Der Hefepilz Candida albicans ist ein harmloser Kommensale auf den Schleimhäuten des Gastrointestinal- und Urogenitaltrakts der meisten gesunden Menschen. Bei einer Störung der natürlichen Mikroflora oder des Wirtsimmunsystems kann der Pilz jedoch auch oberflächliche und sogar systemische Infektionen verursachen. C. albicans weist eine Reihe von Eigenschaften auf, die zur Virulenz des Erregers beitragen. Dazu gehören die Adhärenz an unterschiedliche Wirtsoberflächen, die morphologische Variabilität des Pilzes und die Sekretion von Aspartatproteasen. Die Expression vieler dieser Virulenzfaktoren wird unter anderem durch die Verfügbarkeit von Stickstoff reguliert. Unter Stickstoffmangelbedingungen wechselt C. albicans vom Wachstum als sprossende Hefe zum filamentösen Wachstum, und dieser Wechsel wird durch die Ammoniumpermease Mep2p reguliert. Wie die Induktion des filamentösen Wachstums durch Mep2p kontrolliert wird, ist jedoch weitgehend unbekannt. In der vorliegenden Arbeit wurde eine Mutationsanalyse von Mep2p durchgeführt, um Aminosäuren zu identifizieren, die an der Signalfunktion dieser Permease beteiligt sind. Die C-terminale cytoplasmatische Domäne von Mep2p wird für den Ammoniumtransport nicht benötigt, ist jedoch essentiell für die Signaltransduktion. Progressive C-terminale Verkürzungen von Mep2p zeigten, dass ein MEP2DC433-Allel immer noch in der Lage war, das filamentöse Wachstum zu induzieren, wohingegen die Deletion einer weiteren Aminosäure die Morphogenese blockierte. Das Tyrosin an Position 433 (Y433) ist deshalb die letzte Aminosäure, die für die Signalfunktion von Mep2p essentiell ist. Um besser zu verstehen, wie die Signalaktivität von Mep2p durch die Verfügbarkeit und den Transport von Ammonium reguliert wird, wurden verschiedene hochkonservierte Aminosäuren mutiert, die vermutlich an der Bindung oder dem Transport von Ammonium in die Zelle beteiligt sind. Die Mutation von D180, von dem postuliert wurde, dass es den initialen Kontakt mit extrazellulärem Ammonium ermöglicht, oder der im Transportkanal lokalisierten Histidine H188 und H342 hatte zur Folge, dass Mep2p nicht mehr exprimiert wurde, so dass diese Aminosäuren vermutlich für die Proteinstabilität wichtig sind. Die Mutation von F239, das zusammen mit F126 eine extracytosolische Pforte zur Transportpore bildet, verhinderte trotz korrekter Membranlokalisation sowohl den Ammoniumtransport als auch das filamentöse Wachstum. Allerdings führte auch die Mutation von W167, das vermutlich zusammen mit Y122, F126 und S243 an der Rekrutierung des Ammoniumions an der extrazellulären Seite der Membran beteiligt ist, zur Blockierung des filamentösen Wachstums, obwohl der Ammoniumtransport kaum beeinflusst war. Dies zeigte, dass die intrazelluäre Signaltransduktion durch extrazelluläre Veränderungen in Mep2p beeinflusst werden kann. Die Mutation von Y122 reduzierte die Ammoniumaufnahme weitaus starker als die Mutation von W167, erlaubte jedoch immer noch ein effizientes filamentöses Wachstum. Die Signalaktivität von Mep2p ist deshalb offensichtlich nicht direkt mit der Transportaktivität des Proteins korreliert. Ein wichtiger Aspekt in der Fähigkeit von Mep2p, die Morphogenese zu stimulieren, ist die vergleichsweise starke Expression des Proteins. Um die Regulation der MEP2-Expression aufzuklären, wurden die cis-regulatorischen Sequenzen und die trans-aktivierenden Faktoren, die die MEP2-Induktion unter Stickstoffmangel vermitteln, identifiziert. Eine Promotoranalyse zeigte, dass zwei mutmaßliche Bindungsstellen für GATA-Transkriptionsfaktoren eine zentrale Rolle in der MEP2-Expression haben, da die Deletion oder Mutation dieser GATAA-Sequenzen die Expression von MEP2 stark reduzierte. Um die Rolle der GATA-Transkriptionsfaktoren Gln3p und Gat1p bei der Regulation der MEP2-Expression zu untersuchen, wurden Mutanten hergestellt, in denen die entsprechenden Gene deletiert waren. Die Expression von Mep2p war in gln3D und gat1D Einzelmutanten stark verringert und in gln3D gat1D Doppelmutanten nicht mehr nachweisbar. Die Deletion von GLN3 hatte auch eine starke Reduktion des filamentösen Wachstums zur Folge, die durch die konstitutive Expression von MEP2 unter Kontrolle des ADH1-Promotors aufgehoben wurde. Dagegen hatte die Deletion von GAT1 keinen Einfluss auf das filamentöse Wachstum. Überraschenderweise war das filamentöse Wachstum in den gat1D Mutanten teilweise unabhängig von Mep2p, was darauf hinwies, dass in Abwesenheit von GAT1 andere Signalwege aktiviert werden, die die Morphogenese stimulieren. Diese Ergebnisse zeigten, dass die GATA-Transkriptionsfaktoren Gln3p und Gat1p die Expression der Ammoniumpermease MEP2 kontrollieren und dass Gln3p auch ein wichtiger Regulator des durch Stickstoffmangel induzierten filamentösen Wachstums von