Research Article
20 December 2013

Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme, for Capsule Formation, and for Virulence

ABSTRACT

Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans.

INTRODUCTION

The pathogenic fungus Cryptococcus neoformans causes life-threatening meningoencephalitis in immunocompromised individuals (1). Iron availability influences at least two major virulence traits in this fungus, including the production of a polysaccharide capsule and deposition of melanin in the cell wall (2, 3, 4, 5, 6). Additionally, iron overload exacerbates cryptococcal disease in a mouse model of cryptococcosis (7). Like other pathogens, C. neoformans must compete with mammalian iron sequestration mechanisms and proteins to obtain iron during infection. Iron-sequestering proteins include transferrin, lactoferrin, and ferritin, and they contribute to iron withholding as part of the innate immune system to maintain low iron availability.
C. neoformans has multiple acquisition mechanisms to obtain iron, including a high-affinity uptake system composed of the iron permease Cft1 and the ferroxidase Cfo1, a siderophore uptake pathway, cell surface reductases, and exported reductants such as 3-hydroxyanthranilic acid; in addition, melanin in the cell wall may contribute to ferric iron reduction (3, 8, 9). The Cft1/Cfo1 permease/ferroxidase complex is essential for reductive iron uptake, for iron acquisition from transferrin, and for full virulence in a mouse model of cryptococcosis (10, 11). The CFT1 transcript level is elevated upon iron limitation and during early murine pulmonary infection (12, 13, 14). However, cft1 and cfo1 mutants can obtain iron from siderophores, heme, and hemoglobin, thus indicating that another uptake pathway(s) is needed for these iron sources (10, 11). The mechanisms of heme use have not been identified, but iron acquisition from siderophores is known to require specific transporters such as the ferrioxamine B transporter Sit1, although C. neoformans is unable to produce its own siderophores (15, 16). The regulation of iron uptake mechanisms and sensing in C. neoformans is mediated by the master iron regulator Cir1, as well as several other regulatory proteins, including Nrg1, Rim101, and HapX (17, 18, 19, 20, 21).
Heme is the most abundant iron source in mammalian hosts, and our understanding of pathogen acquisition of iron from this source is based mainly on studies with parasites and pathogenic bacteria (22, 23, 24). Gram-negative bacteria, for example, generally transport heme across the outer membrane through specific receptors or secrete hemophores that bind heme to facilitate uptake (23, 24, 25). Gram-positive bacteria bind and transport heme via cell surface-associated proteins at the cell wall and take up heme via ATP-binding cassette transporters in the plasma membrane (26). Some Gram-positive bacteria also produce hemophores (24). In contrast to the situation in bacterial pathogens and parasites, much less is known about iron acquisition in fungal pathogens of humans.
The pathogenic fungi C. neoformans, Candida albicans, and Histoplasma capsulatum grow well with heme or hemoglobin as a sole iron source in vitro (10, 27, 28). Heme use has been best characterized in C. albicans, and this fungus binds erythrocytes and possesses a hemolytic factor on the cell surface (29, 30, 31, 32, 33). Specific heme/hemoglobin receptors (Rbt5 and Rbt51) in the plasma membrane mediate the uptake of hemoglobin (28, 34). These and other potential hemoglobin receptor proteins, Wap1/Csa1, Csa2, and Pga7, contain a common in fungal extracellular membrane protein domain (28, 35). Furthermore, hemoglobin was found to bind to Rbt5 and to be internalized via an endocytic pathway (34). Once internalized into vacuoles, hemoglobin is likely denatured or hydrolyzed to release heme, and a heme oxygenase (Hmx1) is involved in the further degradation of heme to release iron (30, 36, 37). Overexpression of C. albicans Rbt51 in Saccharomyces cerevisiae enables growth in the presence of hemoglobin, and screening of the deletion collection in this yeast for reduced growth on this iron source revealed roles for a vacuolar ATPase and the ESCRT (endosomal sorting complex required for transport) complexes (34). The ESCRT system is dedicated to the targeting of monoubiquitinated membrane proteins to the vacuole (38). ESCRT mutants of C. albicans were further shown to have defects in iron acquisition from hemoglobin but not the siderophore ferrichrome (28, 34).
A cell surface binding activity for heme has also been reported for the facultative intracellular fungal pathogen H. capsulatum (27). For this pathogen, iron acquisition from heme, siderophores, and iron-binding proteins such as transferrin involves a secreted gamma-glutamyltransferase (Ggt1) and a glutathione-dependent ferric reductase (GSH-FeR) (39, 40). Ggt1 contributes to virulence in H. capsulatum, as do specific siderophores (40, 41, 42).
Given that the mechanisms of heme use in C. neoformans are not known, we constructed and screened an insertion mutant library to identify genes involved in this process. We discovered a role for Vps23, a component of the ESCRT-I complex that is required for intracellular trafficking of endocytic vesicles. Our characterization of vps23 deletion mutants also revealed that Vps23 and ESCRT function contribute to capsule and melanin formation, as well as virulence, in C. neoformans. Overall, this study extends our understanding of iron acquisition in C. neoformans, reveals an additional complexity to the connection between iron and virulence factor production in this pathogen, and underscores the importance of intracellular trafficking in fungal virulence.

