Editor's evaluation: Ferric reductase-related proteins mediate fungal heme acquisition (2022)

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Heme can serve as iron source in many environments, including the iron-poor animal host environment. The fungal pathobiont Candida albicans expresses a family of extracellular CFEM hemophores that capture heme from host proteins and transfer it across the cell wall to the cell membrane, to be endocytosed and utilized as heme or iron source. Here, we identified Frp1 and Frp2, two ferric reductase (FRE)-related proteins that lack an extracellular N-terminal substrate-binding domain, as being required for hemoglobin heme utilization and for sensitivity to toxic heme analogs. Frp1 and Frp2 redistribute to the plasma membrane in the presence of hemin, consistent with a direct role in heme trafficking. Expression of Frp1 with the CFEM hemophore Pga7 can promote heme utilization in Saccharomyces cerevisiae as well, confirming the functional interaction between these proteins. Sequence and structure comparison reveals that the CFEM hemophores are related to the FRE substrate-binding domain that is missing in Frp1/2. We conclude that Frp1/2 and the CFEM hemophores form a functional complex that evolved from FREs to enable extracellular heme uptake. Editor's evaluation This work focuses on the important problem of how a human pathogenic fungus obtains iron during infection. This study uses biochemical and genetic methods to identify the missing link between heme receptors and heme utilization by cells. Specifically, they provide convincing evidence that the ferric reductase-like proteins Frp1 and Frp2 have major roles in iron acquisition from heme, thus identifying a new function for these proteins. Understanding how pathogenic fungi obtain iron and identifying differences in fungal and host iron metabolism can provide valuable leads for drug discovery. https://doi.org/10.7554/eLife.80604.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Hosts and disease-causing fungi are often locked into a battle over resources. The host will attempt to withhold molecules that the fungus needs to survive, while the pathogen will try to find alternative routes to obtain them. Candida albicans, for example, can go after the atoms of iron embedded in the proteins of the organism it infects. To do so it releases molecules known as hemophores, which scavenge the iron-containing heme molecule that equips oxygen-carrying proteins in the blood. Once captured, the heme is carried across the wall that protects C. albicans from the environment and brought to the membrane of the cell. It is then taken in and trafficked inside vesicles to its destination. However, the identity of the molecular actors which help to bridge the internal and external segments of the heme journey remain unclear. Previous studies have shown that the hemophore Pga7 is involved, but this protein is attached to the outside of the cell membrane, where it cannot directly interact with the import machinery. Roy et al. set out to discover this missing link. Examining the genomes of fungal species related to C. albicans highlighted two membrane proteins, Frp1 and Frp2, which could participate in heme uptake. Protein sequence comparison revealed that Frp1 and Frp2 were closely related to ferric reductases, a group of membrane enzymes which can chemically alter extracellular iron prior to uptake. Deleting the genes for Frp1 and Frp2 rendered C. albicans cells incapable of taking in heme. Conversely, a fungal species which cannot normally uptake heme could efficiently internalise these complexes when artificially equipped with Frp1 and Pga7, suggesting that the two proteins work closely together. Finally, protein structure comparisons highlighted that an extracellular domain present in ferric reductases but absent in Frp1 and Frp2 is, in fact, related to Pga7 and other hemophores. This implies that the iron and heme uptake systems may share a common evolutionary origin. Overall, the work by Roy et al. reveals a new family of proteins which allow disease-causing fungi to steal iron from their hosts. This knowledge may be useful to design better anti-fungal treatments. Introduction Candida albicans is a commensal microorganism of the human host (Ghannoum et al., 2010; Nash et al., 2017). It is normally found on mucosal surfaces and on the skin as part of the normal microbial flora. It is also one of the most common human fungal pathogens, causing infections that range from superficial infections such as vulvovaginal candidiasis (Sobel, 2007) to