C. albicans ist. Mutanten, in denen die beiden GATA-Transkriptionsfaktoren Gln3p und Gat1p fehlten, waren nicht mehr in der Lage, in einem Medium zu wachsen, das bovines Serumalbumin (BSA) als einzige Stickstoffquelle enthält. Die Fähigkeit von C. albicans, Proteine als einzige Stickstoffquelle zum Wachstum zu verwenden, wird durch die sekretierte Aspartatprotease Sap2p, die die Proteine zu Peptiden abbaut, und durch Oligopeptidtransporter, die diese Peptide in die Zelle aufnehmen, vermittelt. Der Wachstumsdefekt der gln3D gat1D Doppelmutanten war hauptsächlich durch einen Defekt in der SAP2-Expression verursacht, da die Expression von SAP2 unter Kontrolle des konstitutiven ADH1-Promotors die Fähigkeit zum Wachstum auf BSA wieder herstellte. Es zeigte sich, dass Gln3p und Gat1p die Expression des Transkriptionsfaktors STP1, der für die Induktion von SAP2 in Gegenwart von Proteinen notwendig ist, regulieren. Bei einer Expression von STP1 unter Kontrolle des induzierbaren Tet-Promotors waren Gln3p und Gat1p nicht mehr notwendig für das Wachstum auf Proteinen. Wenn bevorzugte Stickstoffquellen verfügbar sind, wird SAP2 auch in Gegenwart von Proteinen reprimiert, und diese Stickstoff-Katabolitrepression korrelierte mit einer reduzierten STP1-Expression. Die Expression von STP1 unter Kontrolle des Tet-Promotors hob diese Repression auf, was zeigte, dass die Regulation der STP1-Expression durch die GATA-Transkriptionsfaktoren eine Schlüsselrolle sowohl bei der positiven als auch bei der negativen Kontrolle der SAP2-Expression spielt. Eine regulatorische Kaskade, in der die Expression des spezifischen Transkriptionsfaktors Stp1p durch die allgemeinen Regulatoren Gln3p und Gat1p kontrolliert wird, stellt die Expression von SAP2 in C. albicans deshalb unter Stickstoffkontrolle und gewährleistet eine angepasste Expression dieses Virulenzfaktors. Die Ergebnisse dieser Arbeit illustrieren, dass die GATA-Faktoren Gln3p und Gat1p zum Teil überlappende aber auch spezifische Funktionen in der Anpassung von C. albicans an die Verfügbarkeit verschiedener Stickstoffquellen haben. Diese Anpassungsmechanismen spielen auch eine Rolle in der Pathogenität des Pilzes, wobei die relative Bedeutung von Gln3p und Gat1p vom Zielgen und der Stickstoffquelle abhängt. Diese Erkenntnisse geben einen vertieften Eiblick in die molekularen Grundlagen der Anpassung von C. albicans an unterschiedliche Umweltbedingungen. / The yeast Candida albicans is a member of the normal microflora on the mucosal surfaces of the gastrointestinal and urogenital tract in healthy persons. However, it is an opportunistic pathogen that can cause a range of infections from superficial to disseminated, in response to perturbation of the normal microflora or alterations in the host immunity. C. albicans exhibits a variety of characteristics such as adhesion, morphogenetic switching and secreted aspartic protease production that contribute to its virulence. Expression of many of these virulence factors is controlled by the availability of essential element, nitrogen. C. albicans undergoes morphogenetic transition to form filaments under nitrogen starvation conditions and this switch is controlled by the ammonium permease Mep2p. However, little is known about how this signaling function of Mep2p is regulated. Mutational analysis of Mep2p was carried out to identify the residues that confer signaling activity to this permease. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is dispensable for ammonium transport but essential for the signaling activity of Mep2p. In this work, progressive C-terminal truncations analysis demonstrated that a MEP2DC433 allele was still able to induce filamentation while nitrogen starvation-induced filamentous growth was abolished in cells expressing a MEP2DC432 allele. Therefore, tyrosine at position 433 (Y433) is the last amino acid in Mep2p that is essential for signaling. To gain insights into how the signaling activity of Mep2p is regulated by ammonium availability and transport, conserved residues that have been implicated in ammonium binding or uptake were mutated. