Date of Graduation


Document Type


Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level



Biological Sciences


Ines Pinto

Committee Member

David McNabb

Second Committee Member

Robert R. Beitle

Third Committee Member

Ralph Henry


fungus, micronutrient, gene regulation


Candida albicans is a fungal opportunistic human pathogen. Its infections range from surficial infections like skin rash to fatal systemic infections. Filamentation growth mode is associated with C. albicans virulence because it helps penetration of the host’s epithelial cells. The CCAAT-binding factor (CBF) is a conserved heterooligomeric transcription factor found in 30% of eukaryotes genes. In C. albicans it is composed of 4 major subunits, including Hap2, Hap3, Hap4, and Hap5. Hap2 and Hap5 are essential for DNA binding and function. Hap4 has 3 homologous subunits: Hap41 and Hap42 are putative subunits of CBP. Hap43 is the only Hap4 subunit known to associate with the Hap 2/5/31 or 32 complex, and it is involved in gene regulation in iron-dependent genes. Catalase, encoded by the CAT1 gene, is an antioxidant enzyme that detoxifies ROS produced by the host’s immune system, allowing C. albicans to survive in the host cells. Iron is an essential element involved in several physiological processes, including C. albicans virulence. The CAT1 promoter includes 5 putative CCAAT binding sites; the first aim of this work was to investigate the actual binding site that is involved in catalase gene expression. Therefore, I created several plasmids that carry the CAT1 promoter fused to Renilla luciferase as a reporter gene and integrated in the genome. Renilla luciferase activity was measured in iron rich and iron low media, and in response to H2O2 treatment. The two most proximal CCAAT sites were sufficient to provide regulation equivalent to the full promoter, excluding the 3 most upstream sites. Interestingly, both sites appear to cooperate in CAT1 expression, since individual deletions showed partial loss of regulation in rich and low iron conditions. Reporter activity after H2O2 treatment did not show any difference between the two proximal CCAAT sites tested, and increased enzymatic activity was observed in WT and hap5/ strains, suggesting an activation independent of CBF. We concluded that there might be other transcription factors, such as CAP1, that contribute to catalase regulation under peroxide stress.

The second aim was to determine whether the glutamine rich domain present in the Hap5 C-terminus has a transcription activation role. Plasmids expressing wild type Hap5, a Hap5 with a C-terminal truncation, and empty vector were integrated at the ARG4 locus, and I measured CAT1 mRNA expression. The Hap5 truncation had a basic expression in rich medium, confirming the Hap5 glutamine rich domain activation role.

Because the hyphal growth mode is involved in C. albicans virulence, the third aim was to study the possible role of Hap4 subunits in yeast to hypha transition. I monitored Hap 4 subunits’ phenotypes in various filamentation-promoting media. The strain carrying a homozygous hap41/ showed a clear hyphal defective growth similar to hap5/ and hap2/ strains in Spider and M Lee’s media, indicating that Hap41 may associate with CBF under specific physiological conditions, allowing us to establish the parameters for further investigation.