Invasive fungal infections are a significant clinical challenge especially for hospitalized patients as traditional antifungal therapy often fails to resolve these infections. The ability of Candida to undergo yeast-to-hyphae morphological transition is central to this invasive behavior. Morphogenesis is also important for the formation of biofilms which are highly structured communities of microorganisms attached to one another or substratum and embedded within a protective extracellular matrix material. The refractory nature of cells within a biofilm to current antifungal therapies has created a need for alternative antifungal agents for the management of Candida biofilm-related infections. The novel antifungal occidiofungin is a natural product produced by the soil bacteria Burkholderia contaminans shown to be effective against a broad range of fungi including Candida spp. Prior studies have demonstrated that occidiofungin inhibits yeast-to-hyphae morphogenesis in the dimorphic yeast, C. albicans, likely through its impact on disrupting F-actin organization. To extend these findings, the efficacy of occidiofungin on morphogenesis of C. albicans and C. tropicalis strains under different inducing conditions was evaluated. Further, given the role of biofilm on pathogenicity, the anti-biofilm properties of occidiofungin against Candida species was examined using an in vitro static biofilm model developed on a silicon elastomer disk. The accumulated data indicate that occidiofungin inhibits hyphal transformation regardless of the inducing conditions used and prevents hyphal extension when added to cells post switching. Moreover, morphologically switching cells were more sensitive to occidiofungin than their yeast counterpart. In addition, occidiofungin effectively blocks biofilm formation at all stages of development and reduces dispersed cells from the biofilm for both C. albicans and C. tropicalis. Confocal data revealed alterations in actin organization with occidiofungin treatment for both morphologically switching and biofilm cells. These findings correlate with prior observations for occidiofungin activity on yeast form cells indicating the broad activity of occidiofungin against fungi at various stages of pathogenic growth and supports efforts to pursue occidiofungin as a potential therapeutic against Candida based infections.

Antimycotics such as fluconazole are frequently used to treat C. albicans infections of the oral mucosa. Prolonged treatment of the fungal infection with fluconazole pose a risk to resistance development. C. albicans can adapt to these stressful environmental changes by regulation of gene expression or by producing genetically altered variants that arise in the population. Adapted variants frequently carry activating mutations in zinc cluster transcription factors, which cause the upregulation of their target genes, including genes encoding efflux pumps that confer drug resistance. MDR1, regulated by the zinc cluster transcription factor Mrr1, as well as CDR1 and CDR2, regulated by the zinc cluster transcription factor Tac1, are well-known examples of genes encoding efflux pumps that extrude the antimycotic fluconazole from the fungal cell and thus contribute to the survival of the fungus. In this study, it was investigated if C. albicans can develop resistance to the antimicrobial peptide histatin 5, which serves as the first line of defence in the oral cavity of the human host. Recently, it was shown that C. albicans transports histatin 5 outside of the Candia cell via the efflux pump Flu1. As efflux pumps are often regulated by zinc cluster transcription factors, the Flu1 efflux pump could also be regulated by a zinc cluster transcription factor which could in a hyperactive form upregulate the expression of the efflux pump, resulting in increased export of histatin 5 and consequently in histatin 5 resistance. In order to find a zinc cluster transcription factor that upregulates FLU1 expression, a comprehensive library of C. albicans strains containing artificially activated forms of zinc cluster transcription factors was screened for suitable candidates. The screening was conducted on medium containing mycophenolic acid because mycophenolic acid is also a substrate of Flu1 and a strain expressing a hyperactive zinc cluster transcription factor that