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is predicted to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filamentation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate along with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filamentation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filamentation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. An important aspect in the ability of Mep2p to stimulate filamentation in response to nitrogen limitation is its high expression levels. The cis-acting sequences and trans-acting regulators that mediate MEP2 induction in response to nitrogen limitation were identified. Promoter analysis revealed that two putative binding sites for GATA transcription factors have a central role in MEP2 expression, as deletion of the region containing these sites or mutation of the GATAA sequences in the full-length MEP2 promoter strongly reduced MEP2 expression. To elucidate the roles of the GATA transcription factors GLN3 and GAT1 in regulating MEP2 expression, mutants lacking one or both of these transcription factors were constructed. Mep2p expression was strongly reduced in gln3D and gat1D single mutants and virtually abolished in gln3D gat1D double mutants. Deletion of GLN3 strongly inhibited filamentous growth under limiting nitrogen conditions, which could be rescued by constitutive expression of MEP2 from the ADH1 promoter. In contrast, inactivation of GAT1 had no effect on filamentation. Surprisingly, filamentation became partially independent of the presence of a functional MEP2 gene in the gat1D mutants, indicating that the loss of GAT1 function results in the activation of other pathways that induce filamentous growth. These findings demonstrated that the GATA transcription factors Gln3p and Gat1p control expression of the MEP2 ammonium permease and that GLN3 is also an important regulator of nitrogen starvation-induced filamentous growth in C. albicans. C. albicans mutants lacking both the GATA transcription factors Gln3p and Gat1p were unable to grow in a medium containing an alternative nitrogen source, bovine serum albumin (BSA) as the sole nitrogen source. The ability to utilize proteins as sole source of nitrogen for growth of C. albicans is conferred by the secreted aspartic protease Sap2p, which degrades the proteins, and oligopeptide transporters that mediate uptake of the proteolytic products into cell. The growth defect of gln3D gat1D mutants was mainly caused by their inability to express the SAP2 gene, as SAP2 expression from the constitutive ADH1 promoter restored the ability of the mutants to grow on BSA. Expression of STP1, which encodes a transcription factor that is required for SAP2 induction in the presence of proteins, was regulated by Gln3p and Gat1p. Forced expression of STP1 from a tetracycline-inducible promoter bypassed the requirement of the GATA transcription factors for growth of C. albicans on proteins. When preferred nitrogen sources are available, SAP2 is repressed and this nitrogen catabolite repression of SAP2 was correlated with downregulation of STP1 under these conditions. Tetracycline-induced STP1 expression abolished nitrogen catabolite repression of SAP2, demonstrating that regulation of STP1 expression levels by the GATA transcription factors is a key aspect of both positive and negative regulation of SAP2 expression. Therefore, by using a regulatory cascade in which expression of the specific transcription factor Stp1p is controlled by the general regulators Gln3p and Gat1p, C. albicans places SAP2 expression under nitrogen control and ensures proper expression of this virulence determinant. In summary, the present study illustrated how GATA factors, Gln3p and Gat1p, play partially overlapping, but distinct roles, in mediating the appropriate responses of C. albicans to the availability of different nitrogen sources. These responses are also determinants of pathogenicity of the fungus. The relative contributions of Gln3p and Gat1p vary with their target genes and the availability of nitrogen source. Overall, these findings provide us with a better understanding of the molecular basis of some of the important processes that help in adaptation of C. albicans to various environmental conditions. The yeast Candida albicans is a member of the normal microflora on the mucosal surfaces of the gastrointestinal and urogenital tract in healthy persons. However, it is an opportunistic pathogen that can cause a range of infections from superficial to disseminated, in response to perturbation of the normal microflora or alterations in the host immunity. C. albicans exhibits a variety of characteristics such as adhesion, morphogenetic switching and secreted aspartic protease production that contribute to its virulence. Expression of many of these virulence factors is controlled by the availability of essential element, nitrogen. C. albicans undergoes morphogenetic transition to form filaments under nitrogen starvation conditions and this switch is controlled by the ammonium permease Mep2p. However, little is known about how this signaling function of Mep2p is regulated. Mutational analysis of Mep2p was carried out to identify the residues that confer signaling activity to this permease. