Date of Award

Fall 2019

Degree Type

Masters Thesis

Degree Name

Master of Science (MS)

School

Biological, Environmental, and Earth Sciences

Committee Chair

Glen Shearer

Committee Chair School

Biological, Environmental, and Earth Sciences

Committee Member 2

Janet Donaldson

Committee Member 2 School

Biological, Environmental, and Earth Sciences

Committee Member 3

Shahid Karim

Committee Member 3 School

Biological, Environmental, and Earth Sciences

Abstract

Histoplasma capsulatum(Hc) is a pathogenic fungus that causes one of the most common invasive fungal respiratory diseases, Histoplasmosis. Histoplasmaundergoes a dimorphic shift from mold to yeast which is crucial to pathogenesis of the organism.

The thiol specific antioxidant gene, Tsa1,is strongly upregulated in the yeast (pathogenic) morphotype. This data led to the hypothesis that this gene plays a role in protecting Hcfrom host mediated oxidative attack. To characterize Tsa1 function, a knockdown strain (tsa1-RNAi) was created by RNAi gene silencing. Expression of Tsa1in the tsa1-RNAistrain was reduced to 10% that of the wildtype TSA1(+). In this study Hcstrains with 90% Tsa1knockdown have decreased survival after infection in murine macrophages and heightened sensitivity to the oxidizing agents paraquat dichloride and hydrogen peroxide. Flow cytometry of these treated cells strained with the intercellular REDOX indicator dye CM-H2DCFDA shows approximately 2.1 to 3.7 fold greater fluorescence intensity than wild type cells. This result, indicating reduced clearance of ROS in tsa1-RNAiknockdown cells, indicates Tsa1has a role in maintaining redox homeostasis. Catalase and superoxide dismutase, enzymes which help cells survive oxidative stress, show decreased activity in Tsa1knockdown cells as compared to wildtype. Evidence suggests Tsa1protects the Histoplasmayeast in the macrophage environment by resistance against oxidative stress. Research is ongoing to determine HcTsa1protein localization and function. Understanding how these important fungal pathogens evade the host immune system is critical to development of targeted antifungal therapies.

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