Candida albicans is a common opportunistic fungal pathogens of humans. As such, it faces challenges unique to host-dwelling organisms and has evolved mechanisms that allow it to thrive in the constantly changing environment of the human host. In this dissertation we sought to address two such mechanisms of C. albicans biology.

C. albicans can undergo phenotypic switching between two heritable states: white and opaque. This phenotypic plasticity facilitates its colonization in distinct host niches. The master regulator WOR1 is exclusively expressed in opaque phase cells. Positive feedback regulation by Wor1 on the WOR1 promoter is essential for opaque formation, however the underlying mechanism of how Wor1 functions is not clear. In Chapter 3, we use tandem affinity purification coupled with mass spectrometry to identify Wor1-interacting proteins. Tup1 and its associated complex proteins are found as the major factors associated with Wor1. Tup1 occupies the same regions of the WOR1 promoter as Wor1 preferentially in opaque cells. Loss of Tup1 is sufficient to induce the opaque phase, even in the absence of Wor1. This is the first such report of a bypass of Wor1 in opaque formation. These genetic analyses suggest that Tup1 is a key repressor of the opaque state and Wor1 functions to alleviate Tup1 repression at the WOR1 promoter. Opaque cells convert to white en masse at 37°C. However, we find this conversion occurs only in the presence of glycolytic carbon sources, and the opaque state is stabilized when cells are cultured on non-glycolytic carbon sources, even in an MTLa/α background. We further show that temperature and carbon source affect opaque stability by altering the levels of Wor1 and Tup1 at the WOR1 promoter. We propose that Wor1 and Tup1 form the core regulatory circuit controlling the opaque transcriptional program. This model provides molecular insights on how C. albicans adapts to different host signals to undergo phenotypic switching for colonization in distinct host niches.

Iron is an essential molecule involved in a myriad of biological processes. Despite its essential role as a cofactor, excess iron can become toxic through the generation of reactive oxygen species. As a fungal pathogen of humans, C. albicans is subject to a wide range of iron levels encountered in the human host. Consequently, iron homeostasis is essential for survival and is tightly controlled by a regulatory circuit. Glutaredoxins are a conserved family of proteins involved in maintaining cellular redox homeostasis as well as the biogenesis of iron-sulfur clusters, cofactors linked to diverse biological processes including metabolism, DNA maintenance, transcriptional regulation, and protein translation. In Chapter 4, using a combination of functional genetics, molecular biology, and cross-linked tandem affinity purification coupled with mass spectrometry, we assess the function of the conserved monothiol glutaredoxin Grx3. We examined the function of four identified C. albicans glutaredoxins in response to oxidative and iron stresses and found a grx3 mutant to be sensitive to iron level. The grx3 mutant was defective in the expression of the iron-regulatory circuit genes Sfu1, Sef1, and Hap43 in response to iron status. We determined that Grx3 interacts with Sfu1 and regulates its occupancy at SEF1 promoter. Grx3 was also found to contribute to Sef1 nuclear localization and Hap43 activity. Therefore, Grx3 directly controls the activity of the iron homeostasis regulatory circuit. Identification of Grx3 interacting proteins by mass spectrometry uncovered proteins enriched for several functional categories, including those involved in various metabolic and biosynthetic pathways such as redox homeostasis, amino acid and nucleotide metabolism, protein translation, tRNA aminoacylation DNA maintenance and repair, iron-sulfur biogenesis, and iron homeostasis. We validate some of these findings and show that the grx3 mutant is hypersensitive to oxidative, nitrosative and genotoxic stresses and shows decreased translational efficiency compared to wild-type. Finally, we show the grx3 mutant displays decreased virulence in a disseminated infection model. Therefore, we propose C. albicans Grx3 is a global iron sensor critical for both the regulation of the iron homeostasis circuit as well as the functions of iron-sulfur cluster containing proteins involved in a wide array of diverse biological processes.

The aim of the studies presented in this dissertation is to further our knowledge on the pathways responsible for responding to the effects of external pH in Candida albicans. For the majority of this work, we focused on identifying novel genetic pathways responsible for acidic pH repression of the initiation of hyphal morphogenesis, and provided some preliminary studies on pH effects on the C. albicans cell wall.

Chapter 1 presents an introductory overview on the current knowledge of hyphal growth, and how it is regulated by a variety of environmental conditions encountered by the fungus.

In Chapter 2, we showed that translation is important for effective hyphal growth, and inhibition of translation genetically or chemically delays hyphal initiation.

The study in Chapter 3 identified several genes that are responsible for acidic pH repression of hyphal initiation. We focused on the Hog1 stress response pathway and identified Hog1 activity as a requirement for suppressing hyphal initiation in acidic pH and in the presence of stress. We observed that acidic pH (~4) regulates Hog1 by stabilizing Hog1 phosphorylation, different from stress stimulation of Hog1, by suppressing the expression of the tyrosine phosphatase ptp3. Furthermore, stresses that activate Hog1 also reveal an impaired ability to initiate hyphal growth, supporting the role of Hog1 in repressing hyphal initiation. This study also identified Sfl1 as a key hyphal repressor that functions down-stream of the Hog1 pathway. The Hog1-Sfl1 pathway is activated in response to stress parallel to the Nrg1 repressor. Overall, we have identified a new genetic interaction and regulation that’s necessary for hyphal regulation.

