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

Guanggan Hu, Mélissa Caza, Brigitte Cadieux, Vivienne Chan, Victor Liu, James Kronstad

Michael Smith Laboratories, Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, Canada

Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal me-ningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalianhosts 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 onheme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomalsorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect inuptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in theelaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resultedin cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mousemodel of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role fortrafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection betweeniron and virulence factor deployment in C. neoformans.

The pathogenic fungus Cryptococcus neoformans causes life-threatening meningoencephalitis in immunocompromised

individuals (1). Iron availability influences at least two major vir-ulence traits in this fungus, including the production of a polysac-charide capsule and deposition of melanin in the cell wall (2, 3, 4,5, 6). Additionally, iron overload exacerbates cryptococcal diseasein a mouse model of cryptococcosis (7). Like other pathogens, C.neoformans must compete with mammalian iron sequestrationmechanisms 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 im-mune system to maintain low iron availability.

C. neoformans has multiple acquisition mechanisms to obtainiron, including a high-affinity uptake system composed of the ironpermease Cft1 and the ferroxidase Cfo1, a siderophore uptakepathway, cell surface reductases, and exported reductants such as3-hydroxyanthranilic acid; in addition, melanin in the cell wallmay contribute to ferric iron reduction (3, 8, 9). The Cft1/Cfo1permease/ferroxidase complex is essential for reductive iron up-take, for iron acquisition from transferrin, and for full virulence ina mouse model of cryptococcosis (10, 11). The CFT1 transcriptlevel is elevated upon iron limitation and during early murinepulmonary infection (12, 13, 14). However, cft1 and cfo1 mutantscan obtain iron from siderophores, heme, and hemoglobin, thusindicating that another uptake pathway(s) is needed for these ironsources (10, 11). The mechanisms of heme use have not beenidentified, but iron acquisition from siderophores is known torequire specific transporters such as the ferrioxamine B trans-porter Sit1, although C. neoformans is unable to produce its ownsiderophores (15, 16). The regulation of iron uptake mechanismsand sensing in C. neoformans is mediated by the master iron reg-ulator Cir1, as well as several other regulatory proteins, includingNrg1, 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 thissource is based mainly on studies with parasites and pathogenic

bacteria (22, 23, 24). Gram-negative bacteria, for example, gener-ally transport heme across the outer membrane through specificreceptors or secrete hemophores that bind heme to facilitate up-take (23, 24, 25). Gram-positive bacteria bind and transport hemevia cell surface-associated proteins at the cell wall and take upheme via ATP-binding cassette transporters in the plasma mem-brane (26). Some Gram-positive bacteria also produce hemo-phores (24). In contrast to the situation in bacterial pathogens andparasites, much less is known about iron acquisition in fungalpathogens of humans.

The pathogenic fungi C. neoformans, Candida albicans, andHistoplasma capsulatum grow well with heme or hemoglobin as asole iron source in vitro (10, 27, 28). Heme use has been bestcharacterized in C. albicans, and this fungus binds erythrocytesand possesses a hemolytic factor on the cell surface (29, 30, 31, 32,33). Specific heme/hemoglobin receptors (Rbt5 and Rbt51) in theplasma 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 extracellularmembrane protein domain (28, 35). Furthermore, hemoglobinwas found to bind to Rbt5 and to be internalized via an endocyticpathway (34). Once internalized into vacuoles, hemoglobin islikely denatured or hydrolyzed to release heme, and a heme oxy-genase (Hmx1) is involved in the further degradation of heme to

Received 25 September 2012 Returned for modification 11 October 2012Accepted 27 October 2012

Published ahead of print 6 November 2012

Editor: George S. Deepe

Address correspondence to James Kronstad, kronstad@msl.ubc.ca.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01037-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01037-12

292 iai.asm.org Infection and Immunity p. 292–302 January 2013 Volume 81 Number 1

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

release iron (30, 36, 37). Overexpression of C. albicans Rbt51 inSaccharomyces cerevisiae enables growth in the presence of hemo-globin, and screening of the deletion collection in this yeast forreduced growth on this iron source revealed roles for a vacuolarATPase and the ESCRT (endosomal sorting complex required fortransport) complexes (34). The ESCRT system is dedicated to thetargeting of monoubiquitinated membrane proteins to the vacu-ole (38). ESCRT mutants of C. albicans were further shown to havedefects in iron acquisition from hemoglobin but not the sidero-phore ferrichrome (28, 34).

A cell surface binding activity for heme has also been reportedfor the facultative intracellular fungal pathogen H. capsulatum(27). For this pathogen, iron acquisition from heme, sidero-phores, and iron-binding proteins such as transferrin involves asecreted gamma-glutamyltransferase (Ggt1) and a glutathione-dependent ferric reductase (GSH-FeR) (39, 40). Ggt1 contrib-utes to virulence in H. capsulatum, as do specific siderophores(40, 41, 42).

Given that the mechanisms of heme use in C. neoformans arenot known, we constructed and screened an insertion mutant li-brary to identify genes involved in this process. We discovered arole for Vps23, a component of the ESCRT-I complex that is re-quired for intracellular trafficking of endocytic vesicles. Our char-acterization of vps23 deletion mutants also revealed that Vps23and ESCRT function contribute to capsule and melanin forma-tion, as well as virulence, in C. neoformans. Overall, this studyextends our understanding of iron acquisition in C. neoformans,reveals an additional complexity to the connection between ironand virulence factor production in this pathogen, and underscoresthe importance of intracellular trafficking in fungal virulence.

MATERIALS AND METHODSStrains, plasmids, and media. Serotype A strains H99 (MAT�) andKN99a (MATa) (C. neoformans var. grubii) were maintained on YPDmedium (1% yeast extract, 2% peptone, 2% dextrose, 2% agar). Thenourseothricin, neomycin, and hygromycin resistance cassettes werefrom plasmids pCH233, pJAF1, and pJAF15, respectively. YPD mediumplates containing neomycin (200 �g/ml) were used to select vps23 dele-tion transformants. Defined low-iron medium (LIM) (6, 14) and yeastnitrogen base (YNB; pH 7.0) plus 150 �M bathophenanthroline disulfon-ate (BPS) (10) were used as iron-limiting media. YPD and/or YNB liquidmedia or plates (YNB with amino acids) supplemented as indicated wereused for phenotypic characterization. All chemicals were obtained fromSigma-Aldrich unless indicated otherwise. Feroxamine was obtained asdeferoxamine mesylate (Sigma-Aldrich) and chelated with FeCl3.

Agrobacterium tumefaciens-mediated transformation of C. neofor-mans. 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 back-ground). Briefly, Agrobacterium cells were grown overnight with shakingat room temperature in Luria-Bertani medium with kanamycin. Cellswere washed and resuspended in liquid induction medium with 200 �Macetosyringone (AS) at an optical density at 600 nm (OD600) of 0.15 andincubated for 6 h (OD600 of 0.6). C. neoformans H99 cells were grownovernight in YPD medium, washed in induction medium, and resus-pended at 106 or 107/ml. Subsequently, 200 �l of each of the C. neoformansand A. tumefaciens cultures was mixed and plated (without spreading) oninduction agar medium (with AS). The plates were incubated for 2 to 3days before the mixtures were resuspended in liquid YPD medium. Cellswere then plated onto YPD medium with neomycin (200 �g/ml) andcefotaxime (100 �g/ml), and the plates were incubated at 30°C for 3 to 4days.

