Identification of two Cryptococcus neoformans heme transporters involved in Fhb1-mediated nitrosative stress protection in a fission yeast model

Abstract

In Schizosaccharomyces pombe and Cryptococcus neoformans, the flavohemoglobins (flavoHbs) that protect these yeasts against nitrosative stress are Yhb1 and Fhb1, respectively. Although heme is an essential cofactor for flavoHb activity, the molecular circuitry governing the acquisition of exogenous heme by flavoHbs remains largely unknown. Because S. pombe is a powerful model organism for studying heme biology, Fhb1 was heterologously expressed in S. pombe mutant cells lacking Yhb1. Fhb1 expression functionally complements yhb1Δ cells that are defective in nitric oxide (˙NO) detoxification. Moreover, this Fhb1-mediated complementation requires the S. pombe heme transporter Str3 or the heterologous coexpression of two previously uncharacterized C. neoformans transporters, CnStr1 and CnStr3. Consistent with this finding, heterologous expression of CnStr1 and CnStr3 rescues the heme-dependent growth defect of S. pombe cells that are deficient in heme biosynthesis and lack endogenous heme uptake systems when hemin is provided as the sole heme source. Coimmunoprecipitation assays further demonstrate interactions between Fhb1 and S. pombe Str3 or C. neoformans Str3-like proteins when coexpressed in fission yeast cells. In C. neoformans, strains lacking CnStr1 and CnStr3 exhibit a heme-dependent growth defect in the absence of the cell-surface hemophore Cig1. Similar to the C. neoformans fhb1Δ mutant, a cig1Δ Cnstr1Δ Cnstr3Δ triple mutant fails to protect cells from nitrosative stress. Collectively, these findings reveal functional conservation between S. pombe Yhb1 and C. neoformans Fhb1 in their ability to acquire exogenous heme required for activation as defenders against nitrosative stress.

Introduction

Pathogenic and nonpathogenic yeasts occupy diverse ecological niches and have evolved specialized mechanisms to cope with environmental stress. Nitric oxide (˙NO) is a reactive free radical that becomes cytotoxic at elevated intracellular concentrations (Tillmann et al. 2011; Lundberg and Weitzberg 2022). To counter nitrosative stress caused by excess ˙NO, yeasts have developed distinct detoxification mechanisms that promote resistance to reactive nitrogen species (Tillmann et al. 2011; Cánovas et al. 2016).

In Schizosaccharomyces pombe, resistance to cytotoxic ˙NO is mediated primarily by two enzymes: the ˙NO dioxygenase Yhb1 and the S-nitrosoglutathione reductase Fmd2 (Astuti et al. 2016; Ping et al. 2024). However, recent work demonstrated that the flavohemoglobin (flavoHb) Yhb1 is the primary enzyme protecting S. pombe against the ˙NO donor DETANONOate (Ping et al. 2024). FlavoHbs protect microorganisms from nitrosative stress by catalyzing the conversion of toxic ˙NO to inert nitrate (Gardner et al. 1998; Gardner 2005). Like most flavoHbs identified in fungi, S. pombe Yhb1 is composed of three major domains: an N-terminal heme-binding domain followed by C-terminal FAD- and NAD(P)-binding domains.

Heme consists of a protoporphyrin ring with a centrally coordinated iron atom. A defining property of heme is the ability of its bound iron to reversibly cycle between oxidation states (Fe²⁺ to Fe³⁺, and vice versa). This redox versatility of iron makes heme an essential cofactor for numerous enzymes, including peroxidases, catalases and flavoHbs (Severance and Hamza 2009; Reddi and Hamza 2016; Donegan et al. 2019; Shimizu et al. 2019; Swenson et al. 2020; Dutt et al. 2022). In the case of the flavoHb Yhb1, heme is required for enzymatic activity. A Yhb1 mutant carrying a single amino acid substitution (His¹¹⁴ to alanine) that abolishes heme binding fails to protect S. pombe cells from DETANONOate-induced nitrosative stress (El Hammi et al. 2012; Ping et al. 2024).

Owing to the essential role of heme as a cofactor for Yhb1 and other hemoproteins, S. pombe has evolved two different strategies to ensure heme availability. As a heme prototroph, S. pombe possesses a highly conserved 8-step biosynthetic pathway that supports endogenous heme production. In addition to de novo synthesis, S. pombe can also acquire heme from external sources (Labbé et al. 2020). In contrast to the well-characterized enzymes of the heme biosynthetic pathway, the cellular components involved in exogenous heme acquisition remain poorly defined, largely due to the difficulty of uncoupling heme uptake from endogenous biosynthesis. To overcome this limitation, we developed a S. pombe hem1Δ-based system (Mourer et al. 2015; Labbé et al. 2020). Deletion of hem1⁺ (hem1Δ), which encodes the enzyme catalyzing the first step of heme biosynthesis, abolishes endogenous heme production and renders cells fully dependent on exogenous heme uptake. Alternatively, hem1Δ cells can be rescued by supplementation with δ-aminolevulinate (ALA), which bypasses the Hem1-catalyzed step and restores heme biosynthesis beginning at the second enzymatic step, allowing the pathway to proceed through the remaining reactions required for heme production. Using this hem1Δ-based system in ALA-free medium, studies previously identified two independent heme uptake pathways in S. pombe that require Shu1 and Str3, respectively (Mourer et al. 2015; Normant et al. 2018).

Shu1 is a cell-surface glycosylphosphatidylinositol (GPI)-anchored heme-binding protein (Mourer et al. 2015, 2017). Upon exposure to hemin or the fluorescent heme analog zinc mesoporphyrin IX (ZnMP), Shu1 is internalized from the plasma membrane and trafficked to the vacuole (Mourer et al. 2017). Shu1 primarily mediates delivery of ZnMP to the vacuole, from which it is subsequently delivered into the cytosol when the vacuolar transporter Abc3 is present (Mourer et al. 2017). Vacuolar targeting of Shu1 requires Ubi4-dependent ubiquitination, the intracellular receptor Nbr1, and components of the endosomal sorting complex required for transport (ESCRT) machinery, including Hse1 and Sst6 (Mourer et al. 2019).

A second cell-surface transmembrane protein, Str3, restores the heme-dependent growth defect of hem1Δ cells deficient in heme biosynthesis and lacking Shu1 (shu1Δ) (Normant et al. 2018). Although Str3 is classified as a member of the major facilitator superfamily (MFS) of iron-siderophore transporters, it differs from other family members by the presence of an extracellular domain harboring a bipartite heme-binding motif (Normant et al. 2018). This atypical domain binds hemin with a K_D of 6.6 µM, compared with a K_D value of 2.2 µM for Shu1 (Mourer et al. 2015; Normant et al. 2018). Consistent with its lower affinity for hemin, Str3 rescues the hemin-dependent growth defect of a hem1Δ shu1Δ double mutant strain (expressing endogenous str3⁺) only when exogenous hemin is supplied at approximately twice the concentration required to overcome heme deficiency in a hem1Δ single mutant expressing endogenous shu1⁺ (Normant et al. 2018). A second distinguishing feature of Str3 compared with Shu1 is its cell-surface localization, which remains unchanged upon exposure to varying concentrations of hemin (Normant et al. 2021). Using a proximity-dependent biotinylation-based approach coupled to mass spectrometry, the heme-binding proteins Tpx1, Srx1, and Yhb1 were identified as Str3-interacting partners (Normant et al. 2021; Vahsen et al. 2023; Ping et al. 2024). Although Tpx1 and Srx1 are primarily associated with oxidative stress defense, they are also required for the hemin-dependent growth of hem1Δ shu1Δ cells expressing Str3 under microaerobic and low-iron conditions (Normant et al. 2021; Vahsen et al. 2023). Their hemin-mediated interaction with Str3 may serve to protect hemin from degradation, attenuate its intrinsic peroxidase activity, or facilitate its delivery to downstream proteins and cellular compartments. The physiological relevance of the interaction between the hemeprotein Yhb1 and Str3 becomes apparent when Str3 is inactivated in heme biosynthesis-deficient cells expressing Yhb1 under nitrosative stress conditions. Loss of the str3⁺ gene (str3Δ) results in hypersensitivity to the ˙NO donor DETANONOate under hemin-dependent growth conditions (Ping et al. 2024). These findings indicate that the heme transporter Str3 is required for the functional activation of the cytosolic hemeprotein Yhb1, thereby enabling protection against nitrosative stress.

Cryptococcus neoformans is one of the most severe fungal pathogens affecting humans and has been classified by the World Health Organization as a critical-priority fungus threatening human health (Fisher and Denning 2023; Dao et al. 2024; Denning 2024). This pathogen primarily infects immunocompromised individuals, including patients receiving immunosuppressive therapies and those living with HIV/acquired immunodeficiency syndrome (AIDS) (Sati et al. 2023; Tugume et al. 2023). Following inhalation, C. neoformans colonizes the lungs, causing pneumonia, and subsequently disseminates through the bloodstream to the central nervous system, where it frequently causes fatal meningitis (Iyer et al. 2021; Tugume et al. 2023). During infection, C. neoformans relies on enzymatic defense mechanisms to detoxify host-derived ˙NO. A key enzyme involved in ˙NO detoxification is the flavoHb Fhb1 (de Jesús-Berríos et al. 2003). In contrast, the C. neoformans S-nitrosoglutathione reductase Gno1 is unable to compensate for the loss of Fhb1 in protecting fungal cells against the cytotoxic effects of ˙NO (de Jesús-Berríos et al. 2003).