MATERIALS AND METHODS

Strains, plasmids, and media.

Serotype A strains H99 (MATα) and KN99a (MATa) (C. neoformans var. grubii) were maintained on YPD medium (1% yeast extract, 2% peptone, 2% dextrose, 2% agar). The nourseothricin, neomycin, and hygromycin resistance cassettes were from plasmids pCH233, pJAF1, and pJAF15, respectively. YPD medium plates containing neomycin (200 μg/ml) were used to select vps23 deletion transformants. Defined low-iron medium (LIM) (6, 14) and yeast nitrogen base (YNB; pH 7.0) plus 150 μM bathophenanthroline disulfonate (BPS) (10) were used as iron-limiting media. YPD and/or YNB liquid media or plates (YNB with amino acids) supplemented as indicated were used for phenotypic characterization. All chemicals were obtained from Sigma-Aldrich unless indicated otherwise. Feroxamine was obtained as deferoxamine mesylate (Sigma-Aldrich) and chelated with FeCl3.

Agrobacterium tumefaciens-mediated transformation of C. neoformans.

Transformation was performed as previously described (43, 44, 45). Plasmid pPZP-Neo1, which carries a neomycin resistance cassette, was used to transform a C. neoformans cfo1 mutant strain (H99 background). Briefly, Agrobacterium cells were grown overnight with shaking at room temperature in Luria-Bertani medium with kanamycin. Cells were washed and resuspended in liquid induction medium with 200 μM acetosyringone (AS) at an optical density at 600 nm (OD600) of 0.15 and incubated for 6 h (OD600 of 0.6). C. neoformans H99 cells were grown overnight in YPD medium, washed in induction medium, and resuspended at 106 or 107/ml. Subsequently, 200 μl of each of the C. neoformans and A. tumefaciens cultures was mixed and plated (without spreading) on induction agar medium (with AS). The plates were incubated for 2 to 3 days before the mixtures were resuspended in liquid YPD medium. Cells were then plated onto YPD medium with neomycin (200 μg/ml) and cefotaxime (100 μg/ml), and the plates were incubated at 30°C for 3 to 4 days.

Mutant screening and inverse PCR.

A collection of ∼30,000 transformants was screened for growth defects on LIM containing heme as the sole iron source. Briefly, overnight cultures of each strain were grown in YPD medium in a 96-well plate format and 5 μl of each culture was transferred to a well containing 200 μl of LIM (YNB plus 150 μM BPS). These cells were incubated at 30°C for 2 days to reduce the amount of stored intracellular iron. After starvation, 5 μl of each culture was transferred to 96-well plates containing 200 μl of YNB plus 150 μM BPS, YNB plus 150 μM BPS and 10 μM heme, YNB plus 150 μM BPS and 100 μM heme, YNB plus 150 μM BPS and 10 μM FeCl3, or YNB plus 150 μM BPS and 100 μM FeCl3. Growth was determined by measurement of OD600 on a Tecan plate reader after incubation for 3 days at 30°C. Strains with defective growth on heme but not FeCl3 were further confirmed with spot assays on LIM with either 10 or 100 μM heme.
Inverse PCR was used to determine the disruption sites in candidate mutants using genomic DNA and the methods of Zhang and Gurr (46) and Hu et al. (47) for DNA digestion, ligation, and PCR amplification. PCR products were sequenced by Genewiz, and insertion sites were determined by BLAST with the genome sequence database (www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/).

Deletion of VPS23.

The open reading frame of VPS23 in strains H99 and KN99a and the cfo1 mutant (H99 background) was replaced with a neomycin resistance cassette to delete the gene. Briefly, a vps23::NEO deletion allele was constructed by using a modified overlap PCR procedure (13, 48) and the primers listed in Table S1 in the supplemental material. Primers vps23-1/vps23-3 and vps23-4/vps23-6 were used with genomic DNA to obtain the left and right arms for the deletion construct. The selectable marker NEO was amplified from plasmid pJAF1 by using primers vps23-2 and vps23-5. Overlap PCR with primers vps23-1 and vps23-6 yielded a vps23::NEO allele lacking the entire open reading frame (1,850 bp). The resulting PCR product (3,755 bp) was used to transform strains H99 and KN99a and a cfo1 mutant by biolistic transformation (49). Transformants were screened by colony PCR with Extaq polymerase (TaKaRa) using primers vps23-7 and vps23-8 (negative screen) and vps23-9 and hug-Neo (positive screen). Primer vps23-9 was designed from the region upstream of VPS23, and hug-Neo was designed for the NEO gene. Transformants with replacements were confirmed by genomic hybridization analysis as described previously (48). Two independent vps23-9 and vps23-16 mutants in the H99 strain background were constructed and studied further. One vps23 mutant (vps23-9a) in the KN99a background and two double mutants (cfo1 vps23-13 and cfo1 vps23-14b) from the cfo1 background were also analyzed. Mating types of strains were confirmed by mating assays as described previously (50). A similar strategy for gene deletion was used to generate the RIM101 deletion strain, rim101-JK, with the primers listed in Table S1 in the supplemental material. The in vitro phenotypes of the rim101-JK strain matched those of the rim101-AA strain (a gift from J. Andrew Alspaugh), as described by O'Meara et al. (21).

