Ferric reductase related proteins Frp1 and Frp2 mediate reductive heme uptake in the opportunistic pathogen Candida albicans

Heme is an essential cofactor and nutritional source of iron for many pathogenic microbes, including the opportunistic fungal pathogen Candida albicans. C. albicans utilizes common fungal extracellular membrane (CFEM) domain containing hemophores to scavenge heme from the host and deliver it across the cell wall to the cell membrane, where is it subsequently internalized. Previous studies indicated that ferric reductase related proteins Frp1 and Frp2 were required for hemin transport and utilization. However, the molecular mechanism of Frp1 and Frp2 dependent heme uptake remains to be fully elucidated. Motivated by the similarity of Frp1 and Frp2 to ferric iron reductases, we sought to determine if Frp1/2 exhibited heme reductase activity and if heme‑iron oxidation state influenced its uptake in C. albicans. By utilizing ratiometric heme sensors, redox inactive fluorescent ferric and ferrous heme analogs, and a modified pyridine hemochrome method, we determined that C. albicans has a preference for transporting ferrous heme over ferric heme and exhibits cell surface heme reductase activity that is in part dependent on Frp1 and Frp2. Our results are the first to directly demonstrate C. albicans has the capacity to reduce extracellular heme‑iron and that its oxidation state may be important for heme uptake. We further found that another yeast species, Saccharomyces cerevisiae, which poorly utilizes heme iron, also exhibits heme reductase activity, pointing to the broader presence of extracellular heme reduction in the microbial world.