Title

CCAAT-Binding Factor as a Transcriptional Regulator of CYC1 in Candida albicans

Date of Graduation

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

David S. McNabb

Committee Member

Gisela F. Erf

Second Committee Member

Ralph L. Henry

Third Committee Member

Roger E. Koeppe II

Abstract

Candida albicans is a normal commensal of the human flora; however, under appropriate circumstances this organism can become pathogenic to the host and cause life threatening conditions. In fact, Candida species are the fourth most common nosocomial infection in the Unites States with a mortality rate of over 30%

The CCAAT binding factor is a multi-protein transcription factor highly conserved in eukaryotes. It binds specifically to the consensus sequence 5'-CCAAT-3' in promoters and it is one of the most common cisacting elements in eukaryotes promoters. This transcription factor is composed of three DNA binding subunits; Hap2p, Hap3p and Hap5. In yeast and fungi a fourth subunit, termed Hap4, functions as the

effector subunit that regulates the expression of target genes. C. albicans has been found to have multiple genes encoding some of the proteins associated with the CCAAT-binding factor, namely Hap31

and Hap32 as well as Hap41, Hap42, and Hap43. In the studies described in this dissertation, the role of the CCAAT-binding factor in the regulation of CYC1, encoding cytochrome c, was examined. It was

found that the CCAAT-binding factor was the sole transcription factor involved in CYC1 transcriptional regulation. It was found that Hap31 functions to activate CYC1 transcription in an iron rich environment,

while Hap32 represses CYC1 transcription when iron is limited. Thus, the differentially utilization of Hap31 and Hap32 serves as a molecular switch to convert a transcriptional activator to a repressor in

response to environmental signals. It was also shown that HAP32 contains a 118-nucleotide intron that is removed during processing; however, the splicing event was not regulated by iron availability. Additional studies were done to construct a hem1Δ homozygous mutant of C. albicans and this mutant was used to demonstrate that C. albicans can acquire iron, in the form of heme, exogenously. We also examined the growth of the hem1Δ homozygote under anaerobiosis and discovered that the mutant requires a source of unsaturated fatty acids for anaerobic growth; however, it does not require supplementation with

exogenous ergosterol, common in many other fungi. The clinical implications of this finding will be discussed.

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