C. neoformans can acquire heme from external sources (Cadieux et al. 2013; Bairwa et al. 2019). The GPI-anchored, heme-binding mannoprotein Cig1 has been identified as a critical factor for hemin uptake at physiological pH (Cadieux et al. 2013; Li et al. 2025). Recent work showed that Cig1 localizes to the cell periphery and is predominantly associated with the plasma membrane rather than the cell wall (Li et al. 2025). Its presence at the cell surface of C. neoformans makes it a major antigen recognized by the protective host immune system (Li et al. 2025; Avina et al. 2026).

Based on the critical role of Fhb1 in promoting C. neoformans survival by protecting fungal cells against ˙NO, we leveraged the established S. pombe hem1Δ-based system to investigate the heme-dependent activation mechanism of Fhb1 through its heterologous expression in S. pombe cells lacking yhb1⁺ (yhb1Δ). In this context, Fhb1 protected hem1Δ yhb1Δ fmd2Δ mutant cells from nitrosative stress, and this protection was dependent on the presence of the heme transporter Str3. Coimmunoprecipitation (CoIP) assays showed an interaction between Fhb1 and Str3. Heterologous expression of the two C. neoformans Str3-like proteins, CnStr1 and CnStr3, functionally complemented the heme-dependent growth defect of a hem1Δ shu1Δ str3Δ strain. Furthermore, coexpression of either CnStr1 or CnStr3 with Fhb1 enabled growth of heme synthesis-deficient cells supplemented with exogenous hemin in the presence of DETANONOate. Like Str3, both CnStr1 and CnStr3 interacted with Fhb1 in CoIP assays. Consistently, inactivation of these genes in a C. neoformans cig1Δ mutant background resulted in cells that were hypersensitive to ˙NO. Collectively, these findings reveal functional conservation between S. pombe Yhb1 and C. neoformans Fhb1 with respect to their ability to utilize exogenous hemin as the sole source of heme.

Materials and methods

Yeast strains, media, and growth conditions

Genotypic descriptions of the S. pombe and C. neoformans strains used in this study are provided in Table 1. Yeast extract plus supplements (YES) and Edinburgh minimal medium (EMM) were used to grow S. pombe strains under nonselective and selective conditions, respectively (Sabatinos and Forsburg 2010). S. pombe deletion mutant strains were generated using an antibiotic resistance gene disruption cassette engineered for repeated use in fission yeast, as previously described (Gueldener et al. 2002; Iwaki and Takegawa 2004). In the case of S. pombe hem1Δ mutant strains, cells lack Hem1, the first enzyme of the heme biosynthesis pathway, which is responsible for producing δ-ALA (Frontier Speciality Chemicals; #A167), the first porphyrin precursor in the pathway. These strains were maintained alive either by supplementation with ALA or, alternatively, by supplementation with exogenous hemin, thereby forcing reliance on the heme acquisition machinery for growth. In experiments involving exposure to the nitric oxide (˙NO) donor DETANONOate (Cayman Chemicals, Ann Arbor, MI), the pH of YES medium was adjusted to 7.2 using HEPES buffer (1 M HEPES-NaOH, pH 7.5). DETANONOate solutions were freshly prepared as previously described (Ping et al. 2024).

Table 1.

S. pombe strains used in this study.

S. pombe strain	Genotype	Source or reference
FY435	h+ his7-366 leu1-32 ura4-Δ18 ade6-M210	Mourer et al. 2015
TMY17	h+ his7-366 leu1-32 ura4-Δ18 ade6-M210 hem1Δ::loxP shu1Δ::loxP str3Δ::KANr	Normant et al. 2018
FLYPY3	h+ his7-366 leu1-32 ura4-Δ18 ade6-M210 hem1Δ::loxP yhb1Δ::loxP fmd2Δ::KANr	Ping et al. 2024
FLYPY5	h+ his7-366 leu1-32 ura4-Δ18 ade6-M210 hem1Δ::loxP shu1Δ::loxP str3Δ::loxP yhb1Δ::loxP fmd2Δ::loxP	Ping et al. 2024
FLYPY6	h+ his7-366 leu1-32 ura4-Δ18 ade6-M210 hem1Δ::loxP yhb1Δ::loxP fmd2Δ::loxP str3Δ::KANr	This study
C. neoformans strain	Genotype	Source or reference
H99/WT (wild type strain, serotype A)	MATα	J.W. Kronstad collection
FLYPY8	MATα fhb1Δ::HPHr	This study
FLYPY9	MATα cig1Δ::NATr	This study
FLYPY10	MATα str1Δ::HPHr str3Δ::KANr	This study
FLYPY11	MATα cig1Δ::NATr str1Δ::HPHr str3Δ::KANr	This study

For S. pombe liquid proliferation assays, precultures of the indicated strains were initially grown in YES medium supplemented with ALA (25 or 200 μM) and the iron chelator 2,2′-dipyridyl (Dip; 250 μM) (Sigma-Aldrich; #D216305), unless otherwise stated. After preculturing, cells were diluted and transferred to ALA-free medium containing hemin (0.25 or 1 µM) and Dip (25 μM or the indicated concentration), unless otherwise specified. When cultures reached an OD₆₀₀ of 0.8, cells were washed twice and transferred to ALA-free medium (pH 7.2) supplemented with hemin (0.25 or 1 µM), either without DETANONOate or with DETANONOate (3 mM). Cell growth was monitored from this time point (time zero) by measuring OD₆₀₀, as previously described (Ping et al. 2024).

C. neoformans deletion mutant strains were generated using an optimized CRISPR-Cas9 system, as previously described (Huang et al. 2022). In this system, the gene encoding the Cas9 protein was codon optimized for C. neoformans var. grubii strain 99. Editing to create gene disruptions was achieved using a target gene-specific single-guide RNA (sgRNA) expressing cassette together with a resistance gene disruption cassette flanked by short DNA regions homologous to the 5′ and 3′ untranslated regions of the target gene, thereby promoting homologous recombination (Huang et al. 2022).

C. neoformans strains were cultured in YPD (2% Bacto Peptone, 2% dextrose, and 1% yeast extract) or in YNB (6.7 g/L yeast nitrogen base, low copper and iron, without amino acids) (Becton Dickinson; #233520) supplemented with 2% glucose under nonselective conditions. LIM consisted of YNB medium adjusted to pH 7.0 with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and supplemented with Dip (100 µM). For hemin-dependent growth in LIM, exogenous hemin (0.3 μM or otherwise stated) and succinylacetone (SA; 500 μM) (Sigma-Aldrich; #20011), an inhibitor of δ-aminolevulinic acid dehydratase (the second enzyme in the heme biosynthetic pathway), were added. To assess sensitivity to nitrosative stress, the indicated strains were precultured in YPD, washed twice, and diluted 200-fold into a modified LIM medium containing 6.7 g/L yeast nitrogen base (low copper and iron), 2% glucose, and 20 mM HEPES (pH 7.0), supplemented with Dip (100 μM), hemin (0.6 μM), and succinylacetone (500 μM). Cultures were incubated for 17 h in 96-well flat-bottom plates (Grenier) using a SpectraMax M2 plate reader (Molecular Devices) until they reached an optical density at 600 nm (OD₆₀₀) of ∼0.4. At this density, cultures were either left untreated or supplemented with DETANONOate (1 or 3 mM). Cell growth was then monitored by measuring OD₆₀₀ over a 162-h period.

Plasmids

The plasmids pJK-1679yhb1⁺-mCherry, pBPade-785str3⁺-GFP, and pBPGFPalone have been previously described (Normant et al. 2018, 2021; Ping et al. 2024). The cDNA encoding Fhb1 was amplified by reverse transcription PCR (RT-PCR) from mRNA isolated from C. neoformans var. grubii strain 99. The resulting RT-PCR product was digested with NotI and XmaI, and used to replace the equivalent DNA restriction fragment in pJK-1679yhb1⁺-mCherry. The resulting plasmid was designated pJK-1679FHB1-mCherry. The DNA segment corresponding to the yhb1⁺ promoter was subsequently replaced with a SacII-NotI PCR-amplified fragment derived from the S. pombe sod1⁺ promoter, containing the first 860 bp of the 5′ non-coding region. This promoter exchange generated the plasmid pJK-860promsod1-FHB1-mCherry.

The coding sequence of FHB1, excluding the first 654 bp corresponding to the N-terminal 218 amino acids of Fhb1, was amplified by PCR using primers containing NotI and XmaI restriction sites. The resulting PCR product was used to replace the corresponding restriction fragment in pJK-860promsod1-FHB1-mCherry, thereby generating the plasmid pJK-860promsod1-FHB1^Δglobin-mCherry.

PCR amplification was performed using primers designed to introduce ApaI and XmaI restriction sites at the 5′ and 3′ ends of the S. pombe sod1⁺ promoter region, respectively.

The resulting ApaI–XmaI promoter fragment was then used to replace the str3⁺ promoter via the corresponding ApaI and XmaI sites in plasmid pBPade-785str3⁺-GFP. This promoter exchange generated the plasmid pBPade-860promsod1-str3⁺-GFP.

As for FHB1, the cDNAs corresponding to C. neoformans STR1 (CNAG_06761) and STR3 (CNAG_07387) were obtained by RT-PCR from mRNA isolated from C. neoformans var. grubii strain 99. The XmaI-NotI cDNA fragments containing the CnSTR1 and CnSTR3 ORFs were then swapped with the corresponding XmaI-NotI DNA fragment in pBPade-860promsod1-str3⁺-GFP, resulting in plasmids pBPade-860promsod1-CnSTR1-GFP and pBPade-860promsod1-CnSTR3-GFP.

Cladogram analysis

For cladogram analysis, sequences of homologous proteins corresponding to established or predicted MFS-type iron–siderophore-related transporters from S. cerevisiae S288C, S. pombe FY435, and C. neoformans H99 were retrieved from the Saccharomyces Genome Database (SGD), PomBase, and FungiDB. A global multiple sequence alignment, including S. pombe Str1, Str2, and Str3, was performed using MAFFT (version 7.392) with the L-INS-i algorithm (Katoh and Standley 2013). The S. cerevisiae Arn1–Arn4 proteins were included as controls, given the inability of this species to assimilate heme. A cladogram was constructed from the aligned sequences using the Neighbor-Joining method (Saitou and Nei 1987) implemented in MAFFT (version 7.392), and the rooted tree was visualized with ggtree v2.4.1 (Yu 2020).