Mutant screening and inverse PCR. A collection of �30,000 trans-formants was screened for growth defects on LIM containing heme as thesole iron source. Briefly, overnight cultures of each strain were grown inYPD medium in a 96-well plate format and 5 �l of each culture wastransferred to a well containing 200 �l of LIM (YNB plus 150 �M BPS).These cells were incubated at 30°C for 2 days to reduce the amount ofstored intracellular iron. After starvation, 5 �l of each culture was trans-ferred to 96-well plates containing 200 �l of YNB plus 150 �M BPS, YNBplus 150 �M BPS and 10 �M heme, YNB plus 150 �M BPS and 100 �Mheme, YNB plus 150 �M BPS and 10 �M FeCl3, or YNB plus 150 �M BPSand 100 �M FeCl3. Growth was determined by measurement of OD600 ona Tecan plate reader after incubation for 3 days at 30°C. Strains withdefective growth on heme but not FeCl3 were further confirmed with spotassays on LIM with either 10 or 100 �M heme.

Inverse PCR was used to determine the disruption sites in candidatemutants 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 weredetermined 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 H99and KN99a and the cfo1 mutant (H99 background) was replaced with aneomycin resistance cassette to delete the gene. Briefly, a vps23::NEO de-letion 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 genomicDNA to obtain the left and right arms for the deletion construct. Theselectable marker NEO was amplified from plasmid pJAF1 by using prim-ers vps23-2 and vps23-5. Overlap PCR with primers vps23-1 and vps23-6yielded a vps23::NEO allele lacking the entire open reading frame (1,850bp). The resulting PCR product (3,755 bp) was used to transform strainsH99 and KN99a and a cfo1 mutant by biolistic transformation (49).Transformants were screened by colony PCR with Extaq polymerase (Ta-KaRa) using primers vps23-7 and vps23-8 (negative screen) and vps23-9and hug-Neo (positive screen). Primer vps23-9 was designed from theregion upstream of VPS23, and hug-Neo was designed for the NEO gene.Transformants with replacements were confirmed by genomic hybridiza-tion analysis as described previously (48). Two independent vps23-9 andvps23-16 mutants in the H99 strain background were constructed andstudied further. One vps23 mutant (vps23-9a) in the KN99a backgroundand two double mutants (cfo1 vps23-13 and cfo1 vps23-14b) from the cfo1background were also analyzed. Mating types of strains were confirmed bymating assays as described previously (50). A similar strategy for genedeletion was used to generate the RIM101 deletion strain, rim101-JK, withthe primers listed in Table S1 in the supplemental material. The in vitrophenotypes of the rim101-JK strain matched those of the rim101-AA strain (agift from J. Andrew Alspaugh), as described by O’Meara et al. (21).

Overexpression of VPS23 in a vps23 mutant strain. Strains for over-expression of VPS23 and complementation of the vps23 deletion weregenerated by replacing the promoter with that of the elongation factor Igene (CNAG_06125.2). Initially, the promoter was amplified from strainH99 with primers EF1-5R-HindIII and EF1-3F-IF-SpeI (see Table S1 inthe supplemental material). The 1,606-bp product was digested with Hin-dIII and SpeI and cloned into HindIII/SpeI-digested pJAF15 to produceplasmid pEF1pro. The VPS23 fragment was amplified with primersVps23GFP5rP3-SpeI and Vps23comp-IF3Fb-SpeI, and the resulting2,420-bp PCR product was digested with SpeI and cloned into SpeI-di-gested pEF1pro. The plasmid with correct orientation of the Vps23 frag-ment was identified and designated pEF1pro-Vps23-ter. pEF1pro-Vps23-ter was linearized with BglII and introduced into the vps23-9 mutant. Thisconstruct therefore replaced the promoter and complemented the dele-tion mutation. Overexpression was confirmed by quantitative reversetranscription (RT)-PCR as described by Hu et al. (12), using primerslisted in Table S2 in the supplemental material.

Heme Uptake and Virulence in C. neoformans

January 2013 Volume 81 Number 1 iai.asm.org 293

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Toxicity of gallium protoporphyrin (GaPPIX) and manganese pro-toporphyrin (MnPPIX). To test for GaPPIX and MnPPIX toxicity, cellsgrown overnight in YPD medium were washed twice in sterile, low-ironwater (treated with Chelex-100 resin [Bio-Rad]). Tenfold serial dilutionsof the cells were prepared in low-iron water. To ensure exposure to a highconcentration of the expensive chemical GaPPIX, defined LIM agar plus10 �M heme was spread with 200 �l of 10 �M GaPPIX (Frontier Scien-tific), 100 �M MnPPIX (Frontier Scientific), or 10 �M GaCl3 (Sigma-Aldrich) immediately prior to the spotting of 5-�l volumes of dilutions of1 � 106 to 1 � 102 cells/ml. The plates were incubated for 2 days at 30°Cbefore being photographed.

Capsule formation and melanin production. Capsule formation wasexamined by differential interference contrast microscopy after incuba-tion 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 wasexamined with a blot assay performed as described by Yoneda and Doer-ing (51).

FM4-64 staining of the vacuolar membrane. Internalization of thelipophilic dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(dieth-ylamino) phenyl) hexatrienyl) pyridinium dibromide] (T-3166; Invitro-gen, Burlington, Ontario, Canada) was used to visualize the vacuolarmembrane as previously described, with minor modifications (52).FM4-64 was used at a final concentration of 10 �M in phosphate-bufferedsaline (PBS; pH 7.4). Cells were harvested after overnight growth in YPDmedium, stained with FM4-64 for 15 min on ice, washed, and transferredto fresh medium without the stain. The stained cells were incubated in a30°C shaker for 30 min before viewing.

Virulence assays. Virulence was assayed in an inhalation model ofcryptococcosis using female BALB/c mice (4 to 6 weeks old) from CharlesRiver Laboratories (Senneville, Ontario, Canada) as previously described(52). Briefly, C. neoformans strains were grown in 5 ml of YPD medium at30°C overnight, washed twice with PBS (Invitrogen, Burlington, Ontario,Canada), and resuspended in PBS. The BALB/c mice, in groups of 10, wereintranasally inoculated with a suspension of 106 cells in 50 �l. The healthstatus of the mice was monitored daily postinoculation. Mice reaching thehumane endpoint were euthanized by CO2 anoxia. Statistical analyses ofsurvival differences were performed by log rank tests using GraphPadPrism 5 for Windows (GraphPad Software, San Diego, CA). For determi-nation of the fungal load in organs, infected mice were euthanized andorgans were excised, weighed, and homogenized in 1 ml of PBS using aMixerMill (Retsch, Newtown, PA). Serial dilutions of the homogenateswere plated on Sabouraud dextrose agar plates containing 35 �g/ml chlor-amphenicol, and CFU were counted after incubation for 48 h at 30°C. Theprotocols for the virulence assay (protocol A08-0586) were approved bythe University of British Columbia Committee on Animal Care.