upregulates FLU1 expression should exhibit an easily recognisable mycophenolic acid-resistant phenotype. Further, FACS analysis, quantitative real-time RT-PCR analysis, broth microdilution assays as well as histatin 5 assays were conducted to analyse the mechanism and the regulation of histatin 5 resistance. Several zinc cluster transcription factors caused mycophenolic acid resistance and upregulated FLU1 expression. Of those, only hyperactive Mrr1 was able to confer increased histatin 5 resistance. Finding Mrr1 to confer histatin 5 resistance was highly interesting as fluconazole-resistant strains with naturally occurring Mrr1 gain of function mutations exist, which were isolated from HIV-infected patients with oral candidiasis. These Mrr1 gain of function mutations as well as artificially activated Mrr1 cause fluconazole resistance by upregulation of the efflux pump MDR1 and other target genes. In the course of the study, it was found that expression of different naturally occurring MRR1 gain-of-function mutations in the SC5314 wild type background caused increased FLU1 expression and increased histatin 5 resistance. The same was true for fluconazole-resistant clinical isolates with Mrr1 gain of function mutations, which also caused the overexpression of FLU1. Those cells were less efficiently killed by histatin 5 dependent on Mrr1. Surprisingly, FLU1 contributed only little to histatin 5 resistance, rather, overexpression of MDR1 mainly contributed to the Mrr1-mediated histatin 5 resistance, but also additional Mrr1-target genes were involved. These target genes are yet to be uncovered. Moreover, if a link between the yet unknown Mrr1-target genes contributing to fluconazole resistance and increased histatin 5 resistance can be drawn remains to be discovered upon finding of the responsible target genes. Collectively, this study contributes to the understanding of the impact of prolonged antifungal exposure on the interaction between host and fungus. Drug therapy can give rise to resistance evolution resulting in strains that have not only developed resistance to fluconazole but also to an innate host mechanism, which allows adaption to the host niche even in the absence of the drug. / Antimykotika wie Fluconazol werden häufig zur Behandlung von C. albicans Infektionen der Mundschleimhaut verwendet. Dabei stellt eine langzeitige Behandlung der Pilzinfektion mit Fluconazol ein Risiko zur Resistenzentwicklung dar. C. albicans kann sich an solche Umweltveränderungen anpassen, indem es die Genexpression reguliert oder genetisch veränderte Varianten produziert, welche in der Population entstehen. Adaptierte Varianten tragen häufig aktivierende Mutationen in Zink-Cluster-Transkriptionsfaktoren, welche die Hochregulierung der Expression von Genen verursachen, darunter solche, die für Multidrug-Effluxpumpen kodieren und dadurch Antimykotikaresistenz verleihen können. MDR1, reguliert durch den Zink-Cluster-Transkriptionsfaktor Mrr1, sowie CDR1 und CDR2, reguliert durch den Zink-Cluster-Transkriptionsfaktor Tac1, sind bekannte Beispiele für Effluxpumpen, die das Antimykotikum Fluconazol aus der Pilzzelle extrudieren und somit zum Überleben der Pilzzelle beitragen. In dieser Arbeit wurde untersucht, ob C. albicans eine Resistenz gegen das antimikrobielle Peptid Histatin 5 entwickeln kann, das in der Mundhöhle des menschlichen Wirtes als erste Verteidigungsbarriere gegen den Pilz dient. Kürzlich wurde gezeigt, dass C. albicans Histatin 5 über die Effluxpumpe Flu1 aus der Candia-Zelle heraustransportiert (Li et al., 2013). Da Effluxpumpen häufig durch Zink-Cluster-Transkriptionsfaktoren reguliert werden, könnte auch die Flu1-Effluxpumpe durch solch einen Transkriptionsfaktor reguliert werden, der in einer hyperaktiven Form die Expression der Effluxpumpe hochregulieren könnte, was wiederrum zu einem erhöhten Export von Histatin 5 und folglich zur Histatin 5 Resistenz führen könnte. Um einen Zink-Cluster-Transkriptionsfaktor zu finden, der die FLU1-Expression hochreguliert, wurde mit Hilfe einer Bibliothek von C. albicans-Stämmen, die künstlich aktivierte Formen von Zink-Cluster-Transkriptionsfaktoren enthält, nach geeigneten Kandidaten gesucht. Das Screening wurde auf Mycophenolsäure-haltigem Medium durchgeführt, da Mycophenolsäure ebenfalls ein Substrat von Flu1 ist. Folglich sollte ein