Protein extraction and CoIP assays

Whole-cell extracts from the indicated S. pombe strains expressing either mCherry-tagged Yhb1, Fhb1, or Fhb1^Δglobin, or GFP-tagged Str3, CnSTR1, or CnSTR3 were prepared using HEGN₁₀₀ lysis buffer containing 20 mM HEPES (pH 7.9), 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 0.1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and a complete protease inhibitor mixture. Equal amounts of protein extracts (10 to 30 µg) were resolved on 6% or 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels and analyzed by immunoblotting as described previously (Brault et al. 2016). For detection of GFP-tagged proteins, mCherry-tagged proteins, and α-tubulin, the following primary antibodies were used: monoclonal anti-GFP antibody B-2 (Santa Cruz Biotechnology), polyclonal anti-mCherry antibody E5D8F (Cell Signaling Technology), and monoclonal anti-α-tubulin antibody B-5-1-2 (Sigma-Aldrich). After incubation with primary antibodies, membranes were washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Biosciences). Protein signals were detected using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences) and visualized with an ImageQuant LAS 4000 instrument (GE Healthcare).

Coimmunoprecipitation (CoIP) assays were performed using hem1Δ yhb1Δ fmd2Δ str3Δ cells coexpressing Fhb1-mCherry with either Str3-GFP or GFP alone, and hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells coexpressing FHB1-mCherry with either CnSTR1-GFP or CnSTR3-GFP. Cells were cultured in ALA-free medium (YES or EMM without thiamine) for 3 h, followed by treatment with Dip (250 µM) and hemin (10 µM) for an additional 3 h prior to cell extract preparation. EMM lacking thiamine was used for cells coexpressing Fhb1-mCherry with GFP alone because expression of this control protein was driven the thiamine-repressible nmt41 promoter. CoIP assays were conducted as described previously (Ping et al. 2024), except that whole-cell extracts were immunoprecipitated using monoclonal anti-GFP antibodies bound to protein G-Sepharose 4 Fast Flow beads (Sigma, GE17-0618-01). Samples were mixed end-over-end for 2 h at 4 °C. Beads were washed 3 times with HEGN₁₀₀ buffer, transferred to fresh microtubes, and subjected to a final wash. Immunoprecipitates were dissociated from the beads by heating at 65 °C for 30 min, resolved by electrophoresis on 8% and 6% SDS–polyacrylamide gels, and analyzed by immunoblotting as described above for the Western blot procedure. The cell extracts (“Total”) loaded onto gels (10 to 15 µg) represented approximately 5% of the total extracts used in the binding reactions for the CoIP experiments.

Fluorescence microscopy

Fluorescence and differential interference contrast (Nomarski) images of cells were acquired using a Nikon Eclipse E800 epifluorescence microscope (Nikon, Melville, NY) equipped with a Hamamatsu ORCA-ER cooled digital camera (Hamamatsu, Bridgewater, NJ). Cells were visualized at 1000× magnification using two filter sets: 465–495 nm (for GFP signal) and 510–560 nm (for mCherry and ZnMP signals). Representative cell fields presented in this study were obtained from 3 independent experiments. Furthermore, the displayed images reflect protein localization patterns observed in 200 cells analyzed per condition.

ZnMP detection and total heme measurements

For fluorescence microscopy-based visualization of ZnMP in S. pombe cells, liquid cultures of the indicated strains were grown in YES medium supplemented with ALA (25 μM) and Dip (125 μM) to an OD₆₀₀ of 0.5. Cells were transferred to ALA-free medium containing Dip (125 µM) and supplemented with 1 µM hemin. Cultures were then divided into two treatment groups. One group was incubated in the presence of 1 µM hemin without ZnMP, whereas the second group was incubated with 10 µM ZnMP and 1 µM hemin for 90 min. Under the latter condition, ZnMP was provided in excess relative to hemin, thereby ensuring the ZnMP detection resulted from its uptake by Str3, CnStr1, or CnStr3. ZnMP treatment was terminated by adding 5 volumes of ice-cold 5% BSA in phosphate-buffered saline (PBS). Cells were collected by centrifugation, resuspended in ice-cold 2% BSA in PBS, and examined by fluorescence microscopy.

For total heme measurements, all samples were prepared and analyzed following cell culture treatments using a fluorometric assay described previously (Plante et al. 2025).

Results

Fhb1 functionally complements fission yeast cells that are defective in nitric oxide detoxification

S. pombe and C. neoformans share a common strategy to counteract the cytotoxic effects of nitric oxide (˙NO). In both yeasts, ˙NO is detoxified by flavoHbs, specifically Yhb1 in S. pombe and Fhb1 in C. neoformans. The functional monomeric forms of Yhb1 and Fhb1 each contain a single N-terminal heme-binding (globin-like) domain, followed by successive C-terminal FAD- and NAD(P)-binding domains (Fig. 1a). Overall, Yhb1 and Fhb1 share 34.2% amino acid identity and 52.7% similarity. Based on this conservation, we tested whether C. neoformans Fhb1 retains its protective function against nitrosative stress when expressed in a heme biosynthesis-deficient (hem1Δ) S. pombe strain lacking fmd2⁺ (fmd2Δ), which encodes the S-nitrosoglutathione reductase. Under these conditions, S. pombe cells were forced to assimilate exogenous hemin supplied in the growth medium as the sole source of heme, while deletion of fmd2 eliminated any potential contribution of Fmd2-mediated ˙NO detoxification.

Fig. 1.

Fhb1 complements the loss of Yhb1 function and protects fission yeast cells from nitrosative stress. a), S. pombe Yhb1 shares 34.2% sequence identity with the full-length Fhb1 protein from C. neoformans, with an overall amino acid similarity of 52.7%. Conserved predicted domains are indicated, including the globin-like domain (red), the FAD-binding domain (blue), and the NAD-binding domain (orange). Amino acid numbering corresponds to the position relative to the first residue of each protein. b and c), Growth of the indicated strains was assessed in ALA-free medium supplemented with hemin (1 µM). Once cultures reached an OD₆₀₀ of 0.8, they were transferred to ALA-free medium (pH 7.2) containing hemin (1 µM), either without DETANONOate (panel b) or with DETANONOate (3 mM) (panel c). Strain growth was monitored at the specified time points. The strain color codes are as follows: gray, hem1Δ yhb1Δ fmd2Δ carrying the empty vector (v. alone); blue, hem1Δ yhb1Δ fmd2Δ expressing yhb1⁺-mCherry; purple, hem1Δ yhb1Δ fmd2Δ expressing FHB1-mCherry; and orange, hem1Δ yhb1Δ fmd2Δ expressing FHB1^Δglobin-mCherry. The graphs show data from 3 independent experiments performed in biological triplicate, with values presented as the mean ± SD. d) S. pombe hem1Δ yhb1Δ fmd2Δ cells expressing the indicated mCherry-tagged alleles were grown in YES medium supplemented with ALA (200 µM) and Dip (250 µM) and then transferred in ALA-free medium supplemented with hemin (1 µM) for 3 h. Whole cell extracts were prepared and analyzed by immunoblotting using a polyclonal anti-mCherry antibody and a monoclonal anti-α-tubulin antibody. Molecular weight standards (M) are shown to the right of the panel.

As control conditions, we first performed growth assays in an ALA-free medium supplemented with hemin (Fig. 1b). These assays were conducted using hem1Δ yhb1Δ fmd2Δ cells harboring an empty integrative vector or expressing either yhb1⁺-mCherry, FHB1-mCherry, or the FHB1^Δglobin-mCherry allele. In the case of FHB1^Δglobin-mCherry allele, the first 654 nucleotides of the FHB1 coding sequence, encompassing the predicted heme-binding domain, were deleted. All transformants exhibited robust hemin-dependent growth, reaching an OD₆₀₀ of 6.2–7.0 after 26 h (Fig. 1b). The same group of strains was transferred to an ALA-free medium supplemented with hemin (1 µM) and grown to an OD₆₀₀ of ∼0.8. At this 0 h time point, the strains were treated with DETANONOate (3 mM) (Fig. 1c). hem1Δ yhb1Δ fmd2Δ cells containing an empty vector exhibited poor growth upon exposure to DETANONOate, showing 4.3-, 3.5-, and 3.7-fold less growth after 23, 24.5, and 26 h, respectively, compared with hem1Δ yhb1Δ fmd2Δ cells expressing Yhb1-mCherry (Fig. 1c). Similarly, upon DETANONOate treatment, hem1Δ yhb1Δ fmd2Δ cells expressing FHB1^Δglobin-mCherry displayed 3.5-, 3.2-, and 3.3-fold less growth at 23, 24.5, and 26 h, respectively, relative to Yhb1-mCherry-expressing cells (Fig. 1c). By contrast, expression of Fhb1-mCherry in hem1Δ yhb1Δ fmd2Δ cells enabled growth when assayed in ALA-free medium supplemented with hemin and DETANONOate. This functional complementation supported growth at 90.4%, 90.0%, and 94.1% of that observed in Yhb1-mCherry-expressing cells at 23, 24.5, and 26 h, respectively (Fig. 1c). Whole-cell extracts were prepared from strains expressing Yhb1-mCherry, Fhb1-mCherry, or Fhb1^Δglobin-mCherry that were used in the cell proliferation assays and analyzed by immunoblotting. The results showed that mCherry-tagged Yhb1, Fhb1, and FHB1^Δglobin were produced in hem1Δ yhb1Δ fmd2Δ cells (Fig. 1d). Collectively, these findings showed that functional complementation of Yhb1 by Fhb1 confirms Fhb1 as the C. neoformans ortholog of S. pombe Yhb1.

