EUKARYOTIC CELL, Dec. 2009, p. 1901–1908 Vol. 8, No. 121535-9778/09/$12.00 doi:10.1128/EC.00256-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Regulatory Diversity of TUP1 in Cryptococcus neoformans�†Hyeseung Lee, Yun C. Chang, Ashok Varma, and Kyung J. Kwon-Chung*

Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute ofAllergy and Infectious Diseases, NIH, Bethesda, Maryland 20892

Received 31 August 2009/Accepted 2 October 2009

Cryptococcus neoformans serotype A strains, the major cause of cryptococcosis, are distributed worldwide,while serotype D strains are more concentrated in Central Europe. We have previously shown that deletion ofthe global regulator TUP1 in serotype D isolates results in a novel peptide-mediated, density-dependent growthphenotype that mimics quorum sensing and is not known to exist in other fungi. Unlike for tup1� strains ofserotype D, the density-dependent growth phenotype was found to be absent in tup1� strains of serotype Awhich had been derived from several different genetic clusters. The serotype A H99 tup1� strain showed lessretardation in the growth rate than tup1� strains of serotype D, but the mating efficiency was found to besimilar in both serotypes. Deletion of TUP1 in the H99 strain resulted in significantly enhanced capsuleproduction and defective melanin formation and also revealed a unique regulatory role of the TUP1 gene inmaintaining iron/copper homeostasis. Differential expression of various genes involved in capsule formationand iron/copper homeostasis was observed between the wild-type and tup1� H99 strains. Furthermore, the H99tup1� strain displayed pleiotropic effects which included sensitivity to sodium dodecyl sulfate, susceptibility tofluconazole, and attenuated virulence. These results demonstrate that the global regulator TUP1 has patho-biological significance and plays both conserved and distinct roles in serotype A and D strains of C. neoformans.

The fungal Tup1 proteins function as global repressorswhich regulate a large number of genes associated with growth,morphological differentiation, and sexual and asexual repro-duction. As a consequence, tup1 mutants are known to displaynumerous phenotypes (9, 19, 42). The deletion of TUP1 inCandida albicans results in constitutive filamentous growthwith no budding yeast cells and is accompanied by loss ofvirulence (2, 32). In Penicillium marneffei, the only dimorphicspecies known in the genus Penicillium, deletion of the TUP1homolog, tupA, confers reduced filamentation and abnormalityin yeast morphogenesis (38). In the filamentous fungi Aspergil-lus nidulans and Neurospora crassa, deletion of the TUP1 ho-mologs, rcoA and rco-1, respectively, severely affects growthand sexual and asexual reproduction (12, 46).

Cryptococcus neoformans is a bipolar heterothallic basidio-mycetous yeast with two serotypes, A and D, and the functionof Tup1 has been studied only for serotype D strains (26, 27).While disruption of TUP1 in strains of serotype D did notaffect yeast or hyphal cell morphology, it resulted in mating-type-dependent differences, including temperature-dependentgrowth, sensitivity to 0.8 M KCl, and expression of genes inseveral other biological pathways (26). Most importantly,tup1� strains displayed a peptide-mediated quorum-sensing-like phenomenon in both mating types of serotype D strainswhich has not been reported for any other fungal species (27).

According to genome sequence data, the serotype A refer-ence strain H99 shares 95% sequence identity with the sero-

type D reference strain JEC21 (29). However, serotype-specific differences between the two strains have been demon-strated in two major signaling pathways, the pheromone-re-sponsive Cpk1 mitogen-activated protein kinase and cyclicAMP (cAMP) (5, 13, 41, 47). In addition, the high-osmolarityglycerol (HOG) pathway also showed regulatory disparity be-tween the two serotypes (1, 8). Since the regulation of peptide-mediated quorum sensing by TUP1 is reported only for sero-type D strains, we sought to determine whether the deletion ofTUP1 in serotype A strains would have similar consequences.Surprisingly, we found striking differences in the phenotypesmanifested by tup1� strains of the two serotypes. We reporthere the serotype-specific differences in TUP1 regulation be-tween A and D strains and the novel regulatory role of TUP1in maintaining iron/copper homeostasis in C. neoformans.