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is dispensable for ammonium transport but essential for the signaling activity of Mep2p. In this work, progressive C-terminal truncations analysis demonstrated that a MEP2DC433 allele was still able to induce filamentation while nitrogen starvation-induced filamentous growth was abolished in cells expressing a MEP2DC432 allele. Therefore, tyrosine at position 433 (Y433) is the last amino acid in Mep2p that is essential for signaling. To gain insights into how the signaling activity of Mep2p is regulated by ammonium availability and transport, conserved residues that have been implicated in ammonium binding or uptake were mutated. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is predicted to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filamentation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate along with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filamentation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filamentation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. An important aspect in the ability of Mep2p to stimulate filamentation in response to nitrogen limitation is its high expression levels. The cis-acting sequences and trans-acting regulators that mediate MEP2 induction in response to nitrogen limitation were identified. Promoter analysis revealed that two putative binding sites for GATA transcription factors have a central role in MEP2 expression, as deletion of the region containing these sites or mutation of the GATAA sequences in the full-length MEP2 promoter strongly reduced MEP2 expression. To elucidate the roles of the GATA transcription factors GLN3 and GAT1 in regulating MEP2 expression, mutants lacking one or both of these transcription factors were constructed. Mep2p expression was strongly reduced in gln3D and gat1D single mutants and virtually abolished in gln3D gat1D double mutants. Deletion of GLN3 strongly inhibited filamentous growth under limiting nitrogen conditions, which could be rescued by constitutive expression of MEP2 from the ADH1 promoter. In contrast, inactivation of GAT1 had no effect on filamentation. Surprisingly, filamentation became partially independent of the presence of a functional MEP2 gene in the gat1D mutants, indicating that the loss of GAT1 function results in the activation of other pathways that induce filamentous growth. These findings demonstrated that the GATA transcription factors Gln3p and Gat1p control expression of the MEP2 ammonium permease and that GLN3 is also an important regulator of nitrogen starvation-induced filamentous growth in C. albicans. C. albicans mutants lacking both the GATA transcription factors Gln3p and Gat1p were unable to grow in a medium containing an alternative nitrogen source, bovine serum albumin (BSA) as the sole nitrogen source. The ability to utilize proteins as sole source of nitrogen for growth of C. albicans is conferred by the secreted aspartic protease Sap2p, which degrades the proteins, and oligopeptide transporters that mediate uptake of the proteolytic products into cell. The growth defect of gln3D gat1D mutants was mainly caused by their inability to express the SAP2 gene, as SAP2 expression from the constitutive ADH1 promoter restored the ability of the mutants to grow on BSA. Expression of STP1, which encodes a transcription factor that is required for SAP2 induction in the presence of proteins, was regulated by Gln3p and Gat1p. Forced expression of STP1 from a tetracycline-inducible promoter bypassed the requirement of the GATA transcription factors for growth of C. albicans on proteins. When preferred nitrogen sources are available, SAP2 is repressed and this nitrogen catabolite repression of SAP2 was correlated with downregulation of STP1 under these conditions. Tetracycline-induced STP1 expression abolished nitrogen catabolite repression of SAP2, demonstrating that regulation of STP1 expression levels by the GATA transcription factors is a key aspect of both positive and negative regulation of SAP2 expression. Therefore, by using a regulatory cascade in which expression of the specific transcription factor Stp1p is controlled by the general regulators Gln3p and Gat1p, C. albicans places SAP2 expression under nitrogen control and ensures proper expression of this virulence determinant. In summary, the present study illustrated how GATA factors, Gln3p and Gat1p, play partially overlapping, but distinct roles, in mediating the appropriate responses of C. albicans to the availability of different nitrogen sources. These responses are also determinants of pathogenicity of the fungus. The relative contributions of Gln3p and Gat1p vary with their target genes and the availability of nitrogen source. Overall, these findings provide us with a better understanding of the molecular basis of some of the important processes that help in adaptation of C. albicans to various environmental conditions.