Overexpression of VPS23 in a vps23 mutant strain.

Strains for overexpression of VPS23 and complementation of the vps23 deletion were generated by replacing the promoter with that of the elongation factor I gene (CNAG_06125.2). Initially, the promoter was amplified from strain H99 with primers EF1-5R-HindIII and EF1-3F-IF-SpeI (see Table S1 in the supplemental material). The 1,606-bp product was digested with HindIII and SpeI and cloned into HindIII/SpeI-digested pJAF15 to produce plasmid pEF1pro. The VPS23 fragment was amplified with primers Vps23GFP5rP3-SpeI and Vps23comp-IF3Fb-SpeI, and the resulting 2,420-bp PCR product was digested with SpeI and cloned into SpeI-digested pEF1pro. The plasmid with correct orientation of the Vps23 fragment was identified and designated pEF1pro-Vps23-ter. pEF1pro-Vps23-ter was linearized with BglII and introduced into the vps23-9 mutant. This construct therefore replaced the promoter and complemented the deletion mutation. Overexpression was confirmed by quantitative reverse transcription (RT)-PCR as described by Hu et al. (12), using primers listed in Table S2 in the supplemental material.

Toxicity of gallium protoporphyrin (GaPPIX) and manganese protoporphyrin (MnPPIX).

To test for GaPPIX and MnPPIX toxicity, cells grown overnight in YPD medium were washed twice in sterile, low-iron water (treated with Chelex-100 resin [Bio-Rad]). Tenfold serial dilutions of the cells were prepared in low-iron water. To ensure exposure to a high concentration of the expensive chemical GaPPIX, defined LIM agar plus 10 μM heme was spread with 200 μl of 10 μM GaPPIX (Frontier Scientific), 100 μM MnPPIX (Frontier Scientific), or 10 μM GaCl3 (Sigma-Aldrich) immediately prior to the spotting of 5-μl volumes of dilutions of 1 × 106 to 1 × 102 cells/ml. The plates were incubated for 2 days at 30°C before being photographed.

Capsule formation and melanin production.

Capsule formation was examined by differential interference contrast microscopy after incubation for 24 h at 30°C in defined LIM and staining with India ink (6, 14). Melanin production was examined on l-3,4-dihydroxyphenylalanine (l-DOPA) plates containing 0.1% glucose. Capsule shedding from cells was examined with a blot assay performed as described by Yoneda and Doering (51).

FM4-64 staining of the vacuolar membrane.

Internalization of the lipophilic dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide] (T-3166; Invitrogen, Burlington, Ontario, Canada) was used to visualize the vacuolar membrane as previously described, with minor modifications (52). FM4-64 was used at a final concentration of 10 μM in phosphate-buffered saline (PBS; pH 7.4). Cells were harvested after overnight growth in YPD medium, stained with FM4-64 for 15 min on ice, washed, and transferred to fresh medium without the stain. The stained cells were incubated in a 30°C shaker for 30 min before viewing.

Virulence assays.

Virulence was assayed in an inhalation model of cryptococcosis using female BALB/c mice (4 to 6 weeks old) from Charles River Laboratories (Senneville, Ontario, Canada) as previously described (52). Briefly, C. neoformans strains were grown in 5 ml of YPD medium at 30°C overnight, washed twice with PBS (Invitrogen, Burlington, Ontario, Canada), and resuspended in PBS. The BALB/c mice, in groups of 10, were intranasally inoculated with a suspension of 106 cells in 50 μl. The health status of the mice was monitored daily postinoculation. Mice reaching the humane endpoint were euthanized by CO2 anoxia. Statistical analyses of survival differences were performed by log rank tests using GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA). For determination of the fungal load in organs, infected mice were euthanized and organs were excised, weighed, and homogenized in 1 ml of PBS using a MixerMill (Retsch, Newtown, PA). Serial dilutions of the homogenates were plated on Sabouraud dextrose agar plates containing 35 μg/ml chloramphenicol, and CFU were counted after incubation for 48 h at 30°C. The protocols for the virulence assay (protocol A08-0586) were approved by the University of British Columbia Committee on Animal Care.

RESULTS

Insertional mutagenesis screening for mutants unable to use heme.

To identify functions for heme uptake in C. neoformans, we initially constructed a mutant library by Agrobacterium-mediated insertional mutagenesis in a cfo1 mutant lacking the high-affinity iron uptake system. The cfo1 mutant grows poorly under low-iron conditions, but growth is restored by heme (10 μM) or a high concentration of ferric iron (100 μM) (Fig. 1) (10). We reasoned that the lack of Cfo1 would reduce the ability of C. neoformans to use free iron that may be contaminating the heme and block any potential contribution of the reductive uptake system to the use of iron from heme. A stock of ∼30,000 transformants was constructed, and genome hybridization analysis of 16 randomly selected strains using a probe designed from the neomycin resistance cassette revealed that >90% had a single T-DNA insertion event (see Fig. S1 in the supplemental material). We then screened for mutants defective for growth on heme by examining growth (OD600) in low-iron liquid media without and with different levels of heme or FeCl3. This approach yielded 34 mutants with poor growth on heme as the sole iron source, and the phenotypes of these mutants with heme and other iron-related phenotypes were further confirmed with spot assays on solid media (Fig. 1 and data not shown). Genomic DNA was isolated for all candidate mutants, inverse PCR was performed, and the disruption sites were successfully identified for 25 mutants. The analysis of one set of mutants with defects in the VPS23 gene (Fig. 1) is described below, and the analysis of additional mutants will be presented in a subsequent publication.
Fig 1
Fig 1 Growth defect of T-DNA insertion mutants on heme. Tenfold serial dilutions of each strain were spotted on the indicated media, and the plates were incubated at 30°C for 2 days before being photographed. The strains were the parental WT strain H99, the three mutants (272D10, 277A9, and 292E1) with insertions in VPS23, and a mutant with a deletion in the CFO1 gene for the ferroxidase of the high-affinity iron uptake system (10).