MATERIALS AND METHODS

Strains and media. Serotype A strains used in this study included H99 (MAT�)(35), CHC186 (MAT�) (6), VNBt63 (MAT�) (6), WM148 (MAT�) (30), KN99a(MATa) (33), and WSA1156 (MATa) (20). The first four strains were chosenfrom different genetic clusters among the strains of VNI, the global moleculartype within serotype A strains. The other two strains, KN99a and WSA1156, areMATa strains that are isogenic to H99 and were received from J. Heitman andB. Wickes, respectively. Strains HL112 and HL132 are tup1� and tup1��TUP1strains derived from strain H99. HL14 (MAT�) and HL40 (MATa) are serotypeD tup1� strains derived from strains LP1 (MAT�) and LP2 (MATa), respectively,as described before (26).

Yeast extract-peptone-dextrose (YEPD) and RPMI agar were described pre-viously (4). Minimal medium (SD) contains 6.7 g of yeast nitrogen base (Difco)without amino acids and 20 g of glucose per liter. YES medium contains 0.5%(wt/vol) yeast extract plus 3% glucose and, as supplements, 225 �g/ml each ofuracil, adenine, leucine, histidine, and lysine (31). V8 juice agar was used formating assays (24).

Construction of TUP1 deletion strains. A serotype A TUP1 homolog of C.neoformans was identified by BLAST search of the serotype A (H99) genome(http://www.broad.mit.edu/annotation/fungi/cryptococcus_neoformans/index.html). The TUP1 gene was deleted by biolistic transformation in four serotypeA strains of the VNI molecular type with the construct generated by PCR fusionusing a strategy similar to that described for Clostridium difficile (22). The left end

* Corresponding author. Mailing address: Molecular MicrobiologySection, Laboratory of Clinical Infectious Diseases, National Instituteof Allergy and Infectious Diseases, NIH, Bethesda, MD 20892. Phone:(301) 496-1602. Fax: (301) 480-3240. E-mail: june_kwon-chung@nih.gov.

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 9 October 2009.

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of the locus was amplified with primers TND-C1 and TND-C2G418; the rightend of the locus was amplified with primers TND-D1G418 and TND-D2.G418-A1 and G418-B2 were used to amplify the NEO (neomycin phosphotrans-ferase II) selectable marker from the plasmid pJAF1 (a gift from J. Heitman)(see Table S1 in the supplemental material). The upstream and downstreamflanking regions of the TUP1 gene were amplified from the genomic DNA ofeach strain using the same primers. The amplified products were gel purified andused as templates to produce a 4.2-kb tup1::NEO deletion construct containingthe flanking regions of the TUP1 gene connected by the NEO gene. The lineardisruption cassette was then used to homologously integrate into the strains bybiolistic transformation (39). Transformants were screened to identify the tup1�strains by colony PCR. Deletion of TUP1 was confirmed by Southern blothybridization (see Fig. S1 in the supplemental material).

To obtain the H99 TUP1 gene, a 4.8-kb DNA fragment containing the 1.3-kbflanking region on both sides was PCR amplified from H99 genomic DNA,sequenced, and cloned into the pAI3 vector (a gift from J. Heitman) containingthe NAT selectable marker to obtain pHL110. pHL110 was linearized with SmaIand transformed into the H99 tup1� strain by the biolistic method. PCR was usedto identify integrative transformants containing the intact TUP1 gene, and South-ern blot analysis was used to confirm the integration event (see Fig. S1 in thesupplemental material).

Preparation and analysis of nucleic acid. Isolation and analysis of genomicDNA were carried out as described previously (4). For gene expression analysis,overnight cultures of wild-type (H99) and tup1� strains were refreshed andgrown in RPMI for 6 h. RNA was extracted from yeast cells using Trizol(Invitrogen, Carlsbad, CA), treated with RNase-free DNase (Ambion, Austin,TX) for the removal of genomic DNA, and purified with the RNeasy MinElutecleanup kit (Qiagen, Valencia, CA). cDNA was synthesized using a high-capacitycDNA archive kit (Applied Biosystems, Foster City, CA) and used in real-timereverse transcription-PCR (RT-PCR) with TaqMan universal PCR master mixand the ABI Prism 7700 sequence detection system (Applied Biosystems, FosterCity, CA). The primers used in RT-PCR are listed in Table S2 in the supple-mental material. Data were normalized with actin levels and expressed as therelative amount in the tup1� strain compared to that in H99. In addition, thetranscription level of CNAG_03012.2 was normalized with �-tubulin as an inter-nal control.

