Tuesday, May 14, 2024
5/14/2024
Project Summary Copper (Cu) is an essential micronutrient for nearly all eukaryotic organisms. Cells must maintain a careful Cu homeostasis to balance cellular needs while minimizing toxicity due to excess Cu accumulation. Fungi, such as Candida albicans, must acquire, route, store, use, and continually monitor levels of Cu to thrive in human hosts. To survive as a human pathogen, C. albicans must be able to withstand the manipulation of Cu metal concentrations at the host-pathogen interface, forcing the fungi to rely on complicated Cu homeostasis pathways to adapt to Cu-replete and Cu-depleted environments. Although commonly used as a treatment, fluconazole is fungistatic, merely inhibiting growth of C. albicans and increasing the risk of developing resistant strains. Fluconazole induces a Cu-deficient response in C. albicans, despite an increase in total Cu levels compared to untreated cells. The Cu-deficient response includes the induction of the Cu-import gene CTR1, repression of the Cu exporter CRP1, and a switch in expression of cytosolic superoxide dismutase from CuZnSod1 to MnSod3, controlled by the transcription factor Mac1. Mac1 is regulated by Cu levels in the cell, binding Cu ions under Cu- replete conditions and binding DNA under Cu-deficient conditions. Despite understanding the metalloproteins involved in the Cu-deficient response, there are still numerous gaps in our understanding of the response. It is known that Mac1 must lose its Cu ions to bind DNA; however, the molecular basis for activation, including the discrete entity involved, is unknown. The work described within this proposal seeks to further illuminate our understanding of the Cu-deficient pathway of Cu homeostasis in C. albicans by investigating the role of CuZnSod1. CuZnSod1 has been primarily characterized its role as global antioxidant, removing reactive oxygen species; however, there are novel roles proposed for Sod1 in sensing the Cu levels of the cell and participating in the Cu homeostasis response by directly activating Mac1. Here, I aim to probe the location and metalation state of CuZnSod1 under fluconazole-induced Cu-deficient conditions and characterize the conditions for a direct interaction between CuZnSod1 and Mac1, using biophysical techniques. Collectively, this work will allow us further insight into the Cu-deficient homeostasis mechanisms of C. albicans and will allow us in the long-term to apply this understanding to design more effective antifungal treatments exploiting metal-specific weakness to increase potency and decrease changes of developing resistant strains.
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