Stamm mit hyperaktivem Zink-Cluster-Transkriptionsfaktor, welcher die FLU1-Expression hochreguliert, einen leicht erkennbaren Mycophenolsäure-resistenten Phänotyp aufweisen. Weiterhin wurden FACS-Analysen, quantitative real-time RT-PCR-Analysen, Broth microdilution-Assays sowie Histatin 5-Assays durchgeführt, um den Mechanismus und die Regulierung der Histatin-5-Resistenz zu analysieren. Mehrere Zink-Cluster-Transkriptionsfaktoren verursachten Mycophenolsäure-Resistenz und erhöhten die FLU1-Expression. Von diesen war nur hyperaktives Mrr1 in der Lage, eine erhöhte Histatin-5-Resistenz zu verleihen. Das Auffinden von Mrr1 als Regulator der Histatin 5-Resistenz war hochinteressant, da fluconazolresistente Stämme mit natürlich vorkommenden MRR1 gain-of-function Mutationen existieren, die aus HIV-infizierten Patienten mit oropharyngealer Candidiasis isoliert wurden. Diese gain-of-function Mutationen sowie künstlich aktivierendes Mrr1 verursachen Fluconazol-Resistenz durch Hochregulation der Effluxpumpe MDR1 und anderer Zielgene. Im Verlauf der Studie wurde herausgefunden, dass verschiedene natürlich vorkommende MRR1 gain-of-function Mutationen im SC5314 Wildtyp Hintergrund eine erhöhte FLU1-Expression und eine erhöhte Histatin-5-Resistenz verursachten. Das Gleiche galt für Fluconazol-resistente klinische Isolate mit Mrr1 gain-of-function Mutationen, welche die Überexpression von FLU1 verursachten. Zellen dieser Isolate wurden, abhängig von Mrr1, weniger wirksam durch Histatin 5 abgetötet. Überraschenderweise trug FLU1 nur wenig zur Histatin-5-Resistenz bei, vielmehr trug die Überexpression von MDR1 hauptsächlich zur Mrr1-vermittelten Histatin-5-Resistenz bei, aber auch weitere Mrr1-Zielgene waren daran beteiligt. Diese Mrr1-Zielgene gilt es nun noch zu entdecken. Ob ein Zusammenhang zwischen diesen noch unbekannten Mrr1-Zielgenen hergestellt werden kann, die zur Fluconazolresistenz sowie zu einer erhöhten Histatin-5-Resistenz beitragen, wird erst nach dem Auffinden der verantwortlichen Zielgene geprüft werden können. Zusammenfassend trägt diese Studie zum Verständnis der Auswirkungen einer anhaltenden antimykotischen Exposition auf die Interaktion zwischen Wirt und Pilz bei. Eine medikamentöse Therapie kann zu einer Resistenzentwicklung führen, aus der Stämme hervorgehen, welche nicht nur eine Resistenz gegen Fluconazol entwickelt haben, sondern gleichzeitig eine Resistenz gegen einen angeborenen Wirtsabwehrmechanismus, der eine Adaption an die Wirtsnische auch in Abwesenheit des Antimykotikums ermöglicht.

Infections with the opportunistic yeast Candida albicans are frequently treated with the first-line drug fluconazole, which inhibits ergosterol biosynthesis. An alarming problem in clinics is the development of resistances against this azole, especially during long-term treatment of patients. Well-known resistance mechanisms include mutations in the zinc cluster transcription factors (ZnTFs) Mrr1 and Tac1, which cause an overexpression of efflux pump genes, and Upc2, which results in an overexpression of the drug target. C. albicans strains with such gain-of-function mutations (GOF) have an increased drug resistance conferring a selective advantage in the presence of the drug. It was previously shown that this advantage comes with a fitness defect in the absence of the drug. This was observed in different conditions and is presumably caused by a deregulated gene expression. One aim of the present study was to examine whether C. albicans can overcome the costs of drug resistance by further evolution. Therefore, the relative fitness of clinical isolates with one or a combination of different resistance mutations in Mrr1, Tac1 and/or Upc2 was analyzed in competition with the matched fluconazole-susceptible partner. Most fluconazole-resistant isolates had a decreased fitness in competition with their susceptible partner in vitro in rich medium. In contrast, three fluconazole-resistant strains with Mrr1 resistance mutations did not show a fitness defect in competition with their susceptible partner. In addition, the fitness of four selected clinical isolate pairs was examined in vivo in mouse models of gastrointestinal colonization (GI) and disseminated infection (IV). In the GI model all four fluconazole-resistant strains were outcompeted by their respective susceptible partner. In