The Thiol Specific Antioxidant (TSA1) Gene is Required for Survival in Macrophages and Oxidative Stress Resistance in Histoplasma Capsulatum

<p><em>Histoplasma capsulatum</em>(<em>Hc</em>) is a pathogenic fungus that causes one of the most common invasive fungal respiratory diseases, Histoplasmosis. <em>Histoplasma</em>undergoes a dimorphic shift from mold to yeast which is crucial to pathogenesis of the organism.</p> <p>The thiol specific antioxidant gene, <em>Tsa1,</em>is strongly upregulated in the yeast (pathogenic) morphotype. This data led to the hypothesis that this gene plays a role in protecting <em>Hc</em>from host mediated oxidative attack. To characterize <em>Tsa1 </em>function, a knockdown strain (<em>tsa1-RNAi</em>) was created by RNAi gene silencing. Expression of <em>Tsa1</em>in the <em>tsa1-RNAi</em>strain was reduced to 10% that of the wildtype TSA1(+). In this study <em>Hc</em>strains with 90% <em>Tsa1</em>knockdown 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 <em>tsa1-RNAi</em>knockdown cells, indicates <em>Tsa1</em>has a role in maintaining redox homeostasis. Catalase and superoxide dismutase, enzymes which help cells survive oxidative stress, show decreased activity in <em>Tsa1</em>knockdown cells as compared to wildtype. Evidence suggests <em>Tsa1</em>protects the <em>Histoplasma</em>yeast in the macrophage environment by resistance against oxidative stress. Research is ongoing to determine <em>HcTsa1</em>protein localization and function. Understanding how these important fungal pathogens evade the host immune system is critical to development of targeted antifungal therapies.</p>

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

Masters Thesis

Year dc:date.available

2019

Author dc:creator

• Kennedy, Lauren

Contributors dc:contributor

• Glen Shearer
• Janet Donaldson
• Shahid Karim

• yeast
• Histoplasma
• oxidative stress
• Tsa1
• Pathogenic Microbiology

Repository record dc:identifier

https://aquila.usm.edu/masters_theses/701

OAI identifier oai:identifier

oai:aquila.usm.edu:masters_theses-1745