RESULTSInsertional mutagenesis screening for mutants unable to useheme. To identify functions for heme uptake in C. neoformans, weinitially constructed a mutant library by Agrobacterium-mediatedinsertional mutagenesis in a cfo1 mutant lacking the high-affinity

iron uptake system. The cfo1 mutant grows poorly under low-ironconditions, but growth is restored by heme (10 �M) or a highconcentration of ferric iron (100 �M) (Fig. 1) (10). We reasonedthat the lack of Cfo1 would reduce the ability of C. neoformans touse free iron that may be contaminating the heme and block anypotential contribution of the reductive uptake system to the use ofiron from heme. A stock of �30,000 transformants was con-structed, and genome hybridization analysis of 16 randomly se-lected strains using a probe designed from the neomycin resis-tance cassette revealed that �90% had a single T-DNA insertionevent (see Fig. S1 in the supplemental material). We then screenedfor mutants defective for growth on heme by examining growth(OD600) in low-iron liquid media without and with different levelsof heme or FeCl3. This approach yielded 34 mutants with poorgrowth on heme as the sole iron source, and the phenotypes ofthese mutants with heme and other iron-related phenotypes werefurther confirmed with spot assays on solid media (Fig. 1 and datanot shown). Genomic DNA was isolated for all candidate mutants,inverse PCR was performed, and the disruption sites were success-fully identified for 25 mutants. The analysis of one set of mutantswith defects in the VPS23 gene (Fig. 1) is described below, and theanalysis of additional mutants will be presented in a subsequentpublication.

Mutations in the ESCRT-I gene VPS23 cause a growth defecton heme. Three mutants were identified with T-DNA insertionsin the VPS23 gene, which encodes a member of the ESCRT-I pro-tein-sorting complex. The ESCRT complex is composed of fourcore cytoplasmic polyprotein complexes (ESCRT-0, -I, -II, and-III), which are recruited sequentially to ubiquitinated cargo pro-teins at endocytic vesicle membranes and deliver endosomes tothe vacuole (38). In C. neoformans strain H99, VPS23 is annotatedas 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 inSaccharomyces cerevisiae, and 25% identity and 44% similarity toVps23 in Candida albicans. Bioinformatic analysis of the H99 ge-nome and Southern blot analysis demonstrated that only oneVPS23 ortholog exists in C. neoformans (see Fig. S2 in the supple-mental material).

To confirm the phenotypes of the insertion mutants, we gen-erated two independent deletion mutations of VPS23, includingvps23-9 and vps23-16 in the H99 strain (MAT�); one deletionmutation, vps23-9a, in the KN99a strain (MATa); and two doublemutations, vps23 cfo1-13 and vps23 cfo1-14b, in a cfo1 mutantstrain (MAT�, derived from H99) (10). The deletions were con-firmed by colony PCR and DNA hybridization (see Fig. S2 in thesupplemental material). The vps23 mutation was constructed in

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 wereincubated at 30°C for 2 days before being photographed. The strains were the parental WT strain H99, the three mutants (272D10, 277A9, and 292E1) withinsertions in VPS23, and a mutant with a deletion in the CFO1 gene for the ferroxidase of the high-affinity iron uptake system (10).

Hu et al.

294 iai.asm.org Infection and Immunity

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

strains of opposite mating types to collect additional independentmutants. We also complemented the vps23 defect in the vps23-9mutant by introducing the gene under the control of the EF1promoter, and this approach allowed us to also examine the influ-ence of overexpression of the gene. Overexpression of the VPS23gene in the complemented strains was confirmed by quantitativeRT-PCR (see Fig. S3 in the supplemental material).

The vps23 deletion and insertion mutants and the overexpres-sion strains were examined for growth on low-iron YNB mediumwith 150 �M BPS (Fig. 2A). After 3 days of iron starvation, allstrains were unable to grow on this medium, in contrast to theirrobust growth on YNB. The wild-type (WT) strain (H99) grew onlow-iron YNB with 10 �M FeCl3 and all other iron sources. As

expected, the cfo1 mutant strain grew on medium supplementedwith 100 �M FeCl3, heme, or feroxamine but not on mediumsupplemented with 10 �M FeCl3 (Fig. 2B) (10). The vps23 mu-tants grew like the WT strain on low-iron YNB medium supple-mented with 100 �M FeCl3, 10 �M feroxamine, or 2 �M hemo-globin but showed poor growth on low-iron YNB supplementedwith either 10 or 100 �M heme, indicating that Vps23 is requiredfor iron acquisition from heme (Fig. 2A). The VPS23-overexpress-ing strains restored mutant growth to the WT level on heme (Fig.2A). Similar results were observed from spot assays on the definedLIM described by Vartivarian et al. (6) with and without the dif-ferent iron sources (data not shown). Moreover, growth of thecfo1 vps23 double mutants was poor on LIM supplemented with

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 onthe 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 vps23-9mutant, and the overexpression strain were inoculated into liquid YNB medium plus 150 �M BPS without and with supplementation with iron sources. Thecultures were incubated at 30°C, and OD600s were measured. (E) The indicated strains were also tested for growth in the defined LIM supplemented with hemeor FeCl3 by the same method used for panel D.

Heme Uptake and Virulence in C. neoformans

January 2013 Volume 81 Number 1 iai.asm.org 295

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

10 �M FeCl3 or different levels of heme as the iron source (Fig.2B). Growth of the strains in liquid medium (either low-iron YNBor defined low-iron medium) also revealed that the vps23 mutantshad a growth defect with heme or hemoglobin (Fig. 2D and E; datanot shown). In addition, the mutants showed an extended growthlag compared to the WT strain on low-iron media with addition ofFeCl3 and feroxamine (Fig. 2D and E; data not shown). This mayindicate a role for ESCRT function in high-affinity reductive ironuptake, as found in S. cerevisiae (53, 54, 55). The vps23 mutantstrain from the KN99a MATa background also grew poorly withheme but showed WT growth on media with the other ironsources (FeCl3, feroxamine) (Fig. 2C). Taken together, these re-sults reveal that Vps23 is required for robust growth on heme.

Loss of Vps23 reduces susceptibility to noniron metallopro-toporphyrins. Noniron metalloporphyrins (MPs) are taken up bybacteria via heme uptake systems, and they have antibacterial ac-tivity that is thought to result from interference with heme-depen-dent metabolic processes (56, 57). GaPPIX and MnPPIX show thehighest toxicity for bacteria, and we hypothesized that the vps23mutant defect in heme acquisition would reduce susceptibility.Indeed, the vps23 deletion mutants in both the MATa and MAT�backgrounds showed reduced susceptibility to the noniron MPs(Fig. 3A and C). In contrast, the cfo1 mutant, the vps23 cfo1 doublemutant, and a VPS23-overexpressing strain were inhibited like theWT strain (Fig. 3A and B). The double mutant may fail to growbecause Cfo1 is required for the acquisition of inorganic iron con-taminating the heme and/or for another pathway for iron usefrom heme. As a control, none of the strains were inhibited by thepresence of GaCl3, in further support of the requirement for hemeuptake. Overall, these data support the hypothesis that vps23 isinvolved in heme uptake by C. neoformans.