contrast, in the IV model only one out of four fluconazole-resistant isolates did show a slight fitness defect in competition with its susceptible partner during infection of the kidneys. It can be stated, that in the present work the in vitro fitness did not reflect the in vivo fitness and that the overall fitness was dependent on the tested conditions. In conclusion, C. albicans cannot easily overcome the costs of drug resistance caused by a deregulated gene expression. In addition to GOFs in Mrr1, Tac1 and Upc2, resistance mutations in the drug target Erg11 are a further key fluconazole resistance mechanism of C. albicans. Clinical isolates often harbor several resistance mechanisms, as the fluconazole resistance level is further increased in strains with a combination of different resistance mutations. In this regard, the question arises of how strains with multiple resistance mechanisms evolve. One possibility is that strains acquire mutations successively. In the present study it was examined whether highly drug-resistant C. albicans strains with multiple resistance mechanisms can evolve by parasexual recombination as another possibility. In a clonal population, cells with individually acquired resistance mutations could combine these advantageous traits by mating. Thereupon selection could act on the mating progeny resulting in even better adapted derivatives. Therefore, strains heterozygous for a resistance mutation and the mating type locus (MTL) were grown in the presence of fluconazole. Derivatives were isolated, which had become homozygous for the resistance mutation and at the same time for the MTL. This loss of heterozygosity was accompanied by increased drug resistance. In general, strains which are homozygous for one of both MTL configurations (MTLa and MTLα) can switch to the opaque phenotype, which is the mating-competent form of the yeast, and mate with cells of the opposite MTL. In the following, MTLa and MTLα homozygous strains in the opaque phenotype were mated in all possible combinations. The resulting mating products with combined genetic material from both parents did not show an increased drug resistance. Selected products of each mating cross were passaged with stepwise increasing concentrations of fluconazole. The isolated progeny showed high levels of drug resistance and loss of wild-type alleles of resistance-associated genes. In conclusion, selective pressure caused by fluconazole exposure selects for resistance mutations and at the same time induces genomic rearrangements, resulting in mating competence. Therefore, in a clonal population, cells with individually acquired resistance mutations can mate with each other and generate mating products with combined genetic backgrounds. Selection can act on these mating products and highly drug-resistant und thus highly adapted derivatives can evolve as a result. In summary, the present study contributes to the current understanding of the evolution of antifungal drug resistance by elucidating the effect of resistance mutations on the fitness of the strains in the absence of the drug selection pressure and investigates how highly drug-resistant strains could evolve within a mammalian host. / Infektionen mit dem opportunistischen Hefepilz Candida albicans werden häufig mit dem First-Line-Medikament Fluconazol behandelt, welches die Ergosterol-Biosynthese hemmt. Ein besorgniserregendes Problem in der Klinik, insbesondere bei der Langzeitbehandlung von Patienten, ist die Entwicklung von Resistenzen gegen dieses Azol. Zu den bekannten Resistenzmechanismen gehören Resistenzmutationen in den Zink-Cluster-Transkriptionsfaktoren (ZnTFs) Mrr1 und Tac1, die eine Überexpression von Effluxpumpen-Genen bewirken und Resistenzmutationen in Upc2, die zu einer Überexpression des Wirkstofftargets führen. C. albicans Stämme mit solchen Gain-of-Function-Mutationen (GOF) weisen eine erhöhte Medikamentenresistenz auf, was einen selektiven Vorteil in Gegenwart des Medikaments bedeutet. Es wurde zuvor gezeigt, dass dieser Vorteil mit einem Fitnessdefekt in Abwesenheit des Medikaments einhergeht. Dies wurde in verschiedenen Bedingungen nachgewiesen und wird vermutlich durch eine deregulierte Genexpression verursacht. Ein Ziel der vorliegenden Studie war es zu untersuchen, ob C. albicans die Kosten der Medikamentenresistenz durch Evolution