1. Zusammenfassung Candida albicans ist ein opportunistisch pathogener Hefepilz, der sowohl oberflächliche Infektionen der Schleimhaut als auch lebensbedrohliche systemische Infektionen hervorrufen kann. Obwohl die Fähigkeit von C.albicans Infektionen auszulösen weitgehend vom Immunstatus des Wirts abhängt, besitzt der Pilz doch auch spezifische Eigenschaften, die eine Kolonisierung, Disseminierung und Anpassung an unterschiedliche Wirtsnischen ermöglichen und ihn vom harmlosen Kommensalen zum gefährlichen Krankheitsserreger werden lassen. Unter bestimmten Umweltbedingungen geht C.albicans vom Wachstum als sprossende Hefe zum invasiven, filamentösen Wachstum über, das eine wichtige Rolle in der Pathogenität des Pilzes spielt. Stickstoffmangel ist eines der Signale, die das filamentöse Wachstum in C.albicans induzieren, und die Kontrolle der Morphogenese durch die Verfügbarkeit von Stickstoff wurde in dieser Arbeit detailliert untersucht. Ammonium ist für Hefepilze eine bevorzugte Stickstoffquelle, die über spezifische Transporter in die Zelle aufgenommen wird. In der vorliegenden Arbeit konnte gezeigt werden, dass C.albicans zwei Ammoniumpermeasen besitzt, deren Expression durch Stickstoffmangel induziert wird. Während die Deletion von CaMEP1 oder CaMEP2 keinen Einfluss auf das Wachstum bei limitierenden Ammoniumkonzentrationen hatte, konnten mep1 mep2 Doppelmutanten bei Ammoniumkonzentrationen unter 5 mM nicht mehr wachsen. Im Gegensatz zu mep1 Mutanten bildeten mep2 Mutanten unter Stickstoffmangel keine Hyphen mehr und wuchsen ausschließlich in der Hefeform. CaMep2p hat also nicht nur eine Funktion als Ammoniumtransporter, sondern spielt auch eine Rolle bei der Induktion des filamentösen Wachstums. Weitere Experimente zeigten, dass CaMep2p ein weniger effizienter Ammoniumtransporter als CaMep1p ist, dafür aber stärker exprimiert wird, und dass dieser Unterschied wichtig für die Signalfunktion von CaMep2p ist. Durch Deletionsanalysen konnte bewiesen werden, dass die C-terminale, cytoplasmatische Domäne von CaMep2p essentiell für die Induktion des Hyphenwachstums ist, für den Ammoniumtransport jedoch nicht benötigt wird, und diese beiden Funktionen von CaMep2p daher voneinander getrennt werden können. In C.albicans gibt es mindestens zwei Signalwege die das filamentöse Wachstum steuern, eine MAP-Kinase-Kaskade und einen cAMP-abhängigen Signalweg, die in den Transkriptionsfaktoren Cph1p bzw. Efg1p enden. Bei Inaktivierung des einen oder des anderen Signalwegs induziert Stickstoffmangel kein filamentöses Wachstum mehr. Ein hyperaktives CaMEP2 Allel konnte den filamentösen Wachstumsdefekt sowohl von cph1 als auch efg1 Mutanten aufheben, nicht jedoch den einer cph1 efg1 Doppelmutante oder einer Mutante, der das G-Protein Ras1p fehlte, das beide Signalwege aktiviert. Umgekehrt wurde der filamentöse Wachstumsdefekt von mep2 Mutanten durch ein dominant-aktives RAS1 Allel bzw. durch die Zugabe von cAMP aufgehoben. Diese Ergebnisse deuten darauf hin, dass CaMep2p bei Stickstoffmangel sowohl den MAP-Kinase- als auch den cAMP-abhängigen Signalweg aktiviert, um filamentöses Wachstum zu induzieren. In genügend hohen Konzentrationen reprimierte Ammonium das filamentöse Wachstum selbst wenn die Signalwege artifiziell aktiviert waren. Die bevorzugte Stickstoffquelle Ammonium ist deshalb ein Inhibitor der Morphogenese, der durch denselben Transporter in die Zelle aufgenommen wird, der bei Stickstoffmangel das filamentöse Wachstum von C.albicans induziert. Obwohl ein genaues Verständnis der Virulenzmechanismen von C.albicans auch neue Ansätze zur Bekämpfung von Infektionen durch diesen Pilz liefern kann, ist doch die Identifizierung und Charakterisierung von essentiellen Genen als potentielle Ziele für die Entwicklung neuer Antimykotika eine Strategie, die von der pharmazeutischen Industrie favorisiert wird. Aus diesem Grund wurden in Zusammenarbeit mit einem Industriepartner drei Gene von C.albicans ausgewählt, die in anderen Pilzen als essentiell beschrieben wurden, und im Rahmen dieser Arbeit funktionell charakterisiert. RAP1 codiert für das Repressor/Aktivator Protein 1, ein Transkriptionsfaktor und Telomerbindeprotein, das in der Bäckerhefe Saccharomyces cerevisiae essentiell ist. Die Deletion des RAP1 Gens in C.albicans beeinträchtigte jedoch nicht die Lebensfähigkeit der Mutanten, so dass RAP1 kein vielversprechendes Ziel darstellt. CBF1 (centromere binding factor 1) ist in S.cerevisiae wichtig für die korrekte Chromosomenverteilung während der Mitose und außerdem auch für die transkriptionelle Aktivierung der Methioninbiosynthesegene; in den verwandten Hefen Kluyveromyces lactis und Candida glabrata ist CBF1 sogar essentiell. C.albicans