Fhb1-mediated nitrosative stress resistance in S. pombe requires the heme transporter Str3

Our previous work showed that hem1Δ fmd2Δ str3Δ cells expressing Yhb1 exhibit a growth defect under nitrosative stress when incubated in ALA-free medium supplemented with hemin and DETANONOate (Ping et al. 2024). To determine whether Fhb1 similarly requires Str3 to protect cells against nitrosative stress when heterologously expressed in S. pombe, an integrative plasmid encoding mCherry-tagged Fhb1 was transformed either together with a plasmid alone or with a plasmid expressing the str3⁺-GFP allele into hem1Δ yhb1Δ fmd2Δ str3Δ cells.

Strains were grown in ALA-free medium supplemented with hemin (0.25 µM) until they reached an OD₆₀₀ of 0.8. Cultures were then washed and transferred to an ALA-free medium (pH 7.2) containing hemin (0.25 µM). Cells expressing Fhb1-mCherry alone or coexpressing Fhb1-mCherry and Str3-GFP exhibited comparable growth, reaching OD₆₀₀ values of 5.6 and 5.9, respectively, after 48 h (Fig. 2a). When these strains were grown to an OD₆₀₀ of ∼0.8 under the same conditions and subsequently treated with DETANONOate (3 mM), hem1Δ yhb1Δ fmd2Δ str3Δ cells expressing only Fhb1-mCherry displayed markedly impaired growth in the presence of DETANONOate, exhibiting 3.0-, 3.3-, and 3.5-fold reductions in growth after 44, 46, and 48 h, respectively, compared with cells coexpressing Fhb1-mCherry and Str3-GFP (Fig. 2b).

Fig. 2.

Str3 is required for Fhb1-mediated resistance to nitrosative stress in S. pombe. a and b) The indicated strains were grown in ALA-free medium supplemented with hemin (0.25 µM) until they reached an OD₆₀₀ of 0.8. Cultures were then washed and transferred to ALA-free medium (pH 7.2) containing hemin (0.25 µM) without DETANONOate supplementation (unfilled symbols, panel a) or with DETANONOate supplementation (3 mM) (filled symbols, panel b). Growth of the strains was monitored at the indicated time points. Strain color codes are as follows: gray unfilled circle, hem1Δ yhb1Δ fmd2Δ carrying the empty vector (v. alone); purple unfilled square, hem1Δ yhb1Δ fmd2Δ expressing FHB1-mCherry; cyan unfilled square, hem1Δ yhb1Δ fmd2Δ str3Δ carrying FHB1-mCherry and an empty vector; orange unfilled square, hem1Δ yhb1Δ fmd2Δ str3Δ coexpressing FHB1-mCherry and str3⁺-GFP; gray filled circle, hem1Δ yhb1Δ fmd2Δ carrying the empty vector (v. alone); purple filled square, hem1Δ yhb1Δ fmd2Δ expressing FHB1-mCherry; cyan filled square, hem1Δ yhb1Δ fmd2Δ str3Δ carrying FHB1-mCherry and an empty vector; orange filled square, hem1Δ yhb1Δ fmd2Δ str3Δ coexpressing FHB1-mCherry and str3⁺-GFP. The graphs show data from 3 independent experiments performed in biological triplicate, with values presented as the mean ± SD.

In parallel control experiments, hem1Δ yhb1Δ fmd2Δ cells harboring an empty plasmid or expressing Fhb1-mCherry were cultured under identical conditions. Both strains reached an OD₆₀₀ of ∼5.6 after 48 h in the absence of DETANONOate (Fig. 2a). Upon DETANONOate treatment, hem1Δ yhb1Δ fmd2Δ cells carrying the empty plasmid exhibited 3.5-, 3.8-, and 3.9-fold lower growth after 44, 46, and 48 h, respectively, than cells expressing Fhb1-mCherry, which retain an endogenous str3⁺ allele (Fig. 2b). Taken together, these results revealed that Str3 is required for Fhb1-dependent protection of S. pombe cells exposed to nitrosative stress.

C. neoformans Fhb1 interacts with Str3 when heterologously expressed in fission yeast

In iron-starved S. pombe cells supplied with hemin, Yhb1 physically associates with Str3 to form a heteroprotein complex (Ping et al. 2024). To determine whether Fhb1 similarly interacts with Str3 when heterologously expressed in S. pombe, different allele combinations were co-integrated into hem1Δ yhb1Δ fmd2Δ str3Δ cells to coexpress the following pairs: FHB1-mCherry with str3⁺-GFP, FHB1-mCherry with GFP alone, and yhb1⁺-mCherry with str3⁺-GFP. Cotransformed cells were grown in ALA-free medium supplemented with hemin (10 µM) under low-iron conditions (250 µM Dip). After 3 h, total cell extracts were prepared and incubated with protein G-Sepharose beads coupled to anti-GFP antibodies to enrich Str3-GFP and associated proteins. Immunoprecipitation assays showed that both Str3-GFP and GFP alone were consistently retained on the beads, as detected in the immunoprecipitated (IP) fractions (Fig. 3a–c). Immunoblot analysis of bead-associated proteins using an anti-mCherry antibody revealed that Fhb1-mCherry specifically co-immunoprecipitated with Str3-GFP (Fig. 3a). As a positive control, Yhb1-mCherry was also consistently detected in association with Str3-GFP (Fig. 3b). In contrast, Fhb1-mCherry was not detected in the immunoprecipitated fraction from cells coexpressing Fhb1-mCherry and GFP alone, confirming the specificity of the interaction (Fig. 3c). Further validation of assay specificity was provided by the absence of significant α-tubulin signal in all immunoprecipitated fractions (Fig. 3a–c). Collectively, these findings showed that, like Yhb1, C. neoformans Fhb1 interacts with the heme transporter Str3 when heterologously expressed in iron-starved S. pombe cells exposed to exogenous hemin.

Fig. 3.

Fhb1 and Str3 form a heteroprotein complex in iron-starved S. pombe cells grown in the presence of hemin. a–c), hem1Δ yhb1Δ fmd2Δ str3Δ cells coexpressing Fhb1-mCherry with either Str3-GFP (panel a) or GFP alone (panel c) were cultured in an ALA-free medium for 3 h and then treated with Dip (250 µM) and hemin (10 µM) for an additional 3 h before preparing cell extracts. Whole-cell extracts (Total) were subjected to immunoprecipitation (IP) using an anti-GFP antibody coupled to protein G-Sepharose beads. Bound proteins were eluted and analyzed by immunoblot assays using anti-Cherry, anti-GFP, and anti-α-tubulin antibodies. As a positive control (panel b), S. pombe hem1Δ yhb1Δ fmd2Δ str3Δ cells coexpressing Yhb1-mCherry and Str3-GFP, which are known to interact with one another, were analyzed under the same conditions. Aliquots of total cell extracts were included to verify the presence of epitope-tagged proteins prior to IP. Molecular weight standards (M) are shown to the right side of each panel.

CnStr1 and CnStr3 from C. neoformans exhibit cell-surface localization comparable to Str3 when heterologously expressed in fission yeast

Based on the finding that Fhb1 interacts with S. pombe Str3, we searched for Str3-like proteins in C. neoformans. Six homologous proteins were identified, four of which were more closely related to MFS-type iron-siderophore transporters described in S. cerevisiae and S. pombe (Fig. 4a) (Lesuisse et al. 1998; Heymann et al. 1999, 2000a, 2000b; Yun et al. 2000; Kim et al. 2005; Philpott 2006; Plante and Labbé 2019; Mbuya et al. 2025). The remaining two proteins, designated CnStr1 and CnStr3, exhibited the highest sequence similarity to S. pombe Str3 and were therefore selected as candidate orthologs. Using integrative plasmids, S. pombe hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells were cotransformed with distinct pairs of alleles, including FHB1-mCherry and str3⁺-GFP, FHB1-mCherry and CnSTR1-GFP, or FHB1-mCherry and CnSTR3-GFP. All alleles were expressed under the control of the constitutive S. pombe sod1⁺ promoter, except for the str3⁺-GFP allele, which was expressed from its native promoter. Cotransformed strains were cultured in ALA-free medium for 3 h and then treated with hemin (1 µM) and Dip (250 µM) for an additional 3 h prior to whole-cell extract preparation. Immunoblot analysis confirmed the production of all four proteins, including those corresponding to the C. neoformans Str3-like proteins (Fig. 4b).

Fig. 4.

Expression and cellular localization of C. neoformans Fhb1, CnStr1, and CnStr3 in fission yeast. a) Established and predicted MFS-type iron-siderophore transporters from S. cerevisiae (ScArn1, ScArn2, ScArn3, ScArn4), S. pombe (SpStr1, SpStr2), and C. neoformans (CnSit1, CnSit2, CNAG_07751, CNAG_07519) were included for comparison in a phylogenetic cladogram analysis alongside the established S. pombe heme transporter Str3 and the 2 predicted C. neoformans heme transporters CnStr1 and CnStr3. Scale bar 0.1 represents 10 substitutions per 100 residues. b) S. pombe hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells coexpressing FHB1-mCherry and either str3⁺-GFP, CnSTR1-GFP, or CnSTR3-GFP were cultured in an ALA-free medium for 3 h and then treated with hemin (1 µM) and Dip (250 µM) for an additional 3 h before preparing cell extracts. Whole-cell extracts preparations were analyzed by immunoblotting using anti-GFP, anti-mCherry, and anti-α-tubulin antibodies. Molecular weight standards (M) are shown to the right side of each panel. c) Aliquots of the cultures used in panel b were subjected to fluorescence microscopy to visualize the cellular localization of Fhb1-mCherry, Str3-GFP, CnStr1-GFP, and CnStr3-GFP (center left and center right panels). Cell morphology was examined using Nomarski optics (far-left panels). Merged images of Fhb1-mCherry with GFP-tagged Str3, CnStr1, or CnStr3 fluorescent signals are shown in the far-right panels. d) The percentages of cell populations exhibiting peripheral green fluorescence are presented graphically. Results are shown as averages ± SD from 3 independent experiments, each performed in biological triplicate, with at least 200 cells analyzed to assess protein localization.

