Functional Characterization of Histone Deacetylase 2 and Histone Deacetylase 3 in Candida albicans

C. a lbica ns is an opportunistic fungal pathogen which causes infections in humans and contributes significantly to world mortality rates. The phenotypic plasticity of this yeast supports its virulence and different morphologies are triggered by specific environmental cues including temperature, nutritional deprivation and pH. Development of hyphae facilitates the ranslocation of cells through human tissue and white or opaque forms of this yeast have the tendency to form hypha and mate, respectively. Switching between forms is regulated in many ways for example by transcription factors and hypotheses suggest that these may occur though changes to the packaging of DNA. This would be facilitated by modification of the histone proteins that contribute to the condensation of DNA into various chromatin states. It has been shown that deletion of the histone deacetylase HDA1 of C. a lbica ns stunts the yeast to hyphae transition and increases white to opaque switching. In the common model yeast, S. cerevisiae, Hda1 forms a complex with ‘Histone deacetylase’ proteins Hda2 and Hda3. Whether this complex forms in C. a lbica ns is unknown. This thesis evaluates C. a lbica ns Hda1, Hda2 and Hda3 proteins through their expression and separately by in vivo functional analyses. The results offer a plan for soluble expression of full length Hda1 protein as well as a construct for soluble expression of its Arb2 domain. Expression of full length Hda2 and Hda3 yielded insoluble protein. In vivo co-immunoprecipitation experiments unveil an interaction between Hda1 and Hda2 as well as Hda1 and Hda3. Homozygous mutants in these proteins have common and unique phenotypes which are hypothesized to be environmentally dependent. Specifically, the hda2∆∆ and hda3∆∆ homozygous mutants are sensitive to respiratory stressors sodium nitroprusside (SNP) and salicylhydroxamic acid (SHAM), copper and oxidative stress by NaCl but resistant to rapamycin. The hda2∆∆ mutant is resistant to caspofungin and fluconazole while hda3∆∆ is sensitive to zinc. Phenotypes for rapamycin and fluconazole are diminished or not evident in hyphae inducing media (RPMI). Wholeprotein extractions show decreased expression of Hda1 and likely Hda3 in hyphaeinduction conditions. This suggests that changes in protein abundance and environmental conditions lead to different roles, and potentially arrangements, of these proteins in complex. RNAseq results support the observed phenotypes as well as the hypothesis that these proteins form a complex. In summary, this thesis includes evidence of several functional roles for C. albica ns Hda2 and Hda3 proteins including in white-opaque and yeast-hypha morphological switching and gives proof of their interaction with Hda1