kompensieren kann. Daher wurde die relative Fitness von klinischen Isolaten mit einer oder einer Kombination verschiedener Resistenzmutationen in Mrr1, Tac1 und/oder Upc2 im Wettbewerb mit dem zugehörigen Fluconazol-sensitiven Partner analysiert. Die meisten Fluconazol-resistenten Isolate hatten eine verminderte Fitness im Wettbewerb mit ihrem sensitiven Partner in vitro in vollwertigem Medium. Dennoch zeigten drei Fluconazol-resistente Stämme mit Mrr1-Resistenzmutationen keinen Fitnessdefekt im Wettbewerb mit ihrem jeweiligen Partner. Zusätzlich wurde die Fitness von vier ausgewählten klinischen Isolat-Paaren in vivo in Mausmodellen für gastrointestinale Kolonisation (GI) und disseminierte Infektion (IV) untersucht. Im GI-Modell wurden alle vier Fluconazol-resistenten Stämme von ihren sensitiven Partnern überwachsen. Im Gegensatz dazu zeigte im IV-Modell nur einer der vier Fluconazol-resistenten Isolate einen leichten Fitnessdefekt im Wettbewerb mit dem jeweiligen Fluconazol-sensitiven Partner während der Infektion der Nieren. Es kann festgestellt werden, dass in der vorliegenden Arbeit die in vitro-Fitness nicht die in vivo-Fitness widerspiegelt und dass die Gesamtfitness von den getesteten Bedingungen abhängig ist. Zusammenfassend lässt sich sagen, dass C. albicans die Kosten der Medikamentenresistenz, die durch eine deregulierte Genexpression verursacht werden, nur schwer überwinden kann. Neben GOFs in Mrr1, Tac1 und Upc2 sind Resistenzmutationen im Wirkstofftarget Erg11 ein wichtiger Resistenzmechanismus von C. albicans. Klinische Isolate weißen oft mehrere Resistenzmechanismen auf, da die Kombination verschiedener Resistenzmutationen die Fluconazol-Resistenz potenziert. In diesem Zusammenhang stellt sich die Frage, wie sich Stämme mit mehreren Resistenzmechanismen entwickeln. Eine Möglichkeit ist, dass Stämme Mutationen sequenziell erwerben. In der vorliegenden Studie wurde untersucht, ob als weitere Möglichkeit hochresistente C. albicans Stämme mit multiplen Resistenzmechanismen durch parasexuelle Rekombination evolvieren können. In einer klonalen Population könnten Zellen mit individuell erworbenen Resistenzmutationen diese vorteilhaften Eigenschaften durch Paarung kombinieren. Daraufhin könnte Selektionsdruck auf die Matingprodukte wirken und so die Entstehung von besser angepassten Derivaten begünstigen. Daher wurden Resistenzmutation und Mating Type Locus (MTL) heterozygote Stämme in Gegenwart von Fluconazol kultiviert. So konnten Derivate isoliert werden, die homozygot für die Resistenzmutation und gleichzeitig für den MTL geworden waren. Dieser Verlust der Heterozygotie ging mit einer erhöhten Medikamentenresistenz einher. Generell können Stämme, die homozygot für eine der beiden MTL-Konfigurationen (MTLa und MTLα) sind, in den opaque Phänotyp wechseln, der die paarungskompetente Form der Hefe darstellt, und sich mit Zellen des gegensätzlichen MTL paaren. Im Folgenden wurden MTLa und MTLα homozygote Stämme im opaque Phänotyp in allen möglichen Kombinationen verpaart. Die resultierenden Matingprodukte mit kombiniertem genetischem Material beider Elternteile wiesen keine erhöhte Medikamentenresistenz auf. Ausgewählte Paarungsprodukte jeder Kreuzung wurden mit stufenweise ansteigenden Konzentrationen von Fluconazol passagiert. Die isolierten Nachkommen zeigten ein hohes Maß an Medikamentenresistenz und den Verlust von Wildtyp-Allelen der resistenzassoziierten Gene. Zusammenfassend lässt sich sagen, dass der selektive Druck, der durch die Fluconazol-Exposition verursacht wird, für Resistenzmutationen selektiert und gleichzeitig genomische Umlagerungen induziert, die eine Paarung ermöglichen. Daher können sich in einer klonalen Population Zellen mit individuell erworbenen Resistenzmutationen miteinander paaren und Matingprodukte mit kombiniertem genetischem Hintergrund generieren. Auf diese Matingprodukte kann die Selektion wirken, woraufhin sich hochresistente und damit stark an ihre Umwelt angepasste Derivate entwickeln können. Zusammenfassend trägt die vorliegende Studie zum aktuellen Verständnis der Evolution der Antimykotika-Resistenz bei, indem sie den Effekt von Resistenzmutationen auf die Fitness der Stämme in Abwesenheit des Medikamenten-Selektionsdrucks untersucht und aufklärt, wie sich hochgradig resistente Stämme in einem Säugetierwirt entwickeln könnten.