We next examined the subcellular localization of CnSTR1-GFP and CnSTR3-GFP when coexpressed with Fhb1-mCherry in hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells. Fluorescence microscopy of cells coexpressing Fhb1-mCherry and either CnStr1-GFP or CnStr3-GFP revealed that both GFP-tagged proteins were predominantly localized at the cell periphery, similar to the Str3-GFP signal, which served as a plasma membrane marker (Fig. 4c) (Normant et al. 2018). In contrast, the Fhb1-mCherry red fluorescence signal was mainly observed throughout the cytoplasm (Fig. 4c). Collectively, these results indicated that heterologous expression of C. neoformans CnStr1 and CnStr3 in fission yeast leads to their localization at the plasma membrane in the presence of hemin under low-iron conditions.

Expression of CnSTR1 or CnSTR3 complements the heme-dependent growth deficit of S. pombe hem1Δ shu1Δ str3Δ cells

To further investigate the function of CnStr1 and CnStr3 when heterologously expressed in S. pombe, GFP-tagged CnSTR1, CnSTR3, CNAG_07519, and str3⁺ alleles were integrated into hem1Δ shu1Δ str3Δ cells to assess their ability to rescue the hemin-dependent growth defect. As previously reported, when ALA was omitted from the medium and no exogenous hemin was provided as a source of heme, hem1Δ shu1Δ str3Δ cells transformed with an empty plasmid exhibited no significant growth over a 70-h period (OD₆₀₀ of 0.24 after 70 h) (Fig. 5a) (Normant et al. 2018). Similarly, integration of the CnSTR1-GFP, CnSTR3-GFP, CNAG_07519-GFP, or str3⁺-GFP alleles failed to support growth under these conditions (Fig. 5a).

Fig. 5.

Expression of CnSTR1 or CnSTR3 restores the ability of S. pombe hem1Δ shu1Δ str3Δ cells to utilize exogenous hemin as the sole source of heme. hem1Δ shu1Δ str3Δ mutant cells expressing str3⁺-GFP, CnSTR1-GFP, CnSTR3-GFP, or CNAG_07519-GFP were precultured in the presence of ALA (25 µM). During the final 3 h of preculture, Dip was added (250 μM). Cells were then washed and diluted into ALA-free medium containing Dip (25 µM), either without hemin (panel a) or supplemented with hemin (0.25 µM) (panel b). Cell growth was monitored at the indicated time points over a 70-h period. Growth curves from 3 biological replicates are shown, with values presented as the means ± SD. Color codes for hem1Δ shu1Δ str3Δ cells expressing the following alleles are as follows: green, str3⁺-GFP; orange, CnSTR1-GFP; pink, CnSTR3-GFP; blue, CNAG_07519-GFP; and black, empty integrative plasmid (v. alone). The graphs represent data from 3 independent experiments.

In contrast, upon supplementation of ALA-free medium with hemin (0.25 µM), hem1Δ shu1Δ str3Δ cells expressing GFP-tagged CnSTR1, CnSTR3, or str3⁺ alleles regained robust growth, reaching OD₆₀₀ values of 1.27, 1.22, and 1.33, respectively, after 70 h (Fig. 5b). As controls, hemin-supplemented hem1Δ shu1Δ str3Δ cells harboring an empty plasmid or the GFP-tagged CNAG_07519 allele consistently exhibited no significant growth (OD₆₀₀ of 0.23 and 0.24, respectively, after 70 h) compared with cells expressing the GFP-tagged CnSTR1, CnSTR3, or str3⁺ alleles (Fig. 5b). Taken together, these results showed that heterologous expression of CnSTR1 or CnSTR3 rescues the heme-dependent growth defect of S. pombe hem1Δ shu1Δ str3Δ cells when hemin is provided as the sole source of heme.

CnStr1 and CnStr3 promote ZnMP and heme assimilation when expressed in S. pombe hem1Δ shu1Δ str3Δ cells

To validate the role of CnStr1 and CnStr3 in the acquisition of exogenous hemin when heterologously expressed in S. pombe, hem1Δ shu1Δ str3Δ cells harboring an empty plasmid or expressing the CnSTR1-GFP, CnSTR3-GFP or str3⁺-GFP alleles were transferred to ALA-free medium supplemented with Dip and hemin (1 μM) and then subsequently left untreated or supplemented with ZnMP (10 μM) for 90 min. In hem1Δ shu1Δ str3Δ cells expressing the 2 C. neoformans Str3-like proteins (CnStr1-GFP and CnStr3-GFP), ZnMP-associated fluorescence was primarily observed in the cytoplasm, similar to the pattern observed upon reintroduction of the str3⁺-GFP allele (Fig. 6a–d). Furthermore, ZnMP-associated fluorescence was detected in 97 to 99% of the 200 cells examined in each culture (Fig. 6e). In contrast, no detectable ZnMP fluorescence was observed in hem1Δ shu1Δ str3Δ cells transformed with an empty expression plasmid or in cells not exposed to ZnMP (Fig. 6a–d).

Fig. 6.

Heterologous expression of CnSTR1 or CnSTR3 in S. pombe hem1Δ shu1Δ str3Δ cells promotes cytoplasmic accumulation of ZnMP and heme. a–c), hem1Δ shu1Δ str3Δ cells expressing str3⁺-GFP, CnSTR1-GFP, or CnSTR3-GFP alleles were precultured in the presence of ALA (25 µM). The cultures were transferred in ALA-free medium supplemented with hemin (1 µM) and Dip (125 µM) for 90 min. Subsequently, cultures were divided into 2 treatment groups. One group was incubated in the absence of ZnMP, whereas the second group was incubated under the same conditions with ZnMP supplementation (10 µM) for 90 min. Cells were analyzed by fluorescence microscopy to visualize the localization of Str3-GFP, CnStr1-GFP, or CnStr3-GFP (center left), and the accumulation of fluorescent ZnMP (center right). Merged images are shown in the far-right panels, whereas Nomarski optics (far-left) was used to assess cell morphology. White arrowheads indicate the cell periphery. d) As a control, hem1Δ shu1Δ str3Δ cells harboring an empty vector (v. alone) were cultured under identical conditions. e) The percentages of cell populations exhibiting ZnMP-dependent fluorescence are presented graphically. Results are expressed as means ± SD from 3 independent experiments, each conducted in biological triplicate, with at least 200 cells analyzed to assess ZnMP fluorescence detection. f) Following preculture, the indicated strains were transferred to ALA-free medium with Dip (125 µM) and then divided into 2 treatment groups. One group was maintained under the same conditions, whereas the other group was incubated under identical conditions with hemin supplementation (1 µM) for 4 h. Total cellular heme content was measured using a fluorometric-based assay and expressed as picomoles of heme per cell. Results are expressed as means ± SD from 3 independent experiments, each conducted in biological triplicate. Asterisks indicate statistical significance (***P < 0.001; ****P < 0.0001, two-way ANOVA with Tuckey’s multiple comparisons test against the ZnMP-treated strains), whereas ns indicates no significant difference. g) Once precultures of the indicated strains reached mid-logarithmic phase in YES medium containing ALA (25 µM), cells were washed and then diluted 1,000-fold into ALA-free medium supplemented with hemin (1 µM), Dip (125 µM), and 0, 10, 25, 50, or 100 µM MnPPIX. The indicated strains were grown in 96-well plates at 30 °C for 3 d. Strain color codes are as follows: black, hem1Δ shu1Δ str3Δ; green, hem1Δ shu1Δ str3Δ expressing str3⁺-GFP; red, hem1Δ shu1Δ str3Δ expressing CnSTR1-GFP; and, blue, hem1Δ shu1Δ str3Δ expressing CnSTR3-GFP. Results are expressed as means ± SD from 3 independent experiments, each performed in biological triplicate.

Subsequently, experiments were performed to quantify intracellular heme levels in ALA-starved hem1Δ shu1Δ str3Δ cells expressing CnSTR1-GFP, CnSTR3-GFP or str3⁺-GFP. Following preculture, the indicated strains were transferred to ALA-free medium with Dip (125 µM) and then divided into 2 treatment groups. One group was maintained under the same conditions, whereas the other group was incubated under identical conditions with hemin supplementation (1 µM) for 4 h. In the absence of hemin supplementation, intracellular heme levels were measured at 0.12, 0.09, and 0.11 pmol per cell in strains expressing CnStr1-GFP, CnStr3-GFP or Str3-GFP, respectively (Fig. 6f). Upon hemin supplementation, intracellular heme levels increased to 0.81, 0.94, and 1.01 pmol per cell, respectively. In contrast, hem1Δ shu1Δ str3Δ cells harboring an empty plasmid displayed no significant changes in heme levels under the 2 experimental conditions, exhibiting values of 0.11 and 0.13 pmol per cell, respectively (Fig. 6f).