In Chapter 4, we present preliminary data showing that β-Glucans are exposed in acidic pH but live cells are able to protect themselves from that exposure. We have experimental results that exclude the Hog1 and the Cek1MAPK pathways in mediating this protection. Therefore, we propose a genetic screen to identify mutants that are defective in their protection of β-Glucans

Candida albicans, a significant human fungal pathogen, switches between two phenotypic states, white and opaque. The white-opaque switch is a unique and tractable system for the study of epigenetic regulation of gene expression. Switching is controlled by a transcriptional regulatory network of interlocking feedback loops on the master regulator WOR1. Translational regulation of the network, as well as chromatin-level regulation of WOR1, have not been extensively explored. This dissertation investigates the means through which WOR1 expression and the white-opaque phenotype are epigenetically regulated, and identifies several novel mechanisms.

The long 5' untranslated region of WOR1 was identified as a translational regulator of the white-opaque transition. Deletion of the WOR1 5'UTR promoted white-opaque switching and stabilized opaque state. Transcripts containing the WOR1 5'UTR show reduced translational efficiency and lower polysome association, and this repressive effect may exist for additional key regulators of cell fate and morphology with long 5'UTR.

The role of the histone variant H2A.Z in chromatin-level epigenetic regulation of switching was investigated. Deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin, promoted white-opaque switching. Deletion of YNG2, a subunit of NuA4 histone acetyltransferase complex that targets SWR1 activity, produced a similar switching phenotype. Through nucleosome mapping, SWR1 was demonstrated to be required for proper nucleosome positioning and stability on the WOR1 promoter, with effects differing in white and opaque cells. Furthermore, H2A.Z was enriched at the WOR1 promoter in white cells, suggesting stabilization of a repressive chromatin state. An interaction was discovered between swr1 and elevated H3K56ac resulting in a severe synthetic growth defect on nicotinamide, as well as a synergistic effect on white-opaque switching, suggesting altered nucleosome turnover from this interaction.

Finally, the role of the long promoter of WOR1 was investigated. Truncation of the promoter to a 3 kb region, while sufficient to drive opaque-specific gene expression, abrogated switching to opaque, pointing to cis-regulatory elements further upstream. The studies described in this dissertation identify novel translational as well as nucleosome-level regulations of WOR1 expression and white-opaque switching, with further implications for epigenetic regulation.

Candida albicans is a major opportunistic pathogen of humans. In immunocompromised patients, C. albicans becomes pathogenic and can cause Oropharyngeal candidiasis, Vulvovaginal candidiasis and Invasive candidiasis. The mortality rate is up to 40% even with anti-fungal drug treatment. Thus, it is critical to study how the immune system defends against C. albicans infection, and how C. albicans offsets the growth inhibitory effect of the anti-fungal drugs.C. albicans can grow as yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The innate immune response is critical for host defense. However, the relationship between morphogenesis and host immune recognition is not fully elucidated. Dectin-1 is a major C-type lectin receptor that recognizes β-glucan within the fungal cell wall. C. albicans β-glucan is protected by the outer mannan layer of the cell wall. Whether and how β-glucan protection is differentially regulated during hyphal morphogenesis is not fully understood. In Chapter 3, we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with the Yeast Wall Protein Ywp1, controls β-glucan exposure and Dectin-1-dependent immune activation in yeast cells. An eng1 deletion mutant shows enhanced Dectin-1 binding at the septa of yeast cells, while an eng1 ywp1 double mutant, but not an ywp1 single mutant, shows strong overall Dectin-1 binding. Our data suggest that repression of ENG1 and YWP1 expression during hyphal initiation leads to β-glucan exposure. ENG1 expression is also highly regulated and is responsible for β-glucan protection and immune recognition in response to different carbon sources and antifungal drugs. The eng1 mutant modulates virulence in the model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. Mice infected with the eng1 mutant showed delayed mortality compared to male mice infected with the wild-type strain, but earlier mortality in females correlated with a higher renal immune response and lower fungal burden. Thus, Eng1-regulated β-glucan exposure is important for modulating the balance between immune protection and immunopathogenesis during disseminated candidiasis. Fluconazole is the most widely deployed antifungal drug due to its favorable bioavailability and safety profile. It targets the Lanosterol 14α-demethylase (Erg11) in the ergosterol biosynthesis pathway and promotes the accumulation of toxic ergosterol through the Δ5,6- desaturase Erg3. Since fluconazole is fungistatic rather than fungicidal, C. albicans isolates has frequently shown ultra-MIC growth which was characterized as trailing growth or tolerance. High tolerance isolates have shown to cause persistent candidiasis with increased mortality in comparison to sensitive isolates when treated with fluconazole. Little is known about whether and how C. albicans maintains fluconazole tolerance. Here, we show that C. albicans develops stable fluconazole tolerance after single dose of treatment. Tolerance is stably maintained through cell divisions without the selective pressure of fluconazole; and allows the majority of the population to grow in the presence of even higher concentrations of fluconazole without changing the MIC. Fluconazole tolerance can be destabilized by inducing chromatin remodeling, indicating that it is maintained by epigenetic regulation. Thus, tolerance is maintained by a reversible cell state that is established during fluconazole treatment. How epigenetic regulation modulates gene expression to confer the stable fluconazole tolerance in Persisters remained to be studied.