Mutations in the ESCRT-I gene VPS23 cause a growth defect on heme.

Three mutants were identified with T-DNA insertions in the VPS23 gene, which encodes a member of the ESCRT-I protein-sorting complex. The ESCRT complex is composed of four core cytoplasmic polyprotein complexes (ESCRT-0, -I, -II, and -III), which are recruited sequentially to ubiquitinated cargo proteins at endocytic vesicle membranes and deliver endosomes to the vacuole (38). In C. neoformans strain H99, VPS23 is annotated as CNAG_01720.2 on chromosome 11 (GenBank accession no. CP003830.1), and the predicted polypeptide (549 amino acids) displayed 33% identity and 55% similarity to its homologue in Saccharomyces cerevisiae, and 25% identity and 44% similarity to Vps23 in Candida albicans. Bioinformatic analysis of the H99 genome and Southern blot analysis demonstrated that only one VPS23 ortholog exists in C. neoformans (see Fig. S2 in the supplemental material).
To confirm the phenotypes of the insertion mutants, we generated two independent deletion mutations of VPS23, including vps23-9 and vps23-16 in the H99 strain (MATα); one deletion mutation, vps23-9a, in the KN99a strain (MATa); and two double mutations, vps23 cfo1-13 and vps23 cfo1-14b, in a cfo1 mutant strain (MATα, derived from H99) (10). The deletions were confirmed by colony PCR and DNA hybridization (see Fig. S2 in the supplemental material). The vps23 mutation was constructed in strains of opposite mating types to collect additional independent mutants. We also complemented the vps23 defect in the vps23-9 mutant by introducing the gene under the control of the EF1 promoter, and this approach allowed us to also examine the influence of overexpression of the gene. Overexpression of the VPS23 gene in the complemented strains was confirmed by quantitative RT-PCR (see Fig. S3 in the supplemental material).
The vps23 deletion and insertion mutants and the overexpression strains were examined for growth on low-iron YNB medium with 150 μM BPS (Fig. 2A). After 3 days of iron starvation, all strains were unable to grow on this medium, in contrast to their robust growth on YNB. The wild-type (WT) strain (H99) grew on low-iron YNB with 10 μM FeCl3 and all other iron sources. As expected, the cfo1 mutant strain grew on medium supplemented with 100 μM FeCl3, heme, or feroxamine but not on medium supplemented with 10 μM FeCl3 (Fig. 2B) (10). The vps23 mutants grew like the WT strain on low-iron YNB medium supplemented with 100 μM FeCl3, 10 μM feroxamine, or 2 μM hemoglobin but showed poor growth on low-iron YNB supplemented with either 10 or 100 μM heme, indicating that Vps23 is required for iron acquisition from heme (Fig. 2A). The VPS23-overexpressing strains restored mutant growth to the WT level on heme (Fig. 2A). Similar results were observed from spot assays on the defined LIM described by Vartivarian et al. (6) with and without the different iron sources (data not shown). Moreover, growth of the cfo1 vps23 double mutants was poor on LIM supplemented with 10 μM FeCl3 or different levels of heme as the iron source (Fig. 2B). Growth of the strains in liquid medium (either low-iron YNB or defined low-iron medium) also revealed that the vps23 mutants had a growth defect with heme or hemoglobin (Fig. 2D and E; data not shown). In addition, the mutants showed an extended growth lag compared to the WT strain on low-iron media with addition of FeCl3 and feroxamine (Fig. 2D and E; data not shown). This may indicate a role for ESCRT function in high-affinity reductive iron uptake, as found in S. cerevisiae (53, 54, 55). The vps23 mutant strain from the KN99a MATa background also grew poorly with heme but showed WT growth on media with the other iron sources (FeCl3, feroxamine) (Fig. 2C). Taken together, these results reveal that Vps23 is required for robust growth on heme.
Fig 2
Fig 2 Requirement of VPS23 for growth on solid and liquid media with heme as the sole iron source. (A to C) Tenfold serial dilutions of each strain (labeled on the right) were spotted on the indicated media and the plates were incubated at 30°C for 2 days before being photographed. (D) Cells of the WT, the vps23-9 mutant, and the overexpression strain were inoculated into liquid YNB medium plus 150 μM BPS without and with supplementation with iron sources. The cultures were incubated at 30°C, and OD600s were measured. (E) The indicated strains were also tested for growth in the defined LIM supplemented with heme or FeCl3 by the same method used for panel D.