life-threatening systemic infections when the immune system is weakened or when there is damage to epithelial barriers (Kullberg and Arendrup, 2015). Candida species, of which the most prevalent is C. albicans, are responsible for an estimated 750,000 cases of systemic candidiasis annually, with a mortality rate of around 40% (Bongomin et al., 2017; Pfaller et al., 2012). Like most organisms, C. albicans requires iron for its survival. Iron can be found in several oxidation states, of which the more common are Fe2+ and Fe3+. The low solubility of ferric iron (Fe3+) makes iron acquisition challenging in oxidizing environments. Many unicellular organisms express ferric reductases (FREs) on their surface, which can contribute to iron acquisition by reducing ferric iron to its more soluble ferrous (Fe2+) ion and/or extracting it from iron chelates (Kornitzer, 2009; Kosman, 2003; Philpott, 2006). The C. albicans genome contains at least 15 genes encoding FRE-like proteins, including Fre1/Cfl1 and the major iron reductase Fre10 (Baek et al., 2008; Hammacott et al., 2000; Knight et al., 2005). Heme, or Fe-protoporphyrin IX, is a molecule that serves as a cellular cofactor almost universally across life. As such, it is present in many natural environments, and thus, environmental heme can be utilized as an alternative iron source by many organisms, via a large repertoire of heme acquisition systems (Donegan et al., 2019). In animals, HRG-1-like heme transporters conserved from C. elegans to mammals can mediate extracellular heme import or intracellular heme transport (Rajagopal et al., 2008; Reddi and Hamza, 2016) and the human membrane protein FLVCR2 functions as a heme importer (Duffy et al., 2010). For microorganisms, the animal host environment poses a particular challenge for iron acquisition, as the host restricts the availability of free iron by a variety of active mechanisms as part of a defense strategy called 'nutritional immunity' (Ganz, 2018; Weinberg, 1975). In this environment, hemoglobin heme, comprising about two-thirds of the total iron, is therefore expected to be a particularly attractive iron source, and indeed it is utilized by many bacterial pathogens via several well-studied mechanisms that involve distinct extracellular hemophore families and plasma membrane ABC transporters (Cassat and Skaar, 2013; Contreras et al., 2014; Ganz and Nemeth, 2015; Hood and Skaar, 2012; Huang and Wilks, 2017). Fungi have also evolved different mechanisms to internalize heme, involving a membrane heme receptor in the case of Schizosaccharomyces pombe (Labbé et al., 2020; Mourer et al., 2015) and an endocytosis-based mechanism in the case of Cryptococcus neoformans (Bairwa et al., 2019; Bairwa et al., 2017; Cadieux et al., 2013). C. albicans and other Candida spp. express a distinct family of GPI-anchored and soluble extracellular hemophores containing a heme-binding CFEM domain (Roy and Kornitzer, 2019). Pga7 and Rbt5 are GPI-anchored to the cell membrane and the cell wall, respectively, while Csa2 is secreted to the medium (Kuznets et al., 2014; Nasser et al., 2016; Weissman and Kornitzer, 2004). These three CFEM proteins are all able to extract heme from hemoglobin and to transfer it among themselves, consistent with a model where heme captured from hemoglobin or other host heme-binding proteins, such as serum albumin, is transferred across the cell wall to the plasma membrane (Kuznets et al., 2014; Nasser et al., 2016; Pinsky et al., 2020). Heme binding to the CFEM domain involves a unique aspartic acid-mediated heme-iron coordination that renders the heme-binding redox-sensitive: only ferriheme (Fe3+) binds to the CFEM domain (Nasser et al., 2016). Heme utilization in C. albicans requires, in addition to the CFEM hemophores, components of the endocytic pathway and the ESCRT system, and vacuolar function (Weissman et al., 2008), suggesting that it involves endocytosis of the heme to the vacuole. However, GPI-anchored proteins are not known to be subject to ESCRT-mediated endocytosis, suggesting the existence of (an) additional transmembrane protein(s) involved in heme internalization. Here, we describe the identification and characterization of Frp1 and Frp2, plasma membrane proteins related to FREs, that are essential for heme uptake and utilization in C. albicans. Results Phylogenetic profiling for identification of new heme-iron acquisition genes Analysis of genomes from species related to C. albicans indicated that many carry CFEM proteins related to Pga7, Rbt5, and Csa2 (Nasser et al., 2016). We found that across fungal