A vps23 mutant shows aberrant vacuolar staining. The ES-CRT 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 deter-mine whether the Vps23 ortholog in C. neoformans has a similarfunction, we analyzed endocytic transport from the plasma mem-brane to the vacuole by staining with the fluorescent dye FM4-64.In both WT and vps23 mutant cells, FM4-64 was initially observedat the plasma membrane and was rapidly internalized into endo-cytic vesicles. After 75 min, the dye was observed only on thevacuolar membrane in WT cells while migration to the vacuolewas delayed in vps23 mutants (Fig. 4A; see Fig. S4 in the supple-mental material for additional images of cells). In addition,brightly staining spots (caps) of the dye were frequently observedon one side of the vacuole in �90% of the cells of the vps23 mu-tants. This phenomenon is described in S. cerevisiae and C. albi-cans as the vacuolar E-body and likely reflects FM6-64 localizationto a late endocytic-prevacuolar (MVB-like) compartment (58,59). The VPS23-overexpressing strains showed a staining patternlike that of the WT strain (Fig. 4A). Deletion of VPS23 from theKN99a strain (vps23-9a) and from the cfo1 mutant (vps23cfo1-13and cfo1vps23-14b) also caused the occurrence of E bodies (datanot shown). In S. cerevisiae, E bodies (class E compartments) re-sult from missorting of cargo normally destined for the vacuole,causing it to accumulate in abnormal endocytic structures (58). Inthis context, our data suggest that Vps23 in C. neoformans mayalso play a role in MVB transport to the vacuole.

To further probe the connection between Vps23 and intracel-lular trafficking, such as endoplasmic reticulum (ER)-Golgi com-partment transport, we tested the strains for susceptibility tobrefeldin A (BFA; 25 �g/ml) and monensin (0.5 mg/ml). BFA isknown to arrest the anterograde transport of proteins between theER and the Golgi compartment, and monensin is a Na�/H� iono-phore that blocks intracellular transport in both the trans-Golgiand post-Golgi compartments. The vps23 mutants in the strains ofeither mating type background, KN99a or H99, showed increasedsusceptibility to BFA or monensin, a result consistent with Vps23involvement in intracellular trafficking (i.e., endocytosis and ER-Golgi compartment transport) (Fig. 4B). Overexpression ofVPS23 restored the WT level of susceptibility (Fig. 4B).

Vps23 is involved in capsule and melanin elaboration. Wepreviously found that BFA reduced capsule formation in C. neo-formans, and we therefore hypothesized that Vps23 may be in-volved in delivering virulence factors such as capsule and laccase(for melanin production) to the cell surface (13). The Vps23 con-tribution to capsule formation was initially examined in the WT,vps23 mutant, and VPS23-overexpressing strains by staining withIndia ink. Deletion of VPS23 in both H99 and KN99a resulted inreduced capsule formation, and overexpression of VPS23 in vps23mutants resulted in cells with a capsule slightly larger than that ofthe WT strain (Fig. 5A to D). As expected, the cfo1 mutant pro-duced a capsule at the level of the WT strain, but the two cfo1 vps23mutants displayed a marked reduction in capsule size (Fig. 5B).The three independent Agrobacterium-mediated Vps23 insertionmutants were also defective in capsule formation (data notshown). Measurements of capsule size revealed that the capsulereduction in the vps23 and cfo1 vps23 mutants was significantcompared with the WT strain, although the range of capsule sizesof the VPS23-overexpressing strains overlapped that of the WTstrain (Fig. 5D). Overall, the data support the hypothesis thatVps23 is involved in capsule elaboration.

Glucuronoxylomannan (GXM) is the major polysaccharide of

FIG 3 Reduced susceptibility of vps23 mutants to noniron MPs. Tenfold serialdilutions of cells of the indicated strains were spotted onto defined LIM withheme as the iron source. The plates on the right contained GaCl3, GaPPIX, orMnPPIX. The plates were incubated for 2 days at 30°C. (A, B) The mating type� strains in the parental H99 or the cfo1 mutant background were tested forsusceptibility. (C) The vps23 mutant in the mating type a strain (KN99a)background was also tested to confirm the altered susceptibility. Note that 200�l of the noniron MP IX solutions at the indicated concentrations were spreadonto the surface of the plates prior to inoculation.

Hu et al.

296 iai.asm.org Infection and Immunity

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

the capsule that is exported and attached to the cell wall (60). Todetermine whether vps23 mutants have a defect in capsule attach-ment, we used a gel electrophoresis and blotting technique to ex-amine GXM shedding (21, 51). Fungal cells were incubated incapsule-inducing medium, and polysaccharide shed into the me-dium was analyzed for relative abundance by reactivity with ananti-GXM antibody (monoclonal antibody 18B7). As shown inFig. 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 GXMsynthesis but instead causes a defect in capsule attachment to thecell wall. The overexpression of VPS23 resulted in a level of freeGXM similar to that in the WT strain.

C. neoformans cells also produce the virulence factor melaninbecause of deposition of the enzyme laccase in the cell wall. Asshown in Fig. 6, deletion of VPS23 caused reduced melanin for-mation in the single mutants of strains of both mating types. TheWT level of melanin was restored in the overexpression strains.We did note that the growth of the mutants was slightly lower thanthat of the WT strain on the L-DOPA medium used to assay mel-anin formation, and this phenotype could contribute to reducedpigmentation.

Additional phenotypes of vps23 mutants. We next tested theeffects of pH and a high salt concentration on the growth of vps23mutants because loss of ESCRT function influences related pheno-types in S. cerevisiae and C. albicans (59, 61, 62). Compared to the WTand VPS23-overexpressing strains, the vps23 mutant exhibited amore 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 pHwas included as a control (21). The vps23 mutants also showed in-creased sensitivity to 1.5 M NaCl (5 days), 1.5 M KCl, and 100 mMLiCl (10 days) (Fig. 7B and C). However, the mutants grew as well asthe WT on 1.5 M sorbitol, thus indicating that the growth defects on

LiCl, KCl, and NaCl were due to ionic rather than general osmoticstress. Overall, these data indicated that Vps23 in C. neoformansshares functions with orthologs in other fungi.

Vps23 is required for virulence. We hypothesized that dele-tion 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. Toexamine this prediction, a virulence assay was performed in amouse inhalation model of cryptococcosis with WT strain H99,two independent vps23 mutants, and a VPS23-overexpressingstrain. As expected, the mice inoculated with WT cells reached theexperiment endpoint at 18 to 21 days, while mice infected witheither deletion mutant survived for 40 days (Fig. 8). Unexpectedly,overexpression of VPS23 caused a slight increase in virulence, andthe mice infected reached the experimental endpoint at 14 to 18days (Fig. 8). These experiments indicated that VPS23 is requiredfor virulence. To specifically examine whether the vps23 cellscould disseminate beyond the lung, we monitored the numbers offungal cells in the brains and lungs of the mice at the experimentalendpoints. This test revealed that the vps23 mutant cells were ableto reach the brain, although the average fungal load of the twomutants 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 � 370CFU/g). In comparison, the fungal loads in the lung at the end-point for three mice infected were 5.3 � 103 � 8.0 � 103 CFU/gand 2.2 � 103 � 8.2 � 102 CFU/g for the two vps23 mutants versus2.2 � 108 � 6.0 � 107 CFU/g for the WT strain. These data indi-cate that vps23 mutant cells reached the brain in low numbers butfailed to persist in both the lung and the brain.