Assays for mating, melanization, and capsule formation. For mating assays,strains were grown on YEPD agar slants for 2 days. The cells of MATa andMAT� strains were mixed on V8 juice agar medium, incubated, and monitoredfor evidence of mating. Melanin production was estimated after spotting seriallydiluted yeast cells onto norepinephrine-containing medium (2� dilutions start-ing at 1.8 � 105 cells/spot). The plates were then incubated for 2 days at 30°C inthe dark (23). Capsule formation by the yeast cells was determined by micro-scopic examination of slides prepared with India ink.

Virulence study. Female BALB/c mice (6 to 8 weeks old) were injected via thelateral tail vein with 0.2 ml of a suspension of each yeast strain (5 � 106/ml) asdescribed previously (4), and the mortality was monitored. Kaplan-Meier anal-ysis of survival was performed with JMP software for Macintosh (SAS Institute,Cary, NC). To measure the growth rate of each strain in the brain, mice wereinjected with yeast cells (105 cells) as described above, and then three mice peryeast strain were sacrificed at several intervals after injection (at 2 days as thestarting point and at 6, 9, and 13 days postinjection). The brains were homoge-nized with a mortar and pestle, diluted, and then plated onto YEPD agar.Colonies were counted after 2 days of incubation at 30°C.

Spot assay. Exponentially growing cultures (optical density at 600 nm [OD600]of 0.5 to 1.0) were washed, resuspended in 0.9% NaCl, and adjusted to an OD600

of 0.1 for the wild type or 0.2 for the tup1� strain (to compensate for its lowgrowth rate). Adjusted cell suspensions were serially diluted, spotted onto theindicated media, and incubated for 3 to 4 days at 30°C. Limited-iron medium(LIM) was identical to chemically defined medium (34) except that the salts ofpolyvalent metals were dissolved in Chelex-100-treated water (Bio-Rad), andother components were purified by treating with Chelex-100. When more strin-gent control of iron or copper concentration was needed, 0.056 mM ethylenedi-amine-diacetic acid (EDDA) (Sigma-Aldrich) or 1 mM bathocuproine sulfonate(BCS) (Sigma-Aldrich) was added to the medium, respectively. LIM-Fe wasprepared by adding 0.1 mM ferric EDTA (Sigma-Aldrich EDFS) to LIM.

Microarray analysis. The previous microarray study with TUP1 in a serotypeD strain was done with a mini-microarray since a whole-genome cryptococcusarray was not available at that time (26). Recently, a whole-genome array con-taining 7,738 70-mer oligomers was constructed by an academic consortium atthe University of Washington, St. Louis. It has been shown that H99 and JEC21share 95% identity in their genome sequences (29). Although arrays were de-signed based on serotype D strain JEC21, the arrays can be useful to assess the

deletion effect of a specific gene in serotype A as long as the correspondingserotype A wild-type control strain is employed as a reference. RNA was ex-tracted from H99 and HL112 grown in RPMI liquid medium for 3 h and 6 h, andmicroarray analysis was performed as described before (25). Two arrays wereused for each time point, and all the genes whose average expression was affectedby greater than twofold in the tup1� strain compared with the wild-type strainwhen grown for 3 h (group A) or 6 h (group B) in RPMI medium were presented(see Table S3 in the supplemental material).

RESULTS

Differences in growth and quorum-sensing-like phenotypebetween tup1� strains of serotypes A and D. TUP1 deletion inserotype D strains of C. neoformans resulted in growth retar-dation, as reported for tup1� strains of other fungal species,but without any defects in yeast cell morphology or flocculation(26). In order to investigate the effect of TUP1 deletion inserotype A strains, the TUP1 gene was deleted and then com-plemented in strain H99. Although the Tup1 proteins fromserotype A (H99) and D (JEC21) strains share 94% amino acididentity, deletion of the TUP1 gene resulted in distinct pheno-typic differences between the strains. While the H99 tup1�strain showed slight growth retardation compared to the wild-type strain, it was less severe than what had been observed forthe serotype D tup1� strain (see Fig. S2 in the supplementalmaterial) (26). The doubling times of the wild-type H99, tup1�(HL112), and complemented (HL132) strains were 2.23 h,3.9 h, and 2.28 h, respectively, at 30°C and 4.08 h, 5.82 h, and3.78 h, respectively, at 37°C in YES liquid medium. The slightreduction in the growth rate of the tup1� strain was also ob-served on solid agar media such as YES and SD (see Fig. S2 inthe supplemental material, and data not shown). Therefore,TUP1 does not appear to influence cell proliferation in theH99 strain as much as in serotype D strains.