genomes of the Ascomycete clades most closely related to C. albicans, distinct relatives of Rbt5, Pga7, and Csa2 can be identified in almost every genome (Figure 1—figure supplement 1 shows a proximity tree of Rbt5-related proteins from 14 Saccharomycetales genomes). One exception is Meyerozyma (Candida) guilliermondii, which completely lacks any coding sequence related to these CFEM proteins, unlike equally distant relatives of C. albicans such as Debaryomyces hansenii (Kurtzman and Suzuki, 2010). In order to identify additional proteins involved in heme uptake, we applied phylogenetic profiling or pathway co-evolution analysis. Briefly, this method is based on the assumption that, as organisms can acquire new functions and evolve new pathways by gene duplication, acquisition and modification, organisms can similarly lose functions, and their associated genes, when they are no longer required. Genes encoding a single functional pathway are thus expected to be preserved or eliminated in a correlated fashion across genomes, according to whether the function is present or absent in a given organism (Li et al., 2014; Pellegrini et al., 1999). Sequenced fungal genomes currently number well over one thousand, including many tens of Saccharomycetales (Butler et al., 2009; Grigoriev et al., 2014). Two genes whose presence was best correlated with the presence of Rbt5-related CFEM genes were FRP1and FRP2 (see Materials and methods for details). C. albicans Frp1 and Frp2 were previously classified as FREs (Almeida et al., 2009), based on their homology to the main C. albicans FRE Fre10 (Knight and Dancis, 2006), as well as to Cfl1, another C. albicans protein that was experimentally shown to function as an FRE (Hammacott et al., 2000). However, alignment followed by phylogenetic tree building indicates that Frp1 and Frp2 cluster separately from most FRE homologs, including Fre10 and Cfl1 (Figure 1—figure supplement 2A). Notably, Frp1 and Frp2 lack an N-terminal domain present in most other FREs (Figure 1—figure supplement 2B). Frp1 and Frp2 are 36% identical, and out of 13 Saccharomycetales listed in the Candida Gene Order Browser database (cgob3.ucd.ie; Fitzpatrick et al., 2010; Maguire et al., 2013) that have Frp homologs, 12 have an Frp1 ortholog, and 10 have an Frp2 ortholog. Interestingly, FRP1 and FRP2 are adjacent to two CFEM protein genes in the C. albicans genome, FRP1 to PGA7 and FRP2 to CSA1. In both cases, the adjacent genes are arranged head-to-head and share a promoter region. This synteny is conserved across most genomes (75% for FRP1-PGA7 and 70% for FRP2-CSA1; Figure 1—figure supplement 3). Frp1 and Frp2 are required for heme-iron utilization To directly examine the role of Frp1 and Frp2 in heme-iron acquisition, their genes were deleted and the hemoglobin-iron utilization phenotypes were analyzed by growth on YPD plates supplemented with 1 mM bathophenanthroline disulfonate (BPS), an iron chelator, and hemoglobin as iron source. As shown in Figure 1A, the frp1-/- strain was unable to form colonies on the plate with hemoglobin as the sole iron source. Reintegration of a wild-type FRP1 allele completely reverted the growth defect. In contrast, the frp2-/- strain showed no growth defect. We next performed this assay in medium buffered to pH 8.5. On the alkaline plates, we found that the frp1-/- strain was still unable to form colonies, whereas the frp2-/- strain showed a partial defect in heme-iron utilization, and reintegration of the FRP2 wild-type allele complemented this growth defect (Figure 1A). Figure 1 with 4 supplements see all Download asset Open asset FRP1 is essential for hemoglobin-iron acquisition, whereas FRP2 contributes to growth on hemoglobin at alkaline pH. (A) Fivefold dilutions of cultures of the indicated strains were spotted on YPD or YPD pH 8.5, with the indicated supplements, and incubated for 3 days (Hb and BPS plates) or 2 days (YPD plates) at 30°C. WT = KC2, FRP1+/-=KC859, frp1-/-=KC870, frp1-/-<FRP1 >= KC1024, FRP2+/-=KC901, frp2-/-=KC912, frp2-/-<FRP2 >= KC1379. (B) The strains with indicated genotypes in the ccc2-/- background were grown in triplicates in YPD or YPD pH 8.5 media supplemented with 1 mM ferrozine and the indicated amounts of hemoglobin, and incubated at 30°C for 3 days. Each result is the average of three cultures. Standard deviations are indicated by vertical bars. WT = KC811, frp1-/-=KC1146, frp2-/-=KC1414, frp1-/- frp2-/-=KC1412, frp1-/-<FRP1 >= KC1146, frp2-/-<FRP2>=KC1411. (C) The frp1-/- and frp2-/- heme-iron utilization phenotype was compared to that of the CFEM