We next performed cell proliferation assays using the non-iron metalloporphyrin MnPPIX, which is known to be taken up by several microorganisms, including Candida albicans and C. neoformans, through their heme uptake systems (Bairwa et al. 2019; Roy et al. 2022). MnPPIX exerts antifungal activity that is thought to result from interference with essential hemoproteins. The indicated strains were seeded at pre-early logarithmic phase into ALA-free medium supplemented with hemin (1 µM), Dip (125 µM), and 0, 10, 25, 50, or 100 µM MnPPIX and incubated for 3 d. Under these experimental conditions, hem1Δ shu1Δ str3Δ cells expressing CnStr1-GFP, CnStr3-GFP, or Str3-GFP displayed 2.04-, 1.74-, and 2.02-fold lower growth, respectively, in the presence of 50 µM MnPPIX compared to hem1Δ shu1Δ str3Δ cells harboring an empty plasmid (Fig. 6g). Similarly, hem1Δ shu1Δ str3Δ cells expressing CnStr1-GFP, CnStr3-GFP, or Str3-GFP also exhibited increased sensitivity to MnPPIX at lower concentrations, including 10 and 25 µM, further supporting their involvement in the uptake of this compound when heterologously expressed in fission yeast. Taken together, these results revealed that heterologous expression of CnStr1 or CnStr3 in S. pombe hem1Δ shu1Δ str3Δ cells promotes cytoplasmic assimilation of the heme analog ZnMP, increases intracellular heme accumulation upon hemin supplementation, and enhances sensitivity to the cytotoxic effects of non-iron metalloporphyrin MnPPIX.

S. pombe hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells coexpressing FHB1 together with either CnSTR1 or CnSTR3 are protected against nitrosative stress

Based on the finding that heterologous expression of Fhb1 in fission yeast confers protection against nitrosative stress, we next assessed whether this protective effect was maintained when Fhb1-expressing hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells were transformed with either the CnSTR1-GFP or CnSTR3-GFP allele. Transformed cells were grown to an OD₆₀₀ of 0.8 in ALA-free medium supplemented with hemin (0.25 µM), and then transferred to the same medium adjusted to pH 7.2 and supplemented with hemin (0.25 µM) and DETANONOate (3 mM) (Fig. 7). Cell proliferation assays showed that hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells coexpressing Fhb1-mCherry with either CnStr1-GFP or CnStr3-GFP exhibited hemin-dependent growth, with both cultures reaching OD₆₀₀ values of 6.0 after 47 h in the presence of DETANONOate (Fig. 7). As a control, hem1Δ yhb1Δ fmd2Δ cells expressing FHB1-mCherry together with the endogenous str3⁺ gene also grew efficiently under the same nitrosative stress conditions, although to a lesser extent (Fig. 7). In contrast, a pronounced growth defect was observed in DETANONOate-treated hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells expressing CnStr1-GFP or CnStr3-GFP alone, in the absence of Fhb1-mCherry. Under these conditions, cells displayed 4.7- and 5.2-fold reduced growth at 47 h, respectively, compared with cells coexpressing Fhb1-mCherry with either CnStr1-GFP or CnStr3-GFP (Fig. 7). Taken together, these results showed that fission yeast cells lacking Yhb1 and the heme uptake proteins Shu1 and Str3 retain resistance to nitrosative stress when they coexpress specific pairs of C. neoformans proteins, namely Fhb1 with either CnStr1 or CnStr3.

Fig. 7.

Fhb1 together with either CnStr1 or CnStr3 is required to support hemin-dependent growth of S. pombe hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells in the presence of DETANONOate. The S. pombe hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ mutant was cotransformed with the indicated combinations of empty vector (v. alone), FHB1-Cherry, CnSTR1-GFP, and CnSTR3-GFP alleles. In parallel, the hem1Δ yhb1Δ fmd2Δ mutant was transformed with the FHB1-Cherry allele. Strains were grown in ALA-free medium supplemented with hemin (0.25 µM) to an OD₆₀₀ of 0.8, then washed and transferred to ALA-free medium (pH 7.2) containing hemin (0.25 µM) and DETANONOate (3 mM). Growth was monitored at the indicated time points. Strain color codes are as follows: blue, hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ coexpressing FHB1-mCherry and CnSTR1-GFP; green, hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ coexpressing FHB1-mCherry and CnSTR3-GFP; red, hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ expressing CnSTR1-GFP with an empty vector; black, hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ expressing CnSTR3-GFP with an empty vector; and purple, hem1Δ yhb1Δ fmd2Δ expressing FHB1-mCherry. The graph shows data from 3 independent experiments performed in biological triplicate, with values presented as the mean ± SD.

Fhb1 interacts with either CnSTR1 or CnSTR3 when heterologously expressed in fission yeast

Coimmunoprecipitation assays showed that Fhb1-mCherry interacts with S. pombe Str3-GFP when heterologously expressed in fission yeast lacking Yhb1 (Fig. 3a). Based on this finding, we next assessed whether Fhb1-mCherry interacts with either CnStr1-GFP or CnStr3-GFP in hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells coexpressing these C. neoformans proteins. Cells were cultured in ALA-free medium for 3 h and then treated with hemin (10 µM) and Dip (250 µM) for an additional 3 h prior to preparation of whole-cell extracts. Subsequently, extracts were incubated with anti-GFP protein G-Sepharose beads to selectively retain GFP and GFP-tagged CnStr1 and CnStr3 proteins. The immunoprecipitation (IP) fractions consistently contained GFP alone, CnStr1-GFP, or CnStr3-GFP bound to the beads (Fig. 8a–c). Importantly, when extracts from cells expressing CnStr1-GFP or CnStr3-GFP were used, Fhb1-mCherry was detected in the corresponding immunoprecipitated fractions, indicating that Fhb1 associates with either CnStr1 or CnStr3 in a heteroprotein complex when cells were cultured in ALA-free medium supplemented with exogenous hemin under low-iron conditions. In contrast, Fhb1-mCherry was not detected in the immunoprecipitated fraction from cells coexpressing GFP alone (Fig. 8c). As a control, α-tubulin was readily detected in total cell extracts but was not significantly detected in the immunoprecipitated fractions.

Fig. 8.

Fhb1-mCherry associates with either CnStr1-GFP or CnStr3-GFP when expressed in S. pombe under hemin-dependent conditions. a–c), hem1Δ yhb1Δ fmd2Δ shu1Δ str3Δ cells coexpressing FHB1-mCherry together with either CnSTR1-GFP, CnSTR3-GFP, or GFP alone were cultured in an ALA-free medium for 3 h, and subsequently treated with Dip (250 µM) and hemin (10 µM) for an additional 3 h prior to extract preparation. Whole-cell extracts (Total) were incubated with protein G-Sepharose resin coupled to anti-GFP antibodies. After washing, the bound fractions (IP) were analyzed by immunoblotting using anti-GFP and anti-mCherry antibodies to detect CnStr1-GFP, CnStr3-GFP, or GFP alone, and Fhb1-mCherry, respectively. As a control of fractionation, aliquots of both total extracts and immunoprecipitated fractions were probed with an anti-α-tubulin antibody. Aliquots of total protein lysates were also analyzed to confirm the presence of epitope-tagged proteins prior to immunoprecipitation. Molecular weight standards (M) are shown to the right side of each panel.

C. neoformans mutant strains lacking CnStr1, CnStr3, and Cig1 are sensitive to nitrosative stress under hemin-dependent growth conditions

Previous studies established culture conditions to assess hemin-dependent growth of C. neoformans (Vartivarian et al. 1993; Lian et al. 2005; Saikia et al. 2014). Strains were initially cultured in yeast nitrogen base (YNB) medium supplemented with glucose (2%) under nonselective conditions. As a control, we verified that all strains, including the wild-type and mutant strains, grew normally in YNB medium (Fig. 9a). As an additional control, we validated that all strains exhibited a succinylacetone (SA)-mediated inhibitory effect on their growth when this inhibitor of δ-aminolevulinic acid dehydratase was added to the YNB medium (Fig. 9b). When the YNB medium was adjusted to pH 7.0 and supplemented with the iron chelator Dip (100 µM), this modified low-iron medium, referred to as LIM, did not provide sufficient iron to support growth of C. neoformans strains (Fig. 9c), as previously reported (Cadieux et al. 2013).

Fig. 9.

Deletion of the CnSTR1 and CnSTR3 genes in a cig1Δ mutant strain compromises its growth when hemin is provided as the sole source of heme. a) The indicated strains were precultured in YPD medium, then washed, diluted, and transferred to YNB medium supplemented with glucose (2%), where growth was monitored at the indicated time points. b) The same strains were precultured under identical conditions, then diluted and transferred to YNB medium supplemented with glucose (2%) in the absence or presence of succinylacetone (SA; 500 µM). c) Following preculture in YPD medium, the indicated strains were diluted and transferred to iron-depleted LIM medium, in which a growth defect was observed. d) Following preculture as described in panel a, growth of the same set of strains was assessed in LIM medium supplemented with hemin (0.3 µM) and SA (500 µM). Strain color codes are as follows: black, wild-type; purple, fhb1Δ; orange, Cnstr1Δ Cnstr3Δ; green, cig1Δ; and, blue, cig1Δ Cnstr1Δ Cnstr3Δ. The graphs show data from 3 independent experiments performed in biological triplicate, with values presented as the mean ± SD.

To sustain the growth of C. neoformans cells in LIM, exogenous hemin (0.3 µM) was added to enforce reliance on the heme uptake system in the presence of succinylacetone (SA; 500 µM), a metabolic inhibitor that targets ALA dehydratase and blocks endogenous heme biosynthesis at the second enzymatic step of the pathway. Under these conditions, both the wild-type and fhb1Δ mutant strains exhibited robust hemin-dependent growth, reaching OD₆₀₀ values of 1.03 and 1.02, respectively, after 164 h (Fig. 9d). In contrast, the cig1Δ mutant exhibited 9.4- and 7.3-fold reduced growth compared with the wild-type strain after 92 and 120 h, respectively (Fig. 9d). As previously reported (Cadieux et al. 2013), growth of the cig1Δ mutant progressively increased after 120 h, reaching an OD₆₀₀ of 0.59 after 164 h, whereas the wild-type strain had already reached stationary phase at this time point (OD₆₀₀ of 1.03) (Fig. 9d).