cbf1 Mutanten wiesen jedoch keinen erhöhten Chromosomenverlust auf, so dass CBF1 hier offensichtlich keine Rolle bei der Chromosomensegregation spielt. Allerdings waren die Mutanten auxotroph für schwefelhaltige Aminosäuren und generell stark im Wachstum beeinträchtigt, was zeigte, dass Cbf1p für das normale Wachstum von C.albicans wichtig ist. YIL19 ist in S.cerevisiae ein essentielles Gen und hat eine Funktion bei der Reifung der 18S rRNA. YIL19 stellte sich auch in C.albicans als essentiell heraus. Konditionale Mutanten, in denen YIL19 durch induzierbare, FLP-vermittelte Rekombination aus dem Genom deletiert wurde, waren nicht lebensfähig und akkumulierten rRNA Vorstufen. Durch diese Untersuchungen konnte gezeigt werden, dass YIL19 essentiell für diesen wichtigen zellulären Prozess und für die Lebensfähigkeit von C.albicans ist und sich möglicherweise als Ziel für die Entwicklung antifungaler Substanzen eignet. / 1. Summary Candida albicans is an opportunistic human fungal pathogen that causes a variety of infections, ranging from superficial mucosal to deep-seated systemic infections, especially in immunocompromised patients. Although the ability of C.albicans to cause disease largely depends on the immune status of the host, the fungus also exhibits specific characteristics that facilitate colonization, dissemination, and adaptation to different host niches and thereby turn C.albicans from a harmless commensal to an aggressive pathogen. In response to various environmental stimuli C.albicans switches from growth as a budding yeast to invasive filamentous growth, and this morphogenetic switch plays an important role in C.albicans pathogenesis. Nitrogen limitation is one of the signals that induce filamentous growth in C.albicans, and the control of the morphogenetic transition by nitrogen availability was studied in detail in the present work. Ammonium is a preferred nitrogen source for yeasts that is taken up into the cells by specific transporters. It was found in this study that C.albicans possesses two major ammonium transporters, encoded by the CaMEP1 and CaMEP2 genes, expression of which is induced by nitrogen starvation. Whereas mep1 or mep2 single mutants grew as well as the wild-type strain on limiting concentrations of ammonium, deletion of both transporters rendered C.albicans unable to grow at ammonium concentrations below 5 mM. In contrast to mep1 mutants, mep2 mutants failed to filament and grew only in the yeast form under nitrogen starvation conditions, indicating that in addition to its role as an ammonium transporter CaMep2p also has a signaling function in the induction of filamentous growth. CaMep2p was found to be a less efficient ammonium transporter than CaMep1p and to be expressed at much higher levels, a distinguishing feature important for its signaling function. By the construction and analysis of serially truncated versions of CaMep2p, the C-terminal cytoplasmic tail of the protein was shown to be essential for signaling but dispensable for ammonium transport, demonstrating that these two functions of CaMep2p are separable. In C.albicans at least two signal transduction pathways, a MAP kinase cascade and a cAMP-dependent pathway ending in the transcriptional regulators Cph1p and Efg1p, respectively, control filamentous growth, and mutants defective in either one of these pathways are defective for filamentation under nitrogen starvation conditions. A hyperactive CaMEP2 allele rescued the filamentation defect of a cph1 or a efg1 mutant, but not of a cph1 efg1 double mutant or a mutant deleted for RAS1, which acts upstream of and activates both signaling pathways. Conversely, a dominant active RAS1 allele or addition of exogenous cAMP rescued the filamentation defect of mep2 mutants. These results suggest that CaMep2p activates both the MAP kinase and the cAMP pathway in a Ras1p dependent manner to promote filamentous growth under nitrogen starvation conditions. At sufficiently high concentrations, ammonium repressed filamentous growth even when the signaling pathways were artificially activated. Therefore, C.albicans has established a regulatory circuit in which a preferred nitrogen source, ammonium, serves as an inhibitor of morphogenesis that is taken up into the cell by the same transporter that induces filamentous growth in response to nitrogen starvation. Although a detailed understanding of virulence mechanisms of C.albicans may ultimately lead to novel approaches to combat infections caused by this pathogen, the identification and characterization of essential genes as potential targets for the development of antifungal drugs is a strategy favoured by most pharmaceutical companies. Therefore, C.albicans