protein mutants rbt5-/- and pga7-/-. The strains were grown in YPD or YPD pH 8.5, with 1 mM ferrozine and the indicated concentrations of hemoglobin or hemin, and grown and measured as in B. Wild type = KC68, rbt5-/-=KC139, pga7-/-=KC485, frp1-/-=KC923, frp2-/-=KC913. All strains in B and C carry a deletion of the CCC2 gene, which causes a defect in high-affinity iron import and prevents growth in the presence of ferrozine. Figure 1—source data 1 Excel file with data used to make Figure 1B. https://cdn.elifesciences.org/articles/80604/elife-80604-fig1-data1-v2.zip Download elife-80604-fig1-data1-v2.zip Figure 1—source data 2 Excel file with data used to make Figure 1C. https://cdn.elifesciences.org/articles/80604/elife-80604-fig1-data2-v2.zip Download elife-80604-fig1-data2-v2.zip The heme-iron utilization was also tested in liquid medium, using a set of frp1-/-, frp2-/-, and frp1-/-frp2-/- double deletion strains introduced in a background made defective in high-affinity iron acquisition by deletion of the copper transporter CCC2 (Weissman et al., 2002), which makes the cells unable to grow in the presence of the iron chelator ferrozine. Growth of the mutants was assayed in the presence of increasing concentrations of hemoglobin. In unbuffered YPD (pH ~ 6.5), the same picture was seen as on plates, with the frp1-/- mutant unable to utilize hemoglobin-iron and the frp2-/- mutant unaffected (Figure 1B). In alkaline YPD (pH 8.5), the frp1-/- mutant failed to grow, whereas the frp2-/- mutant exhibited reduced growth, suggesting that this mutant is able to utilize hemoglobin, but at a reduced rate (Figure 1B). Reintegration of the wild-type FRP1 and FRP2 genes in the respective mutant backgrounds restored wild-type growth in unbuffered YPD, but did not completely restore growth at pH 8.5. This could be due to haploinsufficiency or to reduced expression of the reintegrated allele under these conditions. We also compared growth of the frp1-/- and frp2-/- mutants to that of the CFEM protein mutants rbt5-/- and pga7-/-, on either hemoglobin or hemin as iron source. As shown in Figure 1C, in all media the frp1-/- mutant was as defective as the pga7-/- mutant, which is lacking the most essential CFEM protein (Kuznets et al., 2014). In YPD, both the frp1-/- and pga7-/- strains were unable to grow on hemoglobin, and the rbt5-/- mutant showed an intermediate phenotype, as noted before (Kuznets et al., 2014; Weissman and Kornitzer, 2004) whereas on hemin, growth of frp1-/- and pga7-/- was partially restored at higher concentrations, to similar extents. At pH 8.5, all mutants exhibited reduced growth on both hemoglobin and hemin, with frp1-/- and pga7-/- again showing the deepest defect (Figure 1C). Since compared to the established FREs, Frp1 and Frp2 are relatively similar to each other, we asked whether differential expression of FRP2 was the reason that it could not replace FRP1, that is, that the frp1-/- strain is unable to utilize hemoglobin even though FRP2 is present in these cells. To answer this, we placed the FRP2 open reading frame under the control of the FRP1 promoter and transformed the resulting plasmid into the frp1-/- and frp2-/- strains. The FRP1p-FRP2 construct could complement the frp2-/- but not the frp1-/- mutant (Figure 1—figure supplement 4), indicating that differential expression is not the reason that Frp2 is inactive in hemoglobin-iron utilization in unbuffered medium. Rather, this suggests that the Frp1 and Frp2 proteins are functionally distinct. Frp1 and Frp2 mediate heme uptake into the cytoplasm To monitor heme uptake by the frp1 and frp2 mutants more directly, we took advantage of a recently developed cytoplasmic heme sensor system, based on a heme-quenchable GFP-cytochrome fusion. In this detection system, an mKATE2 red fluorescent domain linked to the GFP-cytochrome fusion serves as an internal fluorescence control. The ratio of GFP to mKATE2 fluorescence gives a measure of available cytoplasmic heme, with lower ratios indicating stronger GFP quenching due to higher cytochrome occupancy, that is, higher cytoplasmic heme concentrations. To expand the range of testable concentrations, a lower-affinity cytochrome domain mutant (M7A) can be used alongside the high-affinity original sensor (HS1) (Hanna et al., 2016; Weissman et al., 2021). Wild-type, frp1-/-, frp2-/-, and frp1-/- frp2-/-double mutant cells expressing the HS1 and M7A sensors were exposed to increasing hemin concentrations in the medium for 4 hr, and sensor fluorescence was measured. To ensure expression of the heme uptake system, the cells