|M.Sc Student||Yaish Shir|
|Subject||Factors Involved in the Heme Utilization Pathway in|
|Department||Department of Medicine||Supervisor||Professor Daniel Kornitzer|
Candida albicans, a normally harmless commensal organism, can cause life-threatening systemic infections among immunocompromised patients. The human host invests substantial efforts into withdrawing iron from potential pathogens. To overcome the extreme iron limitation in the host, C. albicans, has evolved several mechanisms, including a pathway for heme-iron scavenging that enables access to hemoglobin, the largest iron pool in the human body.
Genetic and biochemical analysis identified RBT5, PGA7 and CSA2, as extracellular GPI-anchored and secreted proteins of the CFEM family that extract heme from hemoglobin and transfer it from one protein to the next across the cell wall. The CFEM domain crystal structure indicated that the CFEM proteins bind heme on the surface of the protein. This enables CFEM proteins to readily bind and release heme, making heme transfer possible. The CFEM domain coordinates heme-iron in a unique manner, with an aspartic residue as its axial ligand. This aspartic acid shows preference to ferric heme over ferrous heme making CFEM heme binding redox-sensitive. Utilization of heme-iron was also found to require endocytosis via the ESCRT pathway. However, the transmembrane protein mediating this endocytosis step remained unidentified.
Here we identify FRP1, a membrane protein, as involved in mediation between the extracellular and endocytic steps. Ferric reductases are heme proteins located on the plasma membrane. C. albicans carries about 15 ferric reductase-encoding genes. Two divergent members of this family, FRP1 and FRP2, were found by phylogenetic profiling to segregate across Ascomycota together with the heme-binding CFEM proteins. Deletion of FRP1, but not FRP2, resulted in a strong reduction in the ability to utilize hemoglobin-iron, supporting a possible function for this protein in the pathway. Re-integration of FRP1 in a knock-out strain restored the ability to utilize hemoglobin-iron.
Growth in different hemoglobin or hemin concentrations showed that strains lacking FRP1 are as deficient in utilizing hemoglobin and hemin as a single iron source as strains lacking PGA7, suggesting a shared role in the pathway.
Frp1-GFP visualization in wild type cells demonstrated differences in Frp1 localization with and without hemoglobin. In the absence of hemoglobin, in addition to the membrane signal, a significant cytoplasmic Frp1-GFP signal was also detectable, whereas in the presence of hemoglobin, this cytoplasmic signal was much reduced. In addition, vacuole signal intensity was stronger in the presence of hemoglobin. The dependence on ESCRT system-mediated endocytosis was supported by Frp1-GFP localization in ESCRT system mutants, since they exhibit a lack of vacuolar signal.
The identification of Frp1 as a membrane protein related to ferric reductases, and the observation that heme binding to CFEM proteins such as Pga7 is redox-sensitive, lead us to suggest that Frp1 is a hemin reductase. By reducing the heme bound to Pga7, it would enable its release from Pga7. The released heme would then bind a heme receptor that would mediate heme internalization to the vacuole via the ESCRT pathway. The localization of Frp1-GFP with and without hemoglobin, and in wild-type cells vs. ESCRT mutants suggests that Frp1 itself could function as such a receptor.