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Exploring Candida biology through integration of proteomic and genetic approaches

Creative Commons 'BY' version 4.0 license

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.

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