Loss of Vps23 reduces susceptibility to noniron metalloprotoporphyrins.

Noniron metalloporphyrins (MPs) are taken up by bacteria via heme uptake systems, and they have antibacterial activity that is thought to result from interference with heme-dependent metabolic processes (56, 57). GaPPIX and MnPPIX show the highest toxicity for bacteria, and we hypothesized that the vps23 mutant defect in heme acquisition would reduce susceptibility. Indeed, the vps23 deletion mutants in both the MATa and MATα backgrounds showed reduced susceptibility to the noniron MPs (Fig. 3A and C). In contrast, the cfo1 mutant, the vps23 cfo1 double mutant, and a VPS23-overexpressing strain were inhibited like the WT strain (Fig. 3A and B). The double mutant may fail to grow because Cfo1 is required for the acquisition of inorganic iron contaminating the heme and/or for another pathway for iron use from heme. As a control, none of the strains were inhibited by the presence of GaCl3, in further support of the requirement for heme uptake. Overall, these data support the hypothesis that vps23 is involved in heme uptake by C. neoformans.
Fig 3
Fig 3 Reduced susceptibility of vps23 mutants to noniron MPs. Tenfold serial dilutions of cells of the indicated strains were spotted onto defined LIM with heme as the iron source. The plates on the right contained GaCl3, GaPPIX, or MnPPIX. The plates were incubated for 2 days at 30°C. (A, B) The mating type α strains in the parental H99 or the cfo1 mutant background were tested for susceptibility. (C) The vps23 mutant in the mating type a strain (KN99a) background was also tested to confirm the altered susceptibility. Note that 200 μl of the noniron MP IX solutions at the indicated concentrations were spread onto the surface of the plates prior to inoculation.

A vps23 mutant shows aberrant vacuolar staining.

The ESCRT complexes function in multivesicular body (MVB) transport to the vacuole, and vacuolar protein sorting (vps) mutants of S. cerevisiae display aberrant vacuolar staining (38, 58). To determine whether the Vps23 ortholog in C. neoformans has a similar function, we analyzed endocytic transport from the plasma membrane to the vacuole by staining with the fluorescent dye FM4-64. In both WT and vps23 mutant cells, FM4-64 was initially observed at the plasma membrane and was rapidly internalized into endocytic vesicles. After 75 min, the dye was observed only on the vacuolar membrane in WT cells while migration to the vacuole was delayed in vps23 mutants (Fig. 4A; see Fig. S4 in the supplemental material for additional images of cells). In addition, brightly staining spots (caps) of the dye were frequently observed on one side of the vacuole in >90% of the cells of the vps23 mutants. This phenomenon is described in S. cerevisiae and C. albicans as the vacuolar E-body and likely reflects FM6-64 localization to a late endocytic-prevacuolar (MVB-like) compartment (58, 59). The VPS23-overexpressing strains showed a staining pattern like that of the WT strain (Fig. 4A). Deletion of VPS23 from the KN99a strain (vps23-9a) and from the cfo1 mutant (vps23cfo1-13 and cfo1vps23-14b) also caused the occurrence of E bodies (data not shown). In S. cerevisiae, E bodies (class E compartments) result from missorting of cargo normally destined for the vacuole, causing it to accumulate in abnormal endocytic structures (58). In this context, our data suggest that Vps23 in C. neoformans may also play a role in MVB transport to the vacuole.
Fig 4
Fig 4 Defective endocytosis and increased susceptibility to trafficking inhibitors for vps23 mutants. (A) The WT, vps23 mutant, and VPS23-overexpressing strains were grown in YPD medium at 30°C overnight, harvested, washed in PBS (pH 7.2), and stained with FM4-64 on ice for 15 min. The cells were then incubated for an additional 30 min at 30°C before viewing. Bar, 5 μm. DIC, differential interference contrast. (B) Tenfold serial dilutions of the indicated strains were spotted onto YNB alone or YNB supplemented with either 20 μg/ml BFA or 0.5 mg/ml monensin. The plates were incubated for 3 days at 30°C.
To further probe the connection between Vps23 and intracellular trafficking, such as endoplasmic reticulum (ER)-Golgi compartment transport, we tested the strains for susceptibility to brefeldin A (BFA; 25 μg/ml) and monensin (0.5 mg/ml). BFA is known to arrest the anterograde transport of proteins between the ER and the Golgi compartment, and monensin is a Na+/H+ ionophore that blocks intracellular transport in both the trans-Golgi and post-Golgi compartments. The vps23 mutants in the strains of either mating type background, KN99a or H99, showed increased susceptibility to BFA or monensin, a result consistent with Vps23 involvement in intracellular trafficking (i.e., endocytosis and ER-Golgi compartment transport) (Fig. 4B). Overexpression of VPS23 restored the WT level of susceptibility (Fig. 4B).

Vps23 is involved in capsule and melanin elaboration.