DISCUSSION

Pathogens have specific mechanisms to overcome iron withhold-ing and proliferate in mammalian hosts. Our overall goals are to

FIG 4 Defective endocytosis and increased susceptibility to trafficking inhibitors for vps23 mutants. (A) The WT, vps23 mutant, and VPS23-overexpressingstrains 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 thenincubated for an additional 30 min at 30°C before viewing. Bar, 5 �m. DIC, differential interference contrast. (B) Tenfold serial dilutions of the indicated strainswere spotted onto YNB alone or YNB supplemented with either 20 �g/ml BFA or 0.5 mg/ml monensin. The plates were incubated for 3 days at 30°C.

Heme Uptake and Virulence in C. neoformans

January 2013 Volume 81 Number 1 iai.asm.org 297

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

define the mechanisms employed by C. neoformans to acquire ironin mammalian hosts and to use the information to develop ap-proaches to block fungal proliferation and pathogenesis. In thisstudy, we used insertional mutagenesis to identify mutants defec-tive in iron acquisition from heme, the most abundant iron sourcein mammals, and we focused on characterizing mutations in thegene for the ESCRT-I complex protein Vps23. This gene contrib-utes to iron acquisition because vps23 mutants displayed a pro-

nounced growth defect on heme and reduced susceptibility to thenoniron MPs GaPPIX and MnPPIX, which have heme uptake-dependent toxicity in bacteria (56, 57). There was also an extendedlag phase for the mutants in medium with iron chloride, and thismay reflect an additional role for the ESCRT pathway in recyclingthe high-affinity, reductive iron permease/ferroxidase proteins(Cft1 and Cfo1) to the plasma membrane under conditions oflow iron availability (10). This idea is based on findings that inS. cerevisiae, iron levels influence endocytic sorting of the ironpermease/ferroxidase proteins Ftr1 and Fet3 (53, 54, 55). Inthis yeast, iron deprivation results in sorting of the proteinsback to the plasma membrane and iron addition causes target-ing to the vacuole for degradation via the ESCRT protein-mediated MVB pathway.

The involvement of ESCRT proteins such as Vps23 in hemeuptake and iron acquisition in C. neoformans indicates that thisfunction is conserved among some pathogenic fungi. That is, mu-tations in VPS23 and other ESCRT pathway genes also causegrowth defects with hemoglobin (�1 �M) as the sole iron sourcein C. albicans (28, 34). Weissman et al. (34) proposed that endo-cytosis of vesicles from the plasma membrane may be a conservedmechanism of iron scavenging from the environment. In C. albi-cans, the plasma membrane proteins Rbt5 and Rbt51 bind hemeand hemoglobin, and heterologous expression of Rbt51 also al-

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°Cfor 48 h, and capsule formation was assessed by India ink staining of the indicated strains. Bar, 5 �m. (D) Fifty cells of each strain were measured to determinethe cell diameter and capsule radius. Each bar represents the average of the 50 measurements with standard deviations. An asterisk indicates that the count isstatistically significantly different from the others by the Student t test (P � 0.05). (E) The electrophoretic mobility and quantity of shed polysaccharide wereassessed as described in Materials and Methods, by using an anti-GXM antibody to detect the capsule.

FIG 6 Reduced melanization in vps23 mutants. Tenfold serial dilutions ofcells of the indicated strains were spotted onto solid L-DOPA plates. The plateswere incubated for 2 days at 30°C prior to being photographed.

Hu et al.

298 iai.asm.org Infection and Immunity

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

lows S. cerevisiae to use iron from hemoglobin (28, 34). Screeningfor mutations in S. cerevisiae that block the use of hemoglobin viaRbt51 revealed a role for vacuolar functions, including vacuolarATPase activity for acidification, ESCRT pathway functions todeliver endocytic vesicles to the vacuole, and HOPS complex pro-teins that mediate vesicle fusion to the vacuole. Although thisscreening only revealed a role for the ESCRT complex II and IIIproteins (Vps22 and Vps32, respectively) in S. cerevisiae, subse-quent analysis of deletion mutants of C. albicans confirmed thatmutations in genes for the ESCRT-I complex, VPS23 and VPS28,

also influenced hemoglobin transport to the vacuole (34). In lightof this study, our finding that Vps23 participates in heme uptakein C. neoformans strongly suggests that the remaining ESCRTcomponents are required for the process and preliminary experi-ments support this idea (G. Hu, unpublished results).

Importantly, Rbt5 and Rbt51 do not contain a cytoplasmicdomain and they are unlikely to be direct targets for the ESCRTpathway. It is possible, therefore, that another transmembranereceptor(s) exists in C. albicans (and in C. neoformans) to facilitatethe transport of heme or hemoglobin to the vacuole via ESCRT-mediated endocytosis. For C. neoformans, we have identified Cig1as a candidate heme-binding protein that is required for growthon heme (B. Cadieux and J. Kronstad, submitted for publication).H. capsulatum may also have a heme/hemoglobin receptor, fur-ther suggesting that the use of these iron sources may be conservedamong fungal pathogens that attack humans (27). However, weshould note that another important human pathogen, Aspergillusfumigatus, is unable to use heme or transferrin as an iron sourceand instead relies mainly on siderophore-based mechanisms ofiron acquisition (63).

In addition to a defect in heme use, the vps23 mutants dis-played a smaller cell-associated capsule and shed more capsularpolysaccharide into the culture supernatant than the WT strain.These observations suggest that Vps23 (and likely the ESCRT-Icomplex) is required for the establishment of the proper capsuleor cell wall structure required for anchoring of the polysaccharideto �-1,3-glucan at the cell surface. That is, the capsule phenotypemay indirectly result from impaired trafficking of materials toproperly construct the cell wall. Alternatively, or in addition, ES-CRT function may contribute to trafficking functions that medi-

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 growthwas 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 spottedonto 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, 3days; KCl, 5 days; NaCl, 5 days; LiCl, 10 days.

FIG 8 Vps23 influences virulence in mice. Ten female BALB/c mice wereinoculated intranasally with each of the strains indicated, and the survival ofthe mice was monitored daily. The mice were each inoculated with 2 � 105

fungal cells. Survival differences between groups of mice were evaluated by logrank tests. The P values for the mice infected with the WT and mutant strainswere statistically significantly different (P � 0.001).