homologs of three genes that are essential in other fungi were selected in collaboration with an industrial partner and functionally characterized in this work. RAP1 encodes the repressor/activator protein 1, a transcription factor and telomere binding protein that is essential for viability in the budding yeast Saccharomyces cerevisiae. However, deletion of the C.albicans RAP1 homolog did not affect viability or growth of the mutants, suggesting that it is not a promising target. CBF1 (centromere binding factor 1) is necessary for proper chromosome segregation and transcriptional activation of methionine biosynthesis genes in S.cerevisiae and is essential for viability in the related yeasts Kluyveromyces lactis and Candida glabrata. Deletion of CBF1 in C.albicans did not result in an increased frequency of chromosome loss, indicating that it has no role in chromosome segregation in this organism. However, the C.albicans cbf1 mutants exhibited severe growth impairment, temperature sensitivity at 42°C, and auxotrophy for sulphur amino acids, suggesting that Cbf1p is a transcription factor that is important for normal growth of C.albicans. YIL19 is an essential gene in S.cerevisiae that is involved in 18S rRNA maturation. YIL19 was found to be an essential gene also in C.albicans. Conditional mutants in which the YIL19 gene could be excised from the genome by inducible, FLP-mediated recombination were non-viable and accumulated rRNA precursors, demonstrating that YIL19 is essential for this important cellular process and for viability of C.albicans and could serve as a target for the development of antifungal drugs.

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: 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

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.

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Gandida albicans strain 105 from a normal human and strain 582 (from the American type Culture Collection) were used for studying the effect in vitro of pH, various amounts of carbon dioxide, nitrogen, and composition of media on filamentation in this yeast-like organism. The yeast phase of the organism was maintained on a glucose, glycine, yeast extract (GGY) medium (1%; 1%; 0.5%) at 37°C. The experiments were conducted on both solid and liquid media. All cultures were incubated at 37°C. for 48 hours. The two strains of c. albicans, although similar to one another in their yeast forms, behaved differently toward the environmental conditions used; strain 582 responded more readily to the factors inducing filament formation than did strain 105. Increasing the pH above 6.5 to 7.0, 7.5 and 8.0 induced maximum filamentation in strain 582, whereas no filaments were produced by strain 105. All the aerobic cultures on solid GGY medium showed alkalinity and were positive for ammonia at the end of the incubation period. In liquid media, no alkalinity was observed at any pH values. Presence of 75% carbon dioxide in the atmosphere increased filamentation in strain 582 to a maximum degree, and induced mycelial formation in strain 105. With 94% or 95% carbon dioxide, growth and filamentation decreased in both strains. None of the CO2 cultures showed alkalinity at the end of the incubation period. Moreover, all the CO2 cultures were negative for ammonia. Growth under nitrogen (9J%) was less than that of the aerobic cultures. However, colonies appeared larger in size. Nitrogen stimulated filamentation in strain 105 only at a pH of 8.0, whereas strain 582 formed a maximum amount of filaments at pH values of 7.0 to 8.0. All the solid cultures under nitrogen showed alkalinity, while the liquid cultures were acid at all pH values. The occurrence of deamination in a medium without glucose in both strains of C. albicans showed that this organism was able to use glycine its source of both nitrogen and carbon. However, only a sparse growth was obtained in a medium lacking glucose. Strain 105 did not form filaments in such a medium, while strain 582 did so. Since more filaments were produced by the latter strain when a fresh subculture on a GGY medium was transferred to a medium without glucose, it was concluded that possibly glucose is required for both growth and filamentation. Comparative studies of the effect of a medium containing mannose with a glucose medium showed the two sugars behaved similarly with regard to fermentation and filament induction in both strains or c. albicans. Under conditions where glucose induced filamentation (e.g., with C02 or N2), mannose also induced filamentation. The decreased growth in the presence of oleic or stearic acid in a concentration of 40 micrograms per liter was