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Access Type

WSU Access

Date of Award

January 2019

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

Robert Akins

Abstract

A thesis presented on the genomic plasticity of yeast species Candida albicans, specifically loss of heterozygosity (LOH) induced in response to stress. C. albicans was the most prevalent yeast species to afflict patients with Candidiasis which can be most harmful when infection was recurrent or when patient was immunocompromised. By understanding the cellular response of this species will give greater insight to how current treatment can lead to resistance, but also how it may result in species will heightened virulent factors that cause even greater negative effects on patients. Further progression of this could lead to increased efficiency of treatment by preventing this process from occurring. The goals of this study was to i) confirm loss of heterozygosity was occurring, but also at which rates across different chromosomal loci, ii) if rates of LOH differ depending on the type of stress and if any loci that have undergone this process show susceptibility, and iii) if colonies that have undergone LOH display phenotypes consistent with increase virulence or resistance.

Confirmation of LOH was done by use of allele-specific qPCR using primers designed specifically to regions where a single-nucleotide polymorphism(SNP) was present and to overcome the limitation of non-specific binding, difference of allele fluorescence detected by qPCR was mathematically determined, in which LOH was predicted if difference exceeded >3.5 cycles. Rates were able to be determined by generating oligo sequences tailored to specific loci, both on the same chromosome and other chromosomes across the Candida genome. Explored here were several chromosome 5 loci, as it has been well-documented this chromosome was a sight of great plasticity, but other locus located on Chromosome 1, 3, and R of Candida genome were explored as well. Confirmation of genomic predictions was accomplished by designing primers located upstream and downstream of SNP region and sent for sequencing.

By comparing rates of LOH in different sample sets which varied in stresses undergone, specifically peroxide only (single stress) and peroxide stress and incubated on flucytosine containing media (double stress), we were able to determine which combination of stresses induced the most LOH. Early and late rising isolates grown on flucytosine media with no initial stress were also analyzed using this methodology to compare rates of genomic alteration.

By modifying media composition of agar plates via addition of drug or percentage of ingredients, insight into resistance and virulence trends were able to be measured for isolates stressed and unstressed to determine if contrast was evident. All samples incubated in modified media were inoculated with low concentration of cells to determine which sample set had the ideal advantage in overcome stress by gained resistance or displayed upregulated virulent factors.

Overall, it was found that in peroxide stressed isolates (single stress), LOH occurred, but in reduced quantities, as in peroxide stressed grown on flucytosine media (double-stress) isolates rates of LOH were increased. Isolates incubated on flucytosine media also showed evidence of LOH, with rates and loci varying for early rising (day 3) and late rising (day 7). Early rising (Day 3) showed loss of heterozygosity primarily at FUR1 locus, as flucytosine applies selective pressure to this region, as Late Rising (Day 7) showed reduced LOH at FUR1, but increased LOH at neighboring loci.

Stressed isolates (peroxide ± Flucytosine and Day 3 & 7 Flucytosine Resistant) also displayed significant phenotypic alterations, both to antifungal drugs such as Terbinafine and Fluconazole, and an increased ability to invade, suggesting an increase resistance and virulence factors.

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