Polymicrobial communities of bacterial and fungal species are present on the skin and mucosal surfaces of the body. Invasive infections caused by Candida species are commonly seen in immunocompromised individuals (HIV, transplants, cancer) and ranks as the third leading cause of infection in hospitalized patients. C. albicans is a polymorphic opportunistic fungus that infects critically ill patients and has the ability to change its morphology from yeast to hyphal form.The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.In this study, we examined two transporter mutants, cdr4 and ssu1, for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. To quantify biomass, wild type and mutant cells were grown overnight at 30˚C in YPD. The cells were washed, counted and diluted to a desired density of 106 cells/ml in medium 199, pH7.5. Cells were added to 96-well plates pre-incubated with 5% fetal bovine serum at densities of 105, 104, and 103 cells/well and allowed to adhere at 37˚C for one hour. The wells were then covered with fresh M199 media containing the QS molecule and monitored for 48 hours at 37˚C. After this time, the media and planktonic cells were removed. The biofilms were fixed with methanol, dried, then stained with 0.05% crystal violet. Bulk biomass was assessed by spectrophotometry. We did observe a difference in biofilm density when incubated in the presence of cyclic-diGMP. We noted that for the wild type and ssu1 strain their biofilms biomass increased by as much as 10% at 104 and 103 cell densities when compared with the control. While the cdr4 strain had a slight reduction in biofilm density when cyclic-diGMP was present. This result also indicates a potentially positive role in which cyclic-diGMP can help C. albicans develop denser biofilms, potentially in the presence of bacteria like P. aeruginsa, which secrete cyclic di-GMP, but kill hyphal forms of C. albicans. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1p, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.Given that mutations in CDR4 and SSU1 impact the QS response in C. albicans, we investigated whether these genes were regulated through two-component signaling by Chk1. To assess CDR4 and SSU1 expression, wild type and mutant strains were grown overnight in YPD media at 30˚C. Cells were then harvested and RNA was obtained by acid phenol extraction. Using RT-PCR, we determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. These results suggest the two genes are downstream targets in a pathway regulated by Chk1p. The finding that QS proceeds through a two-component pathway can be exploited in antifungal drug development. Given that two-component signaling is absent in mammalian cells, development of novel compounds that interfere with this pathway may be a useful alternative for treating patients with candidiasis.

Bacteria and fungi have acquired the ability to interact and survive in many hostile environments both found in nature, as well as, the human body. Candida albicans, an opportunistic fungal pathogen, causes a variety of infections in immunocompromised or immunosuppressed individuals, but also asymptomatically colonizes 80% of the population within the intestinal tract, oral cavity, as well as, the female genitourinary system. A unique capability of C. albicans is the ability to change its morphology from benign circular yeast form, to oval pseudohyphal form, to cylindrical tissue-penetrating hyphal form. Our laboratory has previously identified a bacterium, Alcaligenes faecalis, which displays inhibitory characteristics towards C. albicans. A review of the literature shows that some bacteria have the ability to inhibit C. albicans, but only when in the hyphal form. We therefore wanted to explore if the morphological state of C. albicans dictated the degree of inhibition A. faecalis is able to exude, or simply “does morphology matter?” To determine this effect, a series of both solid and liquid media experiments were performed using a wild-type (able to convert between morphologies) strain of C. albicans, a mutant strain of C. albicans locked into the yeast morphological state, and a mutant strain of C. ablicans locked into the hyphal morphological state. For solid media experiments, the different strains of C. albicans were made into a lawn on agar plates, A. faecalis was spotted onto the lawns, and, after 24 hours, observed for signs of inhibition. For liquid media experiments, C. albicans strains were inoculated alone or co-cultured with A. faecalis for 24 hours and plated to enumerate colony forming units. Our results indicate that: (1) the morphological state of C. albicans is not a determining factor, which is a unique finding compared to other published reports; (2) Both A. faecalis and the closely related A. viscolactis both inhibit C. albicans showing that this is a shared ability among the Alcaligenes genus. (3) that the ability to inhibit C. albicans is thru some form of contact dependent mechanism, as the cell free supernatant of A. faecalis has no inhibitory action. Currently, the exact mechanism for this interaction is unknown, but could be one of the secretion systems bacteria use for interactions with other microbes. As there are very limited treatments for fungal infections and severe side-effects associated with current antifungals, exploiting these mechanisms are medically relevant to human health as they could potentially lead to novel treatments for problematic human fungal pathogens.