Heme Uptake and Virulence in C. neoformans

January 2013 Volume 81 Number 1 iai.asm.org 299

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ate the biosynthesis and/or modification of the capsule polysac-charides GXM and galactoxylomannan such that the moleculesare competent for cell wall attachment. Consistent with this idea,Frases et al. (64) have shown that exopolysaccharide found inculture supernatant has physical and chemical differences fromthe cell-associated material. Similarly, altered polysaccharidechemistry as a result of changes in processing or biosynthesis dur-ing transit to the cell surface might influence self-aggregation ofthe material to build a cell-associated capsule (65). The slightlylarger capsule size that we observed on strains overexpressingVps23 is also consistent with changes in capsule anchoring and/oraggregation.

Capsule polysaccharide is transported to the cell surface in ves-icles via exocytosis, and it is clear that trafficking of polysaccharideand other virulence-related factors such as laccase is important forthe virulence of C. neoformans (60, 66, 67). Our observations thatdeletion of VPS23 caused increased sensitivity to BFA and monen-sin, two intracellular trafficking inhibitors, and that these inhibi-tors reduce capsule size, are consistent with a contribution toproper trafficking (13). Similarly, loss of Vps23 may interrupt theprocessing and/or transport of laccase (resulting in reducedmelanization). Laccase must be loaded with copper during transitthrough the ER-Golgi compartment network, and it must beproperly localized in the cell wall (68). Capsule polysaccharide isalso found in extracellular vesicles, although the extent of the con-tribution of the latter vesicles to capsule formation is not yet clear(67). In the context of trafficking, we note that our preliminaryanalysis of extracellular vesicle production by the vps23 mutantsdid not reveal a defect compared with the WT strain, although amore detailed study is needed (B. Cadieux, unpublished results).In S. cerevisiae, deletion of ESCRT genes (e.g., VPS23, SNF7) andother secretory pathway components (e.g., SEC4) did not com-pletely eliminate the production of extracellular vesicles, althoughtheir composition was altered (69). Finally, with regard to capsuletrafficking, we note that mutants lacking components of otherESCRT 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 resultsfrom (at least) the combined defects in iron acquisition fromheme, reduced capsule size, and impaired melanization. It is in-teresting that the VPS23 deletion and disruption mutants hadboth shared and distinct phenotypes compared with a rim101 mu-tant of C. neoformans (21). The Rim101 (PacC in filamentousfungi) transcription factor regulates the response to extracellularpH and has been characterized in several pathogenic and non-pathogenic fungi, including Aspergillus nidulans, C. albicans, Yar-rowia lipolytica, S. cerevisiae, and C. neoformans (21, 70, 71, 72).The vps23 and rim101 mutants of C. neoformans both displayed aniron-related growth defect, a reduced cell-associated capsule, in-creased capsule shedding, increased sensitivity to alkaline pH, andpoor growth on high concentrations of LiCl, KCl, and NaCl. Theshared phenotypes likely indicate that Vps23 from C. neoformansretains a conserved role in the activation of Rim101, as seen inother fungi (62, 72, 73, 74). In contrast, distinct virulence pheno-types were seen because a rim101 mutant is hypervirulent in ananimal model of cryptococcosis despite the cell-associated capsulereduction (21). As noted, our vps23 mutants are avirulent, sug-gesting a potentially distinct contribution of Vps23 to pathogen-esis that is independent of the Rim101 pathway. Further study isneeded to understand this distinct contribution, particularly in

the context of heme use, and to elucidate the relationship betweenESCRT complex functions and the Rim101 pathway in C. neofor-mans. In addition, it is likely that the ESCRT pathway targets ad-ditional factors that may influence virulence, including othertranscription factors; one important candidate is the major ironregulator Cir1, which controls the expression of iron uptake func-tions, as well as capsule and melanin elaboration.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (R01AI053721) and the Canadian Institutes of Health Research (J.K.). J.K. is aBurroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology.

The content of this report is solely our responsibility and does notnecessarily represent the official views of the National Institute of Allergyand Infectious Diseases or the National Institutes of Health. The fundershad no role in study design, data collection and analysis, the decision topublish, 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 ArturoCasadevall for anticapsule antibody.

REFERENCES1. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG,

Chiller TM. 2009. Estimation of the current global burden of cryptococcalmeningitis among persons living with HIV/AIDS. AIDS 23:525–530.

2. Jacobson ES, Compton GM. 1996. Discordant regulation of phenoloxi-dase and capsular polysaccharide in Cryptococcus neoformans. J. Med. Vet.Mycol. 34:289 –291.

3. Jung WH, Kronstad JW. 2008. Iron and fungal pathogenesis: a case studywith Cryptococcus neoformans. Cell. Microbiol. 10:277–284.

4. Polacheck I, Hearing VJ, Kwon-Chung KJ. 1982. Biochemical studies ofphenoloxidase and utilization of catecholamines in Cryptococcus neofor-mans. J. Bacteriol. 150:1212–1220.

5. Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR.1996. Effect of the laccase gene CNLAC1, on virulence of Cryptococcusneoformans. J. Exp. Med. 184:377–386.

6. Vartivarian SE, Anaissie EJ, Cowart RE, Sprigg HA, Tingler MJ, Jacob-son ES. 1993. Regulation of cryptococcal capsular polysaccharide by iron.J. Infect. Dis. 167:186 –190.

7. Barluzzi R, Saleppico S, Nocentini A, Boelaert JR, Neglia R, Bistoni F,Blasi E. 2002. Iron overload exacerbates experimental meningoencepha-litis by Cryptococcus neoformans. J. Neuroimmunol. 132:140 –146.

8. Jacobson ES, Hong JD. 1997. Redox buffering by melanin and Fe(II) inCryptococcus neoformans. J. Bacteriol. 179:5340 –5346.

9. Jacobson ES, Milhausen SM, Manthey MK. 2003. 3-Hydroxyanthra-nilate in Cryptococcus neoformans: a secreted reductant that does not en-able wood rot. Med. Mycol. 41:309 –320.

10. Jung WH, Hu G, Kuo W, Kronstad JW. 2009. Role of ferroxidases in ironuptake and virulence of Cryptococcus neoformans. Eukaryot. Cell 8:1511–1520.

11. Jung WH, Sham A, Lian T, Singh A, Kosman DJ, Kronstad JW. 2008.Iron source preference and regulation of iron uptake in Cryptococcus neo-formans. PLoS Pathog. 4(2):e45. doi:10.1371/journal.ppat.0040045.

12. Hu G, Cheng PY, Sham A, Perfect JR, Kronstad JW. 2008. Metabolicadaptation in Cryptococcus neoformans during early murine pulmonaryinfection. Mol. Microbiol. 69:1456 –1475.

13. Hu G, Steen BR, Lian T, Sham AP, Tam N, Tangen KL, Kronstad JW.2007. Transcriptional regulation by protein kinase A in Cryptococcus neo-formans. PLoS Pathog. 3(3):e42. doi:10.1371/journal.ppat.0030042.

14. Lian T, Simmer MI, D’Souza CA, Steen BR, Zuyderduyn SD, Jones SJ,Marra MA, Kronstad JW. 2005. Iron-regulated transcription and capsuleformation in the fungal pathogen Cryptococcus neoformans. Mol. Micro-biol. 55:1452–1472.

15. Jacobson ES, Petro MJ. 1987. Extracellular iron chelation in Cryptococcusneoformans. J. Med. Vet. Mycol. 25:415– 418.

