Date of Graduation

5-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

David McNabb

Committee Member

Suresh Kumar

Second Committee Member

Ralph Henry

Third Committee Member

Ines Pinto

Keywords

Biological sciences; Candida albicans; Catalase; Ccat-binding factor; Hap complex; Oxidative stress response; Superoxide dismutases

Abstract

The success of Candida albicans as an opportunistic human pathogen has been attributed to several factors, including the ability to survive in limiting iron environments and the ability to evade the respiratory burst of human macrophages and neutrophils. The goal of this research is to elucidate the role of the CCAAT-binding factor in the oxidative stress response of Candida albicans. Prior whole genome microarray studies performed in our lab compared the gene expression of a wild type Candida albicans strain versus a hap5Δ strain under iron-limiting growth conditions. Among the differentially regulated genes, CTA1, encoding catalase, had a four-fold higher level of expression in the hap5Δ homozygous mutant under iron limitation. The sensitivity of the hap5Δ homozygote to oxidative damage was also assessed by growing the strains in iron replete or iron-limiting conditions and exposing them to various concentrations of hydrogen peroxide. The data obtained from these studies supported our hypothesis that the CCAAT-binding factor was involved in the differential regulation of oxidative stress genes based on iron availability. To further explore the role of the CCAAT-binding factor in the regulation of CTA1, wild type and hap5Δ homozygote were grown in iron replete and iron-limiting conditions, and the total RNA was isolated and Northern blots performed. Our results indicated a CCAAT-binding factor-dependent regulation of CTA1 under iron limitation. The oxidative stress response in most organisms, including Candida albicans, is due to the concerted actions of many gene products. Therefore, we examined the mRNA levels of genes such as superoxide dismutase, thioredoxin, and glutaredoxin, which are known to confer resistance to oxidative stress. Our data indicates that the CCAAT-binding factor in Candida albicans regulates the mRNA expression level of different genes involved in the oxidative stress response. We next performed Northern blots, catalase enzymatic assays and peroxide sensitivity assays to dissect the role of each of the Hap3 and Hap4 subunits towards the iron-dependent regulation of CTA1. Together, our data strongly suggests that the CCAAT-Binding factor not only is essential in the regulation of iron homeostasis, but also regulates the Oxidative Stress Response in Candida albicans.

Available for download on Friday, May 04, 2018

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