One of the striking phenotypes observed previously withserotype D tup1� strains was the inoculum size threshold as aprerequisite for normal growth, which mimics the quorum-sensing phenomenon (27). A cell density of about 5 � 106 wasrequired for the strain HL14, the serotype D tup1� strain, togrow on SD medium (Fig. 1A, left panel). Tests to determinewhether cell density would similarly influence growth of thetup1� serotype A strain, however, did not show a density-dependent growth phenotype in the H99 tup1� strain (HL112)(Fig. 1A, right panel).

To investigate whether the lack of density-dependent growthin H99 is strain dependent or common among serotype Astrains, we deleted TUP1 in several other serotype A strains.Three serotype A strains, CHC186, VNBt63, and WM148, thatare genetically diverse based on their molecular genotype, suchas mini-and macrosatellite DNA, intergenic sequence, and ri-bosomal DNA sequences as well as multilocus sequencing typesequences of marker genes (6), were chosen to construct tup1�strains. TUP1 was also deleted in WSC1156, the MATa strainisogenic to H99. Consistent with the observations for H99,deletion of TUP1 in the backgrounds of all these serotype Astrains caused slight growth retardation but without the den-sity-dependent growth phenotype (data not shown).

In serotype D tup1� strains, the inability to grow at low celldensities could be rescued by supplementing the growth me-dium with the culture filtrate from a high-cell-density tup1�culture. The active molecule in the culture supernatant respon-

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sible for the density-dependent growth phenotype was identi-fied as an oligopeptide, quorum-sensing-like peptide 1 (QSP1)(27). QSP1 is an 11-amino-acid peptide that is processedfrom the CQS1 gene product (Fig. 1B). A CQS1 homolog,CNAG_03012.2, which encodes a hypothetical protein of 45amino acids, is present in the H99 genome. The sequence ofH99 Cqs1 was found to be homologous to that of JEC21 Cqs1,with only two amino acid substitutions (at amino acid positions28 and 34) (Fig. 1B). Deletion of TUP1 results in the transcrip-tional induction of CQS1 in serotype D strains (27). The ex-pression levels of CNAG_03012.2 were measured by real-timeRT-PCR and found to be 2.2- � 0.2-fold higher in HL112 thanin H99, suggesting that the CQS1 homolog in H99 is alsorepressed by Tup1.

The biological activity in the culture filtrate from H99 tup1�(HL112) was determined with regard to the possible accumu-lation of QSPs. Since HL112 did not show the density-depen-dent growth phenotype, the serotype D tup1� strain (HL40)was used for the activity assays. A 25% (vol/vol) mix of theculture filtrate from H99 tup1� combined with fresh mediumwas used in these assays, since a similar proportion of culturefiltrate from serotype D strain HL40 had shown strong biolog-ical activity (27). The HL112 culture filtrate failed to rescue thegrowth of HL40 at low cell density (data not shown). The H99tup1� strain apparently did not produce enough of the quo-rum-sensing-like molecule, or the molecule may not have beenbiologically active. We postulate that strains which can growregardless of cell density do not require a quorum-sensing-likemolecule(s) for cell proliferation at low densities. Therefore,deletion of the TUP1 gene in C. neoformans affects growthdifferently in strains of serotypes A and D, and the density-dependent growth phenotype appears to be serotype D spe-cific.

TUP1 deletion affects mating and capsule formation. One ofthe conserved roles of TUP1 in many fungi, including C. neo-

formans serotype D strains, is the regulation of sexual repro-duction (26, 42, 46). We tested the effect of TUP1 deletion onmating in the serotype A strain H99. Mating of the wild-typeH99 strain with the tester strain KN99a on V-8 juice agarproduced extensive hyphae after 3 days of incubation (Fig. 2A,left panel). In a cross between HL112 (tup1�) and KN99a,however, the production of hyphae was reduced significantly(Fig. 2A, right panel). Similar observations of