Analysis of the impact of CnStr1 and CnStr3 deletion (Cnstr1Δ Cnstr3Δ) revealed that growth of the double mutant in LIM supplemented with hemin (0.3 µM) exhibited slightly lower OD₆₀₀ values than the wild-type strain, reaching OD₆₀₀ values of 0.91, 0.94, 0.95, and 0.96 after 120 h, 140, 148, and 164 h, respectively, compared with the wild-type strain, which reached OD₆₀₀ values of 0.98, 1.02, 1.05, and 1.06 at the corresponding time points (Fig. 9d). Importantly, C. neoformans cells carrying the cig1Δ Cnstr1Δ Cnstr3Δ triple deletion exhibited markedly reduced growth, with OD₆₀₀ values of 0.05, 0.07, 0.08, and 0.09 after 120, 140, 148, and 164 h, respectively, compared with the cig1Δ single mutant, which reached OD₆₀₀ values of 0.13, 0.30, 0.37, and 0.59 at the corresponding time points (Fig. 9d). Collectively, these results revealed that the growth defect phenotype associated with loss of CnSTR1 and CnSTR3 (Cnstr1Δ Cnstr3Δ) is primarily conditional upon deletion of CIG1 (cig1Δ).

Based on the cell proliferation assay results shown in Fig. 9d, we introduced 2 modifications to investigate the effect of DETANONOate exposure on C. neoformans strains. First, following preculture in rich medium, the indicated strains were washed and diluted 200-fold to resume growth at an initial OD₆₀₀ of 0.1 instead of 0.05. Second, the concentration of hemin added to the YNB medium was increased twofold (0.6 µM instead of 0.3 µM), which allowed more uniform growth across all strains and significantly reduced growth differences under nonstress conditions (Fig. 10a). The objective was to ensure that all strains reached approximately the same cell density (OD₆₀₀ of ∼0.4) prior exposure to DETANONOate. At this stage, cultures were either treated with DETANONOate (1 mM) or left untreated to compare their relative growth (Fig. 10, a and b). As expected, fhb1Δ cells exhibited a significant growth defect, with an OD₆₀₀ value of 0.39 after 162 h. (Figure 10b). This DETANONOate-mediated inhibition corresponded to a 2.2-fold reduction in growth compared with the wild-type strain (OD₆₀₀ of 0.87 after 162 h). In addition to the fhb1Δ mutant, the cig1Δ and cig1Δ Cnstr1Δ Cnstr3Δ mutant strains displayed the next most pronounced growth defects, exhibiting 1.5- and 1.7-fold reductions in growth, respectively, compared to the wild-type strain after 162 h (Fig. 10b). In the case of the Cnstr1Δ Cnstr3Δ double mutant, results showed only a slight growth defect upon DETANONOate exposure (OD₆₀₀ of 0.80 after 162 h) compared with the wild-type strain (OD₆₀₀ of 0.87 after 162 h), which was much less severe than that observed for the cig1Δ and cig1Δ Cnstr1Δ Cnstr3Δ mutants (OD₆₀₀ of 0.57 and 0.50, respectively, after 162 h) (Fig. 10b).

Fig. 10.

Effect of the absence of the 3 proteins CnStr1, CnStr3, and Cig1, as well as of Fhb1 on the growth sensitivity of C. neoformans to nitrosative stress. a–c) C. neoformans H99 (wild-type) and the isogenic fhb1Δ, cig1Δ, Cnstr1Δ Cnstr3Δ, and cig1Δ Cnstr1Δ Cnstr3Δ mutant strains were grown in LIM medium (pH 7.0) supplemented with Dip (100 µM), hemin (0.6 µM), and succinylacetone (SA; 500 µM). When cultures reached mid-logarithmic phase (OD₆₀₀ of ∼0.4), they were either left untreated (without DETANONOate supplementation) or treated with 1 or 3 mM DETANONOate. At this stage, growth of the strains was then monitored at the indicated time points. Strain color codes are as follows: black, wild-type; purple, fhb1Δ; green, cig1Δ; orange, Cnstr1Δ Cnstr3Δ; and blue, cig1Δ Cnstr1Δ Cnstr3Δ. The graphs show data from 3 independent experiments performed in biological triplicate, with values presented as the mean ± SD.

In a subsequent series of cell proliferation assays performed under the same conditions, except with 3 mM DETANONOate instead of 1 mM, the Cnstr1Δ Cnstr3Δ double mutant exhibited a much more pronounced defect, showing a 2.1-fold reduction in growth compared to the wild-type strain after 167 h (Fig. 10c). As expected, the cig1Δ, cig1Δ Cnstr1Δ Cnstr3Δ, and fhb1Δ mutants also displayed strong growth defects, with 2.4-, 2.5-, and 2.5-fold reductions in growth, respectively, compared to the wild-type strain after 167 h (Fig. 10c). Taken together, these findings indicated that 1 mM DETANONOate is below the optimal range to clearly reveal defects in nitrosative stress resistance in the absence of CnStr1 and CnStr3. At this concentration, the phenotype is only beginning to emerge, whereas it becomes pronounced at 3 mM DETANONOate.

In parallel experiments, the the wild-type strain, Cnstr1Δ Cnstr3Δ, cig1Δ, and cig1Δ Cnstr1Δ Cnstr3Δ mutants were analyzed for their heme content following transfer into LIM medium supplemented with Dip and SA, in the absence or presence of exogenous hemin, for 48 h. In the absence of hemin supplementation, intracellular heme levels were measured at 0.23, 0.20, 0.21, and 0.23 pmol per cell in the wild-type strain, Cnstr1Δ Cnstr3Δ, cig1Δ, and cig1Δ Cnstr1Δ Cnstr3Δ mutants, respectively (Supplementary Fig. 2). Importantly, upon hemin supplementation, intracellular heme levels reached 2.22 pmol per cell in the wild-type strain, whereas consistently lower heme levels were monitored in the Cnstr1Δ Cnstr3Δ, cig1Δ, and cig1Δ Cnstr1Δ Cnstr3Δ mutants, with values of 1.49, 0.79, and 0.50 pmol per cell, respectively (Supplementary Fig. 2). These results showed that reduced total intracellular heme content in the mutant strains correlates with diminished Fhb1-mediated protection against DETANONOate.

Discussion

In C. neoformans, the flavoHb Fhb1 is a key enzyme for ˙NO detoxification, as its inactivation abolishes cell growth upon exposure to DETANONOate (1 mM), whereas the wild-type strain proliferates normally (de Jesús-Berríos et al. 2003). Although Fhb1 requires heme for its activity, the mechanism by which it acquires exogenous hemin from external sources remains unknown. In this study, we investigated the activation mechanism of Fhb1 through its heterologous expression in heme biosynthesis-deficient S. pombe cells lacking yhb1⁺ (yhb1Δ) as well as the S-nitrosoglutathione reductase gene fmd2⁺ (fmd2Δ). Although previous work showed that Fmd2 does not compensate for the loss of Yhb1 (Ping et al. 2024), we eliminated any potential minor contribution from Fmd2 by using strains deleted for this gene.

To assess the degree of functional conservation between Fhb1 and Yhb1, C. neoformans FHB1 cDNA was expressed in 2 S. pombe strains: hem1Δ yhb1Δ fmd2Δ and hem1Δ yhb1Δ fmd2Δ str3Δ. Heterologous expression of Fhb1 restored resistance to DETANONOate in the presence of the heme transporter Str3, but not in its absence. This growth-based functional assay strongly suggests that Fhb1 functions similarly to Yhb1 when heterologously expressed in S. pombe and requires the Str3 heme uptake protein at the cell surface to acquire its heme cofactor.

Based on these results, 6 candidate MFS-type transporters previously reported to be differentially expressed in response to changes in iron availability in C. neoformans (Jung et al. 2010, 2021; Do et al. 2020) were analyzed for sequence similarity to S. pombe Str3. Two candidates, CnStr1 (CNAG_06761) and CnStr3 (CNAG_07387), were identified as putative functional orthologs, as they exhibited the highest sequence identity and similarity scores, along with the greatest conservation of amino acid residues, relative to S. pombe Str3.

As observed for Str3, membrane topology and structural predictions for CnStr1 and CnStr3 revealed fourteen α-helical transmembrane segments organized into 2 similar bundles, comprising 6 helices in the first bundle and 8 helices in the second, both connected by a central cytoplasmic loop located between transmembrane segments 6 and 7. A distinctive feature of fungal MFS-type transporters involved in the uptake of hemin and siderophore-bound iron is the presence of an atypical, extended C-terminal hydrophilic loop located between the last 2 transmembrane domains (Kim et al. 2005; Normant et al. 2018; Plante and Labbé 2019; Mbuya et al. 2025). In the case of the S. pombe heme transporter Str3, this loop contains a bipartite hemin-binding motif, ⁵³⁰YX₃Y⁵³⁴ and ⁵⁵²SX₄Y⁵⁵⁷, which is absent from the siderophore-iron transporters Str1 and Str2 (Normant et al. 2018). Residues resembling the left-half motif of Str3 (⁵³⁰YX₃Y⁵³⁴) were identified in CnStr1 and CnStr3 at positions corresponding to ⁵³⁴YX₄H⁵³⁹ and ⁵⁴⁴YX₄Y⁵⁴⁹, respectively. The location of these motifs within the final hydrophilic loop is similar to that of the ⁵³⁰YX₃Y⁵³⁴ motif in Str3, except that an additional amino acid residue (X) separates the Tyr and His residues in CnStr1, or the 2 Tyr residues in CnStr3, compared with the spacing observed in Str3. Additional studies will be required to confirm a functional role of ⁵³⁴YX₄H⁵³⁹ and ⁵⁴⁴YX₄Y⁵⁴⁹ motifs found within the final loop of CnStr1 and CnStr3, respectively. Interestingly, these motifs are absent from 4 other putative iron-related MFS-type transporters in C. neoformans: CnSit1, CnSit2, CNAG_07751, and CNAG_07519. Among these, CnSit1 is the only protein that has been functionally characterized as a ferrioxamine B siderophore transporter (Tangen et al. 2007). In contrast, the other 3 proteins have not yet been experimentally characterized in C. neoformans, although they have been predicted to function as siderophore transporters based on sequence analyses (Horianopoulos and Kronstad 2019; Choi et al. 2024).

