Characterization of the ferric reductase gene family in Candida albicans

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Johns Hopkins University
Metals are essential for life, serving as structural cofactors and catalysts in metabolic processes. Nutritional immunity is a host defense mechanism that capitalizes on the essentiality of metals to combat pathogens. Through co-evolution, fungal pathogens have developed strategies to endure metal-depleted conditions. The heme-containing transmembrane ferric reductase domain (FRD) plays a crucial role in maintaining metal homeostasis in pathogenic fungi by converting extracellular Fe+3 or Cu+2 to bioavailable Fe+2 or Cu+1 for cellular uptake. FRDs can also function as NADPH oxidases (NOX), producing reactive oxygen species (ROS) by reducing oxygen to superoxide. The opportunistic fungal pathogen Candida albicans possesses a family of 17 FRDs, of which only 4 have confirmed functions. The goal of this study was to characterize the role of the remaining FRDs and investigate their regulation. We predicted that reductases for Fe+3 and Cu+2 would be induced during metal starvation, and NOXs would be induced when abundant ROS is produced in the hyphal form of C. albicans. We grew wild-type C. albicans in the presence of iron or copper specific metal chelators and induced other cultures to make hyphae, and then analyzed expression of all 17 FRDs by qRT-PCR. We found that 8 FRD genes (FRE1, FRE2, FRE4, FRE5, FRE7, FRE9, FRE10, FRP1) were induced under Fe-starvation conditions, and 2 FRD genes (FRE7, FRE30) were induced under Cu-starvation conditions. 5 FRD genes (FRE2, FRE7, FRE8, FRE17, FRE30) were induced in hyphal cells. To further study our predictions on FRD genes, we conducted ferric reductase and cupric reductase assays on mutants in specific FRD genes and found that several of the genes we predicted to be cupric or ferric reductases showed either an increase or decrease in metalloreductase activity. Additionally, we found that Fe-responsive transcription factor SEF1 regulates the iron starvation induced expression of FRE1, FRE2, FRE4, FRE5, and FRP1. However, Fe-regulation iii of FRE7 and FRE9 occurs independent of SEF1. In addition to intracellular proteins like SEF1, certain unknown small molecule(s) secreted by C. albicans into the media were observed to influence FRD gene expression in response to Fe-starvation. Overall, our studies provide insight into the possible functions of several FRD genes and their regulatory factors.
Ferric reductase, cupric reductase, Candida albicans, NOX, metalloreductase, riboflavin, FRDs