Date of Award

12-2026

Document Type

Thesis

Degree Name

Master of Science (MS)

Program

Clinical Pharmacy and Translational Science

Track

Pharmaceutical Sciences

Research Advisor

Jarrod Fortwendel, PhD

Committee

Brian Peters, PhD Camaron Hole, PhD

Keywords

Aspergillus fumigatus, Regulation of Ace2 and Morphogenesis (RAM) pathway, CotA, fungal morphogenesis, filamentous growth, carbon source utilization, cell wall integrity, stress response

Abstract

Aspergillus fumigatus CotA is a conserved protein kinase and predicted orthologue of Cbk1, a terminal kinase of the Regulation of Ace2 and Morphogenesis (RAM) network. In our previous study, disruption of CotA resulted in carbon source dependent invasive growth with the mutant exhibiting preferential growth on glucose but severely impaired growth on alternate carbon sources. This pathway in other fungi has been implicated in the regulation of morphogenesis, stress responses, and virulence, but this study is the first one to characterize it in the pathogenic filamentous fungus.  In yeast, the RAM network includes the CotA ortholog, Cbk1, and associated proteins, ScMob2, ScTao3, ScHym1, ScSog2, and ScKic1. To determine whether these components contribute to the carbon source dependent phenotype of our cotA-1 mutant, we first identified single copy orthologs for hym1, kic1, sog2, tao3, and mob2 in the A. fumigatus genome through BLAST analyses. Then, using CRISPR/Cas9, we successfully deleted hym1, kic1, and tao3 orthologs. After unsuccessful attempts to delete sog2 and mobB, doxycycline-inducible conditional mutants were generated. We analyzed each strain for growth and morphogenesis in media containing glucose or non-preferred carbon sources (acetate, casamino acids, and ethanol) as the sole source of carbon. Although the deletion of hym1 and repression of either sog2 or mobB exhibited growth defect under all carbon sources, they did not exhibit carbon source dependent phenotype, suggesting that CotA may function independently of these associated proteins. RAM pathway components have been shown to contribute to cell wall integrity, adaptation to elevated CO2 levels, hyphal branching and antifungal susceptibility in other fungal species. Therefore, we assessed sensitivity of RAM mutants under those conditions. Except ∆hym1 to cell wall stress, no mutant displayed significantly increased sensitivity to any of these stresses. Together, these findings reveal a divergence in the organization and function of the RAM network in A. fumigatus, suggesting that CotA-dependent regulation of carbon source utilization and morphogenesis is distinct from the RAM signaling mechanisms characterized in yeast and may reflect fungal-specific adaptations to the filamentous growth and host-associated environment.

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