We previously found that BFA reduced capsule formation in C. neoformans, and we therefore hypothesized that Vps23 may be involved in delivering virulence factors such as capsule and laccase (for melanin production) to the cell surface (13). The Vps23 contribution to capsule formation was initially examined in the WT, vps23 mutant, and VPS23-overexpressing strains by staining with India ink. Deletion of VPS23 in both H99 and KN99a resulted in reduced capsule formation, and overexpression of VPS23 in vps23 mutants resulted in cells with a capsule slightly larger than that of the WT strain (Fig. 5A to D). As expected, the cfo1 mutant produced a capsule at the level of the WT strain, but the two cfo1 vps23 mutants displayed a marked reduction in capsule size (Fig. 5B). The three independent Agrobacterium-mediated Vps23 insertion mutants were also defective in capsule formation (data not shown). Measurements of capsule size revealed that the capsule reduction in the vps23 and cfo1 vps23 mutants was significant compared with the WT strain, although the range of capsule sizes of the VPS23-overexpressing strains overlapped that of the WT strain (Fig. 5D). Overall, the data support the hypothesis that Vps23 is involved in capsule elaboration.
Fig 5
Fig 5 Altered capsule size and polysaccharide shedding of vps23 mutants and VPS23-overexpressing strains. (A to C) Cells were cultured in defined LIM at 30°C for 48 h, and capsule formation was assessed by India ink staining of the indicated strains. Bar, 5 μm. (D) Fifty cells of each strain were measured to determine the cell diameter and capsule radius. Each bar represents the average of the 50 measurements with standard deviations. An asterisk indicates that the count is statistically significantly different from the others by the Student t test (P < 0.05). (E) The electrophoretic mobility and quantity of shed polysaccharide were assessed as described in Materials and Methods, by using an anti-GXM antibody to detect the capsule.
Glucuronoxylomannan (GXM) is the major polysaccharide of the capsule that is exported and attached to the cell wall (60). To determine whether vps23 mutants have a defect in capsule attachment, we used a gel electrophoresis and blotting technique to examine GXM shedding (21, 51). Fungal cells were incubated in capsule-inducing medium, and polysaccharide shed into the medium was analyzed for relative abundance by reactivity with an anti-GXM antibody (monoclonal antibody 18B7). As shown in Fig. 5E, the vps23 mutants (both the vps23 and vps23 cfo1 mutants) produced significantly more free GXM than the WT strain did. This result suggests that deletion of VPS23 may not affect GXM synthesis but instead causes a defect in capsule attachment to the cell wall. The overexpression of VPS23 resulted in a level of free GXM similar to that in the WT strain.
C. neoformans cells also produce the virulence factor melanin because of deposition of the enzyme laccase in the cell wall. As shown in Fig. 6, deletion of VPS23 caused reduced melanin formation in the single mutants of strains of both mating types. The WT level of melanin was restored in the overexpression strains. We did note that the growth of the mutants was slightly lower than that of the WT strain on the l-DOPA medium used to assay melanin formation, and this phenotype could contribute to reduced pigmentation.
Fig 6
Fig 6 Reduced melanization in vps23 mutants. Tenfold serial dilutions of cells of the indicated strains were spotted onto solid l-DOPA plates. The plates were incubated for 2 days at 30°C prior to being photographed.

Additional phenotypes of vps23 mutants.

We next tested the effects of pH and a high salt concentration on the growth of vps23 mutants because loss of ESCRT function influences related phenotypes in S. cerevisiae and C. albicans (59, 61, 62). Compared to the WT and VPS23-overexpressing strains, the vps23 mutant exhibited a more severe growth defect at alkaline pHs (8 and 8.3) (Fig. 7A; P < 0.001). A rim101 mutant that is known to be sensitive to alkaline pH was included as a control (21). The vps23 mutants also showed increased sensitivity to 1.5 M NaCl (5 days), 1.5 M KCl, and 100 mM LiCl (10 days) (Fig. 7B and C). However, the mutants grew as well as the WT on 1.5 M sorbitol, thus indicating that the growth defects on LiCl, KCl, and NaCl were due to ionic rather than general osmotic stress. Overall, these data indicated that Vps23 in C. neoformans shares functions with orthologs in other fungi.
Fig 7
Fig 7 Loss of Vps23 influences growth at alkaline pH and susceptibility to salt stress. (A) Cells of the indicated strains were grown in buffered YNB, and growth was quantified by monitoring the absorbance at 600 nm after 24 h at 30°C. (B, C) Tenfold serial dilutions of cells of the strains indicated on the right were spotted onto solid YNB without or with 1.5 M NaCl, 1.5 M KCl, 1.5 M sorbitol, or 100 mM LiCl. The plates were incubated at 30°C for the following times: sorbitol, 3 days; KCl, 5 days; NaCl, 5 days; LiCl, 10 days.

Vps23 is required for virulence.