Apart from S. pombe Str3 and C. neoformans CnStr1 and CnStr3, other MFS-type proteins have been implicated in heme transport, including the human MFS proteins FLVCR1 (FLVCR1a and FLVCR1b isoforms), and FLVCR2 (Quigley et al. 2004; Keel et al. 2008; Duffy et al. 2010; Chiabrando et al. 2012; Khan and Quigley 2013). FLVCR1a is predicted to contain 12 transmembrane spans and functions as a heme exporter at the plasma membrane (Khan and Quigley 2013). FLVCR1b is a shorter isoform of FLVCR1a that localizes to the mitochondrial membrane (Chiabrando et al. 2012). Although FLVCR1b contains only 6 transmembrane domains, it homodimerizes to form a transporter complex that enables heme export from the mitochondrion (Chiabrando et al. 2012). Overexpression of FLVCR2 in mammalian cell lines and Xenopus laevis oocytes results in a significant increase in the uptake of ZnMP or radiolabeled hemin, supporting a potential role for FLVCR2 in heme import (Duffy et al. 2010). Taken together, these MFS-type transporters identified in fungi and higher eukaryotes that share roles in heme transport may constitute a distinct subgroup of MFS family members with common structural and functional features that warrant further investigation.

In the case of human FLVCR1a, comparative in silico structural analyses with other heme-binding proteins suggest that His¹⁴⁵, Tyr¹⁵³, and His¹⁹⁸ are likely contact residues involved in coordinating heme during transport (Khan and Quigley 2011, 2013). Multiple sequence alignment of S. pombe Str3, C. neoformans CnStr1, CnStr3, and human FLVCR1a revealed that Tyr¹⁵³ is conserved across all these proteins (Supplementary Fig. 1). Accordingly, the aromatic side chain corresponding to Tyr¹⁵³ located at position 108 in Str3, 102 in CnStr3, and 100 in CnStr1, is predicted to be appropriately positioned for heme coordination when these MFS-type transporters adopt the proposed clamp-and-switch configuration that may facilitate heme translocation across the membrane. The two halves of each transporter are predicted to alternate between outward-open (substrate-loaded), occluded (substrate-bound), and inward-open (substrate-released) conformations (Yan 2015; Quistgaard et al. 2016). Further studies will be required to determine whether these conserved Tyr residues in Str3, CnStr1, and CnStr3 play a functional role in heme coordination and to identify additional amino acid residues that cooperate with them in stabilizing heme in the occluded, heme-bound state of these transporters.

Non-iron metalloporphyrins (MPPs) are taken up by bacteria and yeasts through heme uptake systems (Stojiljkovic et al. 1999, 2001; Hu et al. 2013), and they exhibit antibacterial/antifungal activity, likely by interfering with essential hemoproteins or other associated metal-dependent biochemical processes. Previous screens identified C. neoformans mutant strains carrying gene deletions that prevent the cytotoxicity of manganese MPP (MnMPP) (Bairwa et al. 2019). Among these resistant mutants, 1 class of strains harbors a deletion of the STR1 gene (CnSTR1). This finding suggests that, in the absence of the Str1 (CnStr1) protein, MnMPP (or heme) uptake is markedly reduced or blocked, thereby enabling the cnstr1Δ mutant cells to grow in contrast to the wild-type strain expressing CnSTR1. The identification of CnStr1 through this large-scale approach is consistent with our results, supporting a role for this protein in heme acquisition to sustain C. neoformans cell growth.

The cell surface-anchored heme-binding protein Cig1 plays a major role in heme acquisition by C. neoformans (Cadieux et al. 2013; Li et al. 2025; Perfect and Kronstad 2025). At physiological pH, Cig1 is required for maximal hemin-dependent growth. Accordingly, loss of Cig1 (cig1Δ) results in a growth defect at pH 7.2 compared with the isogenic wild-type strain H99 when cells rely exclusively on exogenous hemin as the sole source of iron (Cadieux et al. 2013). In this study, we found that heterologous expression of CnStr1 or CnStr3 in S. pombe hem1Δ shu1Δ str3Δ cells enabled growth in the presence of exogenous hemin as the sole source of heme. Moreover, these CnStr1 or CnStr3-expressing cells were competent to acquire the heme analog ZnMP. Based on these findings, a double-deletion C. neoformans Cnstr1Δ Cnstr3Δ strain was created and assessed for hemin-dependent growth. Surprisingly, the Cnstr1Δ Cnstr3Δ double mutant exhibited only a slight decrease in growth when hemin was provided as the sole source of heme. In contrast, the Cnstr1Δ Cnstr3Δ cig1Δ triple mutant strain displayed a more pronounced growth defect than the limited growth observed in the cig1Δ single mutant. These genetic data suggest that CIG1 is epistatic to CnSTR1 and CnSTR3, and functions upstream of these genes in the exogenous heme acquisition pathway. One potential model is that Cig1 functions as a heme receptor at the plasma membrane, where heme is subsequently transferred, by a mechanism that remains to be defined, either to CnStr1, CnStr3, or to an alternative uptake pathway that allows heme translocation across the membrane. Although the equilibrium dissociation constant (K_D) of Cig1 for heme has not yet been determined, absorbance spectroscopy analyses have shown that recombinant Cig1 binds hemin (Cadieux et al. 2013). Moreover, transcriptomic studies have shown that Cig1 is among the most strongly induced cryptococcal genes under iron-limiting conditions, supporting its prominent role in the early stages of heme acquisition (Jung et al. 2006, 2010; Li et al. 2019, 2025; Yu et al. 2021).

In C. albicans, two ferric reductase-related proteins, Frp1 and Frp2, participate in heme acquisition (Roy et al. 2022). Frp1 functions across a broad range of growth conditions, whereas Frp2 promotes heme uptake specifically under neutral and alkaline pH conditions (Roy et al. 2022). Their proposed mode of action involves the reduction of ferriheme (Fe³⁺-heme) to ferroheme (Fe²⁺-heme), thereby facilitating the release of Fe²⁺-heme from the redox-sensitive heme-binding domain of the cell-surface receptor Pga7. Following this release, Frp1 and Frp2 may also act as client heme-binding proteins that mediate intracellular heme trafficking from the cell surface to endosomal-vacuole compartments (Roy et al. 2022). A distinguishing feature of these 2 ferric reductases is the absence of a Cys-rich N-terminal domain found in 12 of the 17 ferric reductase-like proteins in C. albicans. In C. neoformans, 8 putative ferric reductases have been identified, including Fre2 (CNAG_06821.7), which is required for optimal hemin-dependent growth (Saikia et al. 2014). Similar to Frp1 and Frp2 in C. albicans, CnFrp2 lacks a Cys-rich N-terminal domain and may share functional properties with these proteins. However, unlike frp1Δ and frp2Δ mutants in C. albicans and the C. neoformans cig1Δ mutant strain, loss of CnFre2 does not confer resistance to the toxic heme analog gallium protoporphyrin IX (Ga-PPIX) (Saikia et al. 2014). Future studies will likely clarify whether any of the 8 ferric reductases in C. neoformans contribute to heme acquisition, as previously reported for C. albicans.

Because a clear understanding of the molecular circuitry governing the acquisition of exogenous heme at the cell surface of C. neoformans is still lacking, elucidating the respective roles and potential functional relationships among Cig1, CnStr1, and CnStr3 in heme uptake, as well as determining whether they preferentially interact with hemoproteins such as Fhb1, represents an important direction for future investigation.

Data availability

All data are included in the present manuscript. Strains and plasmids used for this study are available from the corresponding author upon reasonable request. The authors state that all results obtained for confirming the conclusions presented in the article are represented fully within the article.

Supplemental material available at GENETICS online.

Funding

This study was supported in part by the Canadian Institutes of Health Research (CIHR, grant #202303PJ4-501788-MID-CFDA-36776) and the Natural Sciences and Engineering Research Council of Canada (NSERC, grant #RGPIN-2020/2025-04802) awarded to S.L.

Author contributions

Conceptualization, F.L.Y.P., S.P., G.B., V.C., and S.L.; Methodology, F.L.Y.P., S.P., G.B., V.C., and S.L.; Validation, F.L.Y.P., S.P., and S.L.; Formal Analysis, F.L.Y.P., S.P., G.B., V.C., and S.L.; Investigation, F.L.Y.P., S.P., G.B., V.C., and S.L.; Resources, F.L.Y.P., S.P., G.B., V.C., and S.L.; Writing—original draft preparation F.L.Y.P. and S.L.; Review and editing, F.L.Y.P., S.P., G.B., V.C., and S.L.; Supervision, S.P., and S.L.; Project administration, S.L.; Funding acquisition, S.L. All authors have read, reviewed and approved the final version of the manuscript.

Ethical statement

No ethical approval was required for this study as it did not involve human or animal subjects. Only laboratory yeast strains were used in this study.

Copyright statement

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives Licence (CC BY-NC-ND), which permits users to copy, distribute and transmit an article as long as the author is attributed. The article is not used for commercial purposes, and the work is not modified or adapted in any way.