No description available.

<p dir="ltr">The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. In general, few conserved functions of Cdc14 phosphatase have been defined. Here, I present mechanistic biochemical and phenotypic characterization of Cdc14 phosphatases in fungi. I have demonstrated that fungal Cdc14 phosphatases possess an invariant motif in the disordered C-terminal tail that is required for full enzymatic activity. This motif, termed substrate-like catalytic enhancer (SLiCE), functions during the rate-limiting step of Cdc14-directed catalysis, by binding to the active site and supporting phospho-enzyme hydrolysis. Adjacent to the SLiCE motif exists a conserved minimal Cdk consensus motif that likely serves a regulatory function as phosphorylation of this site inhibits Cdc14 activity in vitro. Vertebrate Cdc14 enzymes also possess a distinct, but mechanistically similar SLiCE motif, which may be the first described biochemical difference between Cdc14 enzymes. Moreover, the vertebrate SLiCE motif lacks an adjacent Cdk consensus motif, which may point to differences in how Cdc14 activity is regulated in higher eukaryotes.</p><p dir="ltr">Mutation of this motif in vivo served as a tool to discover biological processes that require high Cdc14 activity. In S. cerevisiae strains expressing this hypomorphic mutant allele (cdc14hm), I discovered a novel sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. This sensitivity was also observed in the distantly related fungi Schizosaccharomyces pombe deletion strain and the human fungal pathogen Candida albicans hypomorphic and deletion strains, suggesting that this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. I also revealed that high Cdc14 activity is required for C. albicans ability to develop hyphae, which is an important virulence trait. This led to our determination that high Cdc14 activity is critical for virulence in two animal models of invasive candidiasis. Together, these results argue that Cdc14 would be an excellent antifungal drug target for the treatment of invasive Candida infections and sensitization to existing antifungal drugs.</p><p dir="ltr">Lastly, I implemented the auxin-inducible degradation system in C. albicans. Using this system, we were able to deplete Cdc14 and other target protein levels to >95% within minutes. Depletion of Cdc14 was robust enough to phenocopy gene deletions, confirming previous results and demonstrating the utility of rapid target protein inactivation. This system will serve as a powerful tool for future functional characterization of Cdc14 in C. albicans and other pathogenic fungal species.</p>

No description available.

No description available.

Polymicrobial interactions are an important, yet understudied area of research. Candida albicans is the most common human fungal pathogen. The bacterial genus, Enterobacter, is a source of nosocomial acquired infections and increased drug resistance. Our lab has previously discovered that Enterobacter preferentially adheres to C. albicans hyphae. From an E. cloacae transposon library screen, six candidates displayed reduction in C. albicans attachment. These candidates were identified genetically and characterized for involvement in attachment to C. albicans. A fluorescent plasmid was introduced into E. cloacae to measure and observe adherence to C. albicans in planktonic and biofilm growth. In vivo experiments using Caenorhabditis elegans showed no significant differences in microbial burden or nematode survivability exposed to Candida and Enterobacter. Candida-Enterobacter co-infections were observed microscopically within C. elegans. This study highlights the complex dynamics of C. albicans-E. cloacae interactions, underscoring the importance of understanding polymicrobial relationships in research and clinical settings.