We hypothesized that deletion of VPS23 would negatively impact cryptococcal virulence, given that the mutation caused a reduction in capsule attachment, melanin formation, and growth in heme as an iron source. To examine this prediction, a virulence assay was performed in a mouse inhalation model of cryptococcosis with WT strain H99, two independent vps23 mutants, and a VPS23-overexpressing strain. As expected, the mice inoculated with WT cells reached the experiment endpoint at 18 to 21 days, while mice infected with either deletion mutant survived for 40 days (Fig. 8). Unexpectedly, overexpression of VPS23 caused a slight increase in virulence, and the mice infected reached the experimental endpoint at 14 to 18 days (Fig. 8). These experiments indicated that VPS23 is required for virulence. To specifically examine whether the vps23 cells could disseminate beyond the lung, we monitored the numbers of fungal cells in the brains and lungs of the mice at the experimental endpoints. This test revealed that the vps23 mutant cells were able to reach the brain, although the average fungal load of the two mutants in each of three mice was lower than that of WT cells (19 ± 3.3 and 120 ± 21 CFU/g versus the WT load of 230 ± 370 CFU/g). In comparison, the fungal loads in the lung at the endpoint for three mice infected were 5.3 × 103 ± 8.0 × 103 CFU/g and 2.2 × 103 ± 8.2 × 102 CFU/g for the two vps23 mutants versus 2.2 × 108 ± 6.0 × 107 CFU/g for the WT strain. These data indicate that vps23 mutant cells reached the brain in low numbers but failed to persist in both the lung and the brain.
Fig 8
Fig 8 Vps23 influences virulence in mice. Ten female BALB/c mice were inoculated intranasally with each of the strains indicated, and the survival of the mice was monitored daily. The mice were each inoculated with 2 × 105 fungal cells. Survival differences between groups of mice were evaluated by log rank tests. The P values for the mice infected with the WT and mutant strains were statistically significantly different (P < 0.001).