16. Tangen KL, Jung WH, Sham AP, Lian T, Kronstad JW. 2007. The iron-and cAMP-regulated gene SIT1 influences ferrioxamine B utilization,melanization and cell wall structure in Cryptococcus neoformans. Microbi-ology 153:29 – 41.

Hu et al.

300 iai.asm.org Infection and Immunity

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

17. Cramer KL, Gerrald QD, Nichols CB, Price MS, Alspaugh JA. 2006.Transcription factor Nrg1 mediates capsule formation, stress response,and pathogenesis in Cryptococcus neoformans. Eukaryot. Cell 5:1147–1156.

18. Jung WH, Sham A, White R, Kronstad JW. 2006. Iron regulation of themajor virulence factors in the AIDS-associated pathogen Cryptococcusneoformans. PLoS Biol. 4(12):e410. doi:10.1371/journal.pbio.0040410.

19. Jung WH, Saikia S, Hu G, Wang J, Fung CK, D’Souza C, White R,Kronstad JW. 2010. HapX positively and negatively regulates the tran-scriptional response to iron deprivation in Cryptococcus neoformans. PLoSPathog. 6(11):e1001209. doi:10.1371/journal.ppat.1001209.

20. Kronstad J, Saikia S, Nielson ED, Kretschmer M, Jung W, Hu G,Geddes JM, Griffiths EJ, Choi J, Cadieux B, Caza M, Attarian R. 2012.Adaptation of Cryptococcus neoformans to mammalian hosts: integratedregulation of metabolism and virulence. Eukaryot. Cell 11:109 –118.

21. O’Meara TR, Norton D, Price MS, Hay C, Clements MF, Nichols CB,Alspaugh JA. 2010. Interaction of Cryptococcus neoformans Rim101 andprotein kinase A regulates capsule. PLoS Pathog. 6(2):e1000776. doi:10.1371/journal.ppat.1000776.

22. Toh SQ, Glanfield A, Gobert GN, Jones MK. 2010. Heme and blood-feeding parasites: friends or foes? Parasit. Vectors 3:108. doi:10.1186/1756-3305-3-108.

23. Wandersman C, Delepelaire P. 2004. Bacterial iron sources: from sidero-phores to hemophores. Annu. Rev. Microbiol. 58:611– 647.

24. Wandersman C, Delepelaire P. 2012. Haemophore functions revisited.Mol. Microbiol. 85:618 – 631.

25. Mazmanian SK, Skaar EP, Gaspar AH, Humayun M, Gornicki P,Jelenska J, Joachmiak A, Missiakas DM, Schneewind O. 2003. Passage ofheme-iron across the envelope of Staphylococcus aureus. Science 299:906 –909.

26. Wilks A, Burkhard KA. 2007. Heme and virulence: how bacterial patho-gens regulate, transport and utilize heme. Nat. Prod. Rep. 24:511–522.

27. Foster LA. 2002. Utilization and cell-surface binding of heme by Histo-plasma capsulatum. Can. J. Microbiol. 48:437– 442.

28. Weissman Z, Kornitzer D. 2004. A family of Candida cell surface haem-binding proteins involved in haemin and haemoglobin-iron utilization.Mol. Microbiol. 53:1209 –1220.

29. Moors MA, Stull TL, Blank KJ, Buckley HR, Mosser DM. 1992. A rolefor complement receptor-like molecules in iron acquisition by Candidaalbicans. J. Exp. Med. 175:1643–1651.

30. Pendrak ML, Chao MP, Yan SS, Roberts DD. 2004. Heme oxygenase inCandida albicans is regulated by hemoglobin and is necessary for metab-olism of exogenous heme and hemoglobin to alpha-biliverdin. J. Biol.Chem. 279:3426 –3433.

31. Pendrak ML, Krutzsch HC, Roberts DD. 2000. Structural requirementsfor hemoglobin to induce fibronectin receptor expression in Candida al-bicans. Biochemistry 39:16110 –16118.

32. Pendrak ML, Yan SS, Roberts DD. 2004. Sensing the host environment:recognition of hemoglobin by the pathogenic yeast Candida albicans.Arch. Biochem. Biophys. 426:148 –156.

33. Watanabe T, Takano M, Murakami M, Tanaka H, Matsuhisa A, NakaoN, Mikami T, Suzuki M, Matsumoto T. 1999. Characterization of ahaemolytic factor from Candida albicans. Microbiology 145:689 – 694.

34. Weissman Z, Shemer R, Conibear E, Kornitzer D. 2008. An endocyticmechanism for haemoglobin-iron acquisition in Candida albicans. Mol.Microbiol. 69:201–217.

35. Kulkarni RD, Kelkar HS, Dean RA. 2003. An eight-cysteine-containingCFEM domain unique to a group of fungal membrane proteins. TrendsBiochem. Sci. 28:118 –121.

36. Navarathna DH, Roberts DD. 2010. Candida albicans heme oxygenaseand its product CO contribute to pathogenesis of candidemia and altersystemic chemokine and cytokine expression. Free Radic. Biol. Med. 49:1561–1573.

37. Santos R, Buisson N, Knight S, Dancis A, Camadro JM, Lesuisse E.2003. Haemin uptake and use as an iron source by Candida albicans: roleof CaHMX1-encoded haem oxygenase. Microbiology 149:579 –588.

38. Hurley JH, Emr SD. 2006. The ESCRT complexes: structure and mech-anism of a membrane-trafficking network. Annu. Rev. Biophys. Biomol.Struct. 35:277–298.

39. Timmerman MM, Woods JP. 1999. Ferric reduction is a potential ironacquisition mechanism for Histoplasma capsulatum. Infect. Immun. 67:6403– 6408.

40. Zarnowski R, Cooper KG, Brunold LS, Calaycay J, Woods JP. 2008.

Histoplasma capsulatum secreted gamma-glutamyltransferase reducesiron by generating an efficient ferric reductant. Mol. Microbiol. 70:352–368.

41. Hilty J, Smulian GA, Newman SL. 2011. Histoplasma capsulatum utilizessiderophores for intracellular iron acquisition in macrophages. Med. My-col. 49:633– 642.

42. Hwang LH, Mayfield JA, Rine J, Sil A. 2008. Histoplasma requires SID1,a member of an iron-regulated siderophore gene cluster, for host coloni-zation. PLoS Pathog. 4(4):e1000044. doi:10.1371/journal.ppat.1000044.

43. Idnurm A, Giles SS, Perfect JR, Heitman J. 2007. Peroxisome functionregulates growth on glucose in the basidiomycete fungus Cryptococcusneoformans. Eukaryot. Cell 6:60 –72.

44. Idnurm A, Reedy JL, Nussbaum JC, Heitman J. 2004. Cryptococcusneoformans virulence gene discovery through insertional mutagenesis. Eu-karyot. Cell 3:420 – 429.

45. Walton FJ, Idnurm A, Heitman J. 2005. Novel gene functions requiredfor melanization of the human pathogen Cryptococcus neoformans. Mol.Microbiol. 57:1381–1396.