attributed to the toxic effect of the fatty acids. Moreover, it was noted that the two acids had different effects on filamentation in the two strains. Oleic acid in a solid GGY medium induced hyphal formation in strain 105 only under nitrogen; without glucose, oleic acid did not bring about filamentation under any of the atmospheric conditions tested. In liquid media, oleic acid induced filamentation for strain 105 only when glucose was omitted. With strain 562, oleic acid promoted filamentation in both liquid and solid media with or without glucose, except for solid cultures incubated under nitrogen in the absence or glucose. Stearic acid did not stimulate filamentation in strain 105 under any conditions, but did increase hypha! formation in strain 582. In the presence of stearic acid, maximum filamentation occurred in aerobic cultures wnen glucose was absent. Although maximum filamentation occurred with an increase in the pH of the medium under aerobic conditions, in the presence of 75% C02, under nitrogen or in the presence of stearic acid in a medium without glucose, yeast cells were also present, indicating that this Y to f transformation was not complete. / 2031-01-01

Orientador: Marlise Inêz Klein / Resumo: O objetivo foi avaliar quais são os mecanismos de Streptococcus mutans e de Candida albicans que contribuem para aumentar a patogenicidade do biofilme misto. Biofilmes mistos e simples das cepas S. mutans UA159 (Sm) e C. albicans SC5314 (Ca) foram formados sobre discos de hidroxiapatita com película salivar, na presença de sacarose. O pH do meio de cultura foi aferido em diversas fases de desenvolvimento do biofilme. Após 43h de crescimento, foram realizadas análises bioquímicas (peso seco, proteinas, composição da matriz extracelular) e de população microbiana. A estrutura dos biofilmes foi avaliada por microscopia confocal em 19 e 43h. A expressão de genes de vias metabólicas de ambas espécies foi verificada em 28h. Os dados foram avaliados por métodos estatísticos considerando α=0,05. Verificou-se diferença do pH do meio para os três biofilmes nos tempos 19, 27 e 43h (p<0,001; ANOVA dois critérios, Tukey). Biofilmes de Sm e misto foram mais ácidos em 19 e 43h, enquanto biofilmes de Ca mantiveram o pH neutro (p>0,05). As quantidades do peso seco e de proteínas de biofilme misto foram maiores comparadas aos biofilmes simples, e menores para Ca (p=0,001; ANOVA um critério). Não houve diferença na quantidade de exopolissacarídeos solúveis entre biofilmes Sm e misto, porém o biofilme Ca apresentou menor quantidade (p<0,001; Kruskal-Wallis, Dunn). Houve maior quantidade de exopolissacarídeos insolúveis em biofilme misto (p=0,002). Verificou-se mesmo comportamento populacional pa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective was to evaluate the mechanisms of Streptococcus mutans and Candida albicans that contribute to increasing the pathogenicity of the mixed-species biofilm. Mixed and single-species biofilms of the strains S. mutans UA159 (Sm) and C. albicans SC5314 (Ca) were formed on saliva-coated hydroxyapatite discs in the presence of sucrose. The pH values of the culture media were verified at distinct developmental phases of biofilms. After 43h of growth, the biofilms were subjected to distinct assays biochemical (dry weight, proteins, the composition of extracellular matrix) and microbiological (colony forming units), analyzes. The biofilm's structure was verified via confocal microscopy at 19 and 43h. Gene expression of metabolic ways from both species was evaluated at 28h. The data were assessed by statistical methods (α=0,05). There was a difference in the media pH for the three biofilms at times 19, 27 and 43h (p<0,001; two-way ANOVA, Tukey). Sm and mixed-species biofilms were acidic at 19 and 43h, while Ca biofilms maintained a neutral pH (p>0,05). The amounts of dry weight and proteins were higher for mixed-species biofilm compared to singlespecies biofilms, being lower for Ca (p=0,001; one-way ANOVA). The quantity of soluble exopolysaccharides was similar for Sm and mixed-species biofilms but Ca presented a lower amount than those biofilms (p<0,001; Kruskal-Wallis, Dunn). There was a higher amount of insoluble exopolysaccharides in mixed-species biofilm (p=0,002). There was no difference in Sm population in single- and mixed-species biofilms (p=0,404; Mann Whitney); however, the mixed-species biofilm presents a higher population of Ca versus the single-species biofilm (p<0,001; t-Test). The threedimensional structure analysis showed larger microcolonies in mixed-species biofilms versus Sm biofilm, and absence of these structures in Ca biofilm...(Complete abstract electronic access below) / Mestre