Date of Award

Summer 2025

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

First Advisor

Caryn Outten

Abstract

The pathogenic fungi Aspergillus fumigatus requires metals to survive and infect humans, but metal overload can be detrimental. Metallothioneins (MTs) are small, cysteine-rich proteins that alleviate metal toxicity by binding excess metals (typically Zn or Cu) for storage or delivery. A recent report indicates that the Fe-S cluster binding glutaredoxin GrxD and the putative Cu-MT CmtA were interacting under high iron conditions in A. fumigatus. We hypothesize that this MT may have a role in binding iron or Fe-S clusters under iron overload conditions to prevent iron toxicity. We also aim to characterize this new MT by determining its metal binding properties. The purpose of this project was to investigate the interaction of CmtA and GrxD in vitro, characterize metal-binding properties of CmtA, and to investigate the role of MTs in Fe homeostasis in vivo. Our results showed that CmtA binds to a [2Fe-2S] cluster evident from UV-visible absorption, CD, EXAFS, Mossbauer, and EPR spectroscopy. GrxD and CmtA do not form a complex, but GrxD is able to transfer the [2Fe-2S] to CmtA. CmtA not only binds to Fe, but it is able to bind to Cu and Zn. Spectroscopy and ESI-MS confirmed that CmtA binds to these metals, as Cu6-CmtA, Cu10-CmtA, Zn5-CmtA, and Fe5-CmtA species. In terms of binding affinity, Cu binds the tightest, followed by Zn, which is then followed by Fe. In vivo results did not conclude whether CmtA and other MTs can help with alleviating Fe toxicity in S. cerevisiae. These studies have provided insight into the function of MTs in iron homeostasis and may explain how pathogens survive in metal-stressed environments and maintain their pathogenicity.

Rights

© 2025, David Eap

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