DISCUSSION

Pathogens have specific mechanisms to overcome iron withholding and proliferate in mammalian hosts. Our overall goals are to define the mechanisms employed by C. neoformans to acquire iron in mammalian hosts and to use the information to develop approaches to block fungal proliferation and pathogenesis. In this study, we used insertional mutagenesis to identify mutants defective in iron acquisition from heme, the most abundant iron source in mammals, and we focused on characterizing mutations in the gene for the ESCRT-I complex protein Vps23. This gene contributes to iron acquisition because vps23 mutants displayed a pronounced growth defect on heme and reduced susceptibility to the noniron MPs GaPPIX and MnPPIX, which have heme uptake-dependent toxicity in bacteria (56, 57). There was also an extended lag phase for the mutants in medium with iron chloride, and this may reflect an additional role for the ESCRT pathway in recycling the high-affinity, reductive iron permease/ferroxidase proteins (Cft1 and Cfo1) to the plasma membrane under conditions of low iron availability (10). This idea is based on findings that in S. cerevisiae, iron levels influence endocytic sorting of the iron permease/ferroxidase proteins Ftr1 and Fet3 (53, 54, 55). In this yeast, iron deprivation results in sorting of the proteins back to the plasma membrane and iron addition causes targeting to the vacuole for degradation via the ESCRT protein-mediated MVB pathway.
The involvement of ESCRT proteins such as Vps23 in heme uptake and iron acquisition in C. neoformans indicates that this function is conserved among some pathogenic fungi. That is, mutations in VPS23 and other ESCRT pathway genes also cause growth defects with hemoglobin (<1 μM) as the sole iron source in C. albicans (28, 34). Weissman et al. (34) proposed that endocytosis of vesicles from the plasma membrane may be a conserved mechanism of iron scavenging from the environment. In C. albicans, the plasma membrane proteins Rbt5 and Rbt51 bind heme and hemoglobin, and heterologous expression of Rbt51 also allows S. cerevisiae to use iron from hemoglobin (28, 34). Screening for mutations in S. cerevisiae that block the use of hemoglobin via Rbt51 revealed a role for vacuolar functions, including vacuolar ATPase activity for acidification, ESCRT pathway functions to deliver endocytic vesicles to the vacuole, and HOPS complex proteins that mediate vesicle fusion to the vacuole. Although this screening only revealed a role for the ESCRT complex II and III proteins (Vps22 and Vps32, respectively) in S. cerevisiae, subsequent analysis of deletion mutants of C. albicans confirmed that mutations in genes for the ESCRT-I complex, VPS23 and VPS28, also influenced hemoglobin transport to the vacuole (34). In light of this study, our finding that Vps23 participates in heme uptake in C. neoformans strongly suggests that the remaining ESCRT components are required for the process and preliminary experiments support this idea (G. Hu, unpublished results).
Importantly, Rbt5 and Rbt51 do not contain a cytoplasmic domain and they are unlikely to be direct targets for the ESCRT pathway. It is possible, therefore, that another transmembrane receptor(s) exists in C. albicans (and in C. neoformans) to facilitate the transport of heme or hemoglobin to the vacuole via ESCRT-mediated endocytosis. For C. neoformans, we have identified Cig1 as a candidate heme-binding protein that is required for growth on heme (B. Cadieux and J. Kronstad, submitted for publication). H. capsulatum may also have a heme/hemoglobin receptor, further suggesting that the use of these iron sources may be conserved among fungal pathogens that attack humans (27). However, we should note that another important human pathogen, Aspergillus fumigatus, is unable to use heme or transferrin as an iron source and instead relies mainly on siderophore-based mechanisms of iron acquisition (63).
In addition to a defect in heme use, the vps23 mutants displayed a smaller cell-associated capsule and shed more capsular polysaccharide into the culture supernatant than the WT strain. These observations suggest that Vps23 (and likely the ESCRT-I complex) is required for the establishment of the proper capsule or cell wall structure required for anchoring of the polysaccharide to α-1,3-glucan at the cell surface. That is, the capsule phenotype may indirectly result from impaired trafficking of materials to properly construct the cell wall. Alternatively, or in addition, ESCRT function may contribute to trafficking functions that mediate the biosynthesis and/or modification of the capsule polysaccharides GXM and galactoxylomannan such that the molecules are competent for cell wall attachment. Consistent with this idea, Frases et al. (64) have shown that exopolysaccharide found in culture supernatant has physical and chemical differences from the cell-associated material. Similarly, altered polysaccharide chemistry as a result of changes in processing or biosynthesis during transit to the cell surface might influence self-aggregation of the material to build a cell-associated capsule (65). The slightly larger capsule size that we observed on strains overexpressing Vps23 is also consistent with changes in capsule anchoring and/or aggregation.
Capsule polysaccharide is transported to the cell surface in vesicles via exocytosis, and it is clear that trafficking of polysaccharide and other virulence-related factors such as laccase is important for the virulence of C. neoformans (60, 66, 67). Our observations that deletion of VPS23 caused increased sensitivity to BFA and monensin, two intracellular trafficking inhibitors, and that these inhibitors reduce capsule size, are consistent with a contribution to proper trafficking (13). Similarly, loss of Vps23 may interrupt the processing and/or transport of laccase (resulting in reduced melanization). Laccase must be loaded with copper during transit through the ER-Golgi compartment network, and it must be properly localized in the cell wall (68). Capsule polysaccharide is also found in extracellular vesicles, although the extent of the contribution of the latter vesicles to capsule formation is not yet clear (67). In the context of trafficking, we note that our preliminary analysis of extracellular vesicle production by the vps23 mutants did not reveal a defect compared with the WT strain, although a more detailed study is needed (B. Cadieux, unpublished results). In S. cerevisiae, deletion of ESCRT genes (e.g., VPS23, SNF7) and other secretory pathway components (e.g., SEC4) did not completely eliminate the production of extracellular vesicles, although their composition was altered (69). Finally, with regard to capsule trafficking, we note that mutants lacking components of other ESCRT complexes (e.g., Vps22 in ESCRT-II and Snf7 in ESCRT-III) also had a reduced capsule size (G. Hu, unpublished results).
The lack of virulence of the vps23 mutants most likely results from (at least) the combined defects in iron acquisition from heme, reduced capsule size, and impaired melanization. It is interesting that the VPS23 deletion and disruption mutants had both shared and distinct phenotypes compared with a rim101 mutant of C. neoformans (21). The Rim101 (PacC in filamentous fungi) transcription factor regulates the response to extracellular pH and has been characterized in several pathogenic and nonpathogenic fungi, including Aspergillus nidulans, C. albicans, Yarrowia lipolytica, S. cerevisiae, and C. neoformans (21, 70, 71, 72). The vps23 and rim101 mutants of C. neoformans both displayed an iron-related growth defect, a reduced cell-associated capsule, increased capsule shedding, increased sensitivity to alkaline pH, and poor growth on high concentrations of LiCl, KCl, and NaCl. The shared phenotypes likely indicate that Vps23 from C. neoformans retains a conserved role in the activation of Rim101, as seen in other fungi (62, 72, 73, 74). In contrast, distinct virulence phenotypes were seen because a rim101 mutant is hypervirulent in an animal model of cryptococcosis despite the cell-associated capsule reduction (21). As noted, our vps23 mutants are avirulent, suggesting a potentially distinct contribution of Vps23 to pathogenesis that is independent of the Rim101 pathway. Further study is needed to understand this distinct contribution, particularly in the context of heme use, and to elucidate the relationship between ESCRT complex functions and the Rim101 pathway in C. neoformans. In addition, it is likely that the ESCRT pathway targets additional factors that may influence virulence, including other transcription factors; one important candidate is the major iron regulator Cir1, which controls the expression of iron uptake functions, as well as capsule and melanin elaboration.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (R01 AI053721) and the Canadian Institutes of Health Research (J.K.). J.K. is a Burroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology.
The content of this report is solely our responsibility and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript.
We thank Alex Idnurm and Joe Heitman for plasmids; Joyce Wang, Chunmei Li, and Madeleine Ennis for technical assistance; and Arturo Casadevall for anticapsule antibody.

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cover image Infection and Immunity
Infection and Immunity
Volume 81Number 1January 2013
Pages: 292 - 302
Editor: George S. Deepe
PubMed: 23132495

History

Received: 25 September 2012
Returned for modification: 11 October 2012
Accepted: 27 October 2012
Published online: 20 December 2013

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Guanggan Hu
Michael Smith Laboratories, Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada
Mélissa Caza
Michael Smith Laboratories, Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada
Brigitte Cadieux
Michael Smith Laboratories, Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada
Vivienne Chan
Michael Smith Laboratories, Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada
Victor Liu
Michael Smith Laboratories, Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada
James Kronstad
Michael Smith Laboratories, Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada

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George S. Deepe
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Notes

Address correspondence to James Kronstad, [email protected].

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