed to assess the antibiofilm activity of these biomaterials. Inhibition percentages of biofilm formation of Candida tropicalis isolates were determined by the minor antibiofilm concentration (MAC) assay. Scanning electron microscopy was applied to verify the inhibition of biofilm formation on these biomaterials. The cytotoxicity of IS and the cell adhesion and biocompatibility of human mesenchymal stem cells (hMSC) of the HDPE, PHB and PLA films was studied. Histopathologic assays were performed with these biomaterials on ear pigskin to verify the occurrence of microscopic lesions. The HDPE, PHB and PLA films were characterized to understand their surface morphology, the presence of IS on the surface and the physicochemical properties thereof. The polymer films with different concentrations of the IS C16MImCl and C16MImMeS presented antibiofilm activity against isolates of Candida tropicalis, C. parapsilosis and C. albicans. The MAC assay enabled to determine that an increasing IS (C16MImCl and C16MImMeS) content in the PLA and PHB biomaterials promoted, in general, an increase in the inhibition percentage of biofilm formation, which was not observed for the HDPE films. The SEM micrographs showed the effective prevention of biofilm formation on the IS containing biomaterials. Lower concentrations of the IS showed no cytotoxicity. The biomaterials presented good cell adhesion percentages and biocompatibility with hMSC. Furthermore, no acute microscopic lesions were identified in the histopathological evaluation after contact between the biomaterials and ear pigskin. In combination with the good morphological, physicochemical and mechanical properties, these biomaterials can be considered as promising for use in the manufacturing of medical devices.

A natamicina é um composto antifúngico bastante utilizado na indústria alimentar, pois é seguro para o consumo e não afeta a qualidade dos alimentos. Recentemente surgiu uma preocupação em relação ao seu uso aditivo em bebidas e iogurtes, uma vez que a natamicina poderia exercer pressão seletiva e desencadear o desenvolvimento de resistência aos polienos in vivo na microbiota comensal do hospedeiro. Diante dessa incerteza, abordamos o possível desenvolvimento da resistência contra a natamicina. Uma seleção de 17 isolados de Candida spp. foi submetida a concentrações crescentes de natamicina durante um tempo prolongado. A exposição à natamicina provocou um aumento de MIC em 14 dos 17 isolados testados e o desenvolvimento de resistência à natamicina foi evidenciado em 5 destes 14 isolados. Dezesseis isolados apresentaram aumento de MIC para anfotericina B e 4 destes isolados mostraram resistência à anfotericina B. A capacidade de formação de biofilmes desses isolados também foi realizada e 3 isolados apresentaram aumento da capacidade de formação de biofilmes após indução de resistência à natamicina. Foi possível evidenciar que a resistência adquirida à natamicina foi maior em isolados que apresentavam baixa suscetibilidade antes da exposição à natamicina. Em vista dos resultados acima mencionados, ressalta-se a importância do monitoramento da natamicina aditivada nos alimentos, pois seus níveis residuais em concentrações elevadas podem exercer uma pressão seletiva sobre as espécies de Candida constituintes da microbiota comensal humana gastrointestinal, no que tange a sua virulência e resistência. / Natamycin is a antifungal compound used in the food industry because it is safe for consumption and has no effect on quality of foods. Recently a concern has arisen over its additive use in beverages and yogurts since natamycin could exert selective pressure and trigger the development of polyene resistance in vivo in the commensal microbiota of host. In view of this uncertainty, we address the possible development of resistance against the natamycin. A selection of 17 isolates of Candida spp. was subjected to increasing concentrations of natamycin for prolonged time. The natamycin-exposure caused an increase of M.I.C. in 14 out of 17 tested isolates and the development of natamycin resistance was evidenced in 5 of these 14 isolates. Sixteen isolates showed increased of M.I.C to amphotericin B and 4 of these isolates showed resistance to amphotericin B. The biofilm forming capacity of the isolates also was performed and 3 isolates presented increasing the capacity to form biofilms after induction of natamycin resistance. It was possible to show that acquired natamycin resistance was increased in isolates that showed low susceptibility before natamycin exposure. In view of the aforementioned results, the importance of the monitoring of natamycin in food is highlighted, since its residual levels in high concentrations may exert a selective pressure on Candida species constituting the gastrointestinal human commensal microbiota, in relation to its virulence and resistance.