were grown with 1 mM ferrozine, which imposes iron limitation and activates the heme-uptake genes. The medium was buffered to pH 8.5, a condition where Frp2 was shown to be required as well (Figure 1). As can be seen in Figure 2 (bottom panel), the low-affinity cytoplasmic sensor showed minimal occupancy in the absence of external hemin ratio of and in the presence of hemin, only the wild-type cells showed a but in sensor The wild-type HS1 in contrast, showed a fluorescence ratio of in all strains in the absence of hemin to the medium, indicating a partial occupancy which the free heme in the In medium supplemented with 10 hemin, in wild-type cells sensor occupancy indicating an in cytoplasmic hemin The in occupancy was in the frp1-/- mutant, and still in the frp2-/- and frp1-/- frp2-/- even when exposed to and hemin in the medium. while cellular heme concentrations do with extracellular heme, the in intracellular heme is reduced in the frp1-/- and frp2-/- and almost in the frp1-/- frp2-/- The reduced in sensor occupancy in the frp2-/- mutant exposed to external hemin compared to the frp1-/- mutant suggests that Frp2 is more important for heme into the cytoplasm Frp1 under these in of Frp1 being for heme utilization even at pH 8.5 (Figure 1). Figure 2 Download asset Open asset The frp1-/- and frp2-/- mutants are defective in heme uptake from the The high-affinity HS1 and low-affinity M7A heme sensors were used to monitor heme into the cytoplasm in the wild-type and frp1-/- frp2-/- strains grown for 4 in YPD medium at pH 8.5 with 1 mM ferrozine, and with the indicated concentrations of hemin Each data is the average of three different each measured and the that are different from the hemin reading with Figure data 1 Excel file with data used to make Figure Download FRP1 and FRP2 are required for uptake and The are heme that have a of the iron at the of the Many are toxic to and this on the bacterial pathways et al., 2017; et al., 2017; et al., 2012; et al., et al., 2014). Two and were also found to be toxic to the pathogenic fungus C. and their on of the same required for heme uptake in that organism (Bairwa et al., 2019; et al., 2015). We tested of these two as well as of and in YPD medium supplemented with 1 mM ferrozine to the heme-uptake and found that all of are toxic to C. albicans. To the roles of Frp1 and Frp2 in uptake of these we then tested the frp1-/-, frp2-/-, and double mutants for and sensitivity in YPD medium and in YPD medium buffered to pH (Figure In YPD medium, the frp1-/- mutant showed to all of the tested The frp2-/- mutant showed to and to a to but not to or to At pH the frp2-/- strain but not the frp1-/- strain showed to The frp2-/- strain also showed to and and to a so did the frp1-/- strain was not toxic at pH In Frp1 and Frp2 were required for of at least of the in either unbuffered YPD medium (pH ~ or in medium buffered to pH with Frp1 being more important for in unbuffered medium and Frp2 for at pH Figure 3 with 2 supplements see all Download asset Open asset Frp1 and Frp2 participate in the uptake of heme (A) FRP1 and FRP2 are required for sensitivity to toxic heme The indicated strains were in YPD medium with different concentrations of as and grown in plates at 30°C for 2 days. The the of and the deviations are indicated by vertical bars. The strains used are (B) Expression of either FRP1 or FRP2 is to enable uptake. strain strain that a single FRP1 gene under the promoter and strain that a single FRP2 gene under the promoter were grown in YPD to then exposed to 1 mM for 10 and by = Figure data 1 Excel file with data used to make Figure Download In of the essential role of the CFEM hemophore Pga7 in heme acquisition, and of the that CFEM hemophores can and to we tested whether Pga7 is required for As shown in Figure supplement no could be in the sensitivity of pga7-/- PGA7 indicating that Pga7 is not involved in the uptake of these we Frp1 and Frp2 can mediate uptake of is a fluorescent that heme and that used to heme uptake pathways in and fungal cells et al., 2017; et al., We found that in a wild-type iron the of the cells to and the addition of hemin to the medium efficiently suggesting that is taken by the same pathway as heme (Figure supplement We also noted that unlike in pombe and where the fluorescent is in C. albicans, the fluorescent associated with Since in cells in medium, uptake is due to lack of expression of the heme-uptake system genes, we could whether Frp1 or Frp2 were to mediate uptake by expressing under these conditions. 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