46. Zhang Z, Gurr SJ. 2000. Walking into the unknown: a ‘step down’ PCR-based technique leading to the direct sequence analysis of flankinggenomic DNA. Gene 253:145–150.

47. Hu G, Kamp A, Linning R, Naik S, Bakkeren G. 2007. Complementationof Ustilago maydis MAPK mutants by a wheat leaf rust, Puccinia triticinahomolog: potential for functional analyses of rust genes. Mol. Plant Mi-crobe Interact. 20:637– 647.

48. Hu G, Kronstad JW. 2006. Gene disruption in Cryptococcus neoformansand Cryptococcus gattii by in vitro transposition. Curr. Genet. 49:341–350.

49. Davidson RC, Cruz MC, Sia RA, Allen B, Alspaugh JA, Heitman J. 2000.Gene disruption by biolistic transformation in serotype D strains of Cryp-tococcus neoformans. Fungal Genet. Biol. 29:38 – 48.

50. Erke KH. 1976. Light microscopy of basidia, basidiospores, and nuclei inspores and hyphae of Filobasidiella neoformans (Cryptococcus neoformans).J. Bacteriol. 128:445– 455.

51. Yoneda A, Doering TL. 2008. Regulation of Cryptococcus neoformanscapsule size is mediated at the polymer level. Eukaryot. Cell 7:546 –549.

52. Hu G, Kronstad JW. 2010. A putative P-type ATPase, Apt1, is involved instress tolerance and virulence in Cryptococcus neoformans. Eukaryot. Cell9:74 – 83.

53. Felice MR, De Domenico I, Li L, Ward DM, Bartok B, Musci G, KaplanJ. 2005. Post-transcriptional regulation of the yeast high affinity irontransport system. J. Biol. Chem. 280:22181–22190.

54. Strochlic TI, Schmiedekamp BC, Lee J, Katzmann DJ, Burd CG. 2008.Opposing activities of the Snx3-retromer complex and ESCRT proteinsmediate regulated cargo sorting at a common endosome. Mol. Biol. Cell19:4694 – 4706.

55. Strochlic TI, Setty TG, Sitaram A, Burd CG. 2007. Grd19/Snx3p func-tions as a cargo-specific adapter for retromer-dependent endocytic recy-cling. J. Cell Biol. 177:115–125.

56. Stojiljkovic I, Evavold BD, Kumar V. 2001. Antimicrobial properties ofporphyrins. Expert Opin. Investig. Drugs 10:309 –320.

57. Stojiljkovic I, Kumar V, Srinivasan N. 1999. Non-iron metalloporphy-rins: potent antibacterial compounds that exploit haem/Hb uptake sys-tems of pathogenic bacteria. Mol. Microbiol. 31:429 – 442.

58. Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH. 1992. Mor-phological classification of the yeast vacuolar protein sorting mutants:evidence for a prevacuolar compartment in class E vps mutants. Mol. Biol.Cell 3:1389 –1402.

59. Cornet M, Bidard F, Schwarz P, Da Costa G, Blanchin-Roland S,Dromer F, Gaillardin C. 2005. Deletions of endocytic components VPS28and VPS32 affect growth at alkaline pH and virulence through bothRIM101-dependent and RIM101-independent pathways in Candida albi-cans. Infect. Immun. 73:7977–7987.

60. Yoneda A, Doering TL. 2006. A eukaryotic capsular polysaccharide issynthesized intracellularly and secreted via exocytosis. Mol. Biol. Cell 17:5131–5140.

61. Kullas AL, Li M, Davis DA. 2004. Snf7p, a component of the ESCRT-IIIprotein complex, is an upstream member of the RIM101 pathway in Can-dida albicans. Eukaryot. Cell 3:1609 –1618.

62. Xu W, Smith FJ, Jr, Subaran R, Mitchell AP. 2004. Multivesicularbody-ESCRT components function in pH response regulation in Saccha-romyces cerevisiae and Candida albicans. Mol. Biol. Cell 15:5528 –5537.

63. Haas H. 2012. Iron—a key nexus in the virulence of Aspergillus fumigatus.Front. Microbiol. 3:28. doi:10.3389/fmicb.2012.00028.

Heme Uptake and Virulence in C. neoformans

January 2013 Volume 81 Number 1 iai.asm.org 301

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

64. Frases S, Nimrichter L, Viana NB, Nakouzi A, Casadevall A. 2008.Cryptococcus neoformans capsular polysaccharide and exopolysaccharidefractions manifest physical, chemical, and antigenic differences. Eukaryot.Cell 7:319 –327.

65. Nimrichter L, Frases S, Cinelli LP, Viana NB, Nakouzi A, Travassos LR,Casadevall A, Rodrigues ML. 2007. Self-aggregation of Cryptococcus neo-formans capsular glucuronoxylomannan is dependent on divalent cations.Eukaryot. Cell 6:1400 –1410.

66. Panepinto J, Komperda K, Frases S, Park YD, Djordjevic JT, CasadevallA, Williamson PR. 2009. Sec6-dependent sorting of fungal extracellularexosomes and laccase of Cryptococcus neoformans. Mol. Microbiol. 71:1165–1176.

67. Rodrigues ML, Nakayasu ES, Oliveira DL, Nimrichter L, NosanchukJD, Almeida IC, Casadevall A. 2008. Extracellular vesicles produced byCryptococcus neoformans contain protein components associated with vir-ulence. Eukaryot. Cell 7:58 – 67.

68. Waterman SR, Hacham M, Panepinto J, Hu G, Shin S, Williamson PR.2007. Cell wall targeting of laccase of Cryptococcus neoformans duringinfection of mice. Infect. Immun. 75:714 –722.

69. Oliveira DL, Nakayasu ES, Joffe LS, Guimarães AJ, Sobreira TJ,Nosanchuk JD, Cordero RJ, Frases S, Casadevall A, Almeida IC, Nim-richter L, Rodrigues ML. 2010. Characterization of yeast extracellularvesicles: evidence for the participation of different pathways of cellulartraffic in vesicle biogenesis. PLoS One 5(6):e11113. doi:10.1371/journal.pone.0011113.

70. Davis DA. 2009. How human pathogenic fungi sense and adapt to pH: thelink to virulence. Curr. Opin. Microbiol. 12:365–370.

71. Liu OW, Chun CD, Chow ED, Chen C, Madhani HD, Noble SM. 2008.Systematic genetic analysis of virulence in the human fungal pathogenCryptococcus neoformans. Cell 135:174 –188.

72. Peñalva MA, Tilburn J, Bignell E, Arst HN, Jr. 2008. Ambient pH generegulation in fungi: making connections. Trends Microbiol. 16:291–300.

73. Peñalva MA, Arst HN, Jr. 2004. Recent advances in the characterizationof ambient pH regulation of gene expression in filamentous fungi andyeasts. Annu. Rev. Microbiol. 58:425– 451.

74. Wolf JM, Johnson DJ, Chmielewski D, Davis DA. 2010. The Candidaalbicans ESCRT pathway makes Rim101-dependent and -independentcontributions to pathogenesis. Eukaryot. Cell 9:1203–1215.

Hu et al.

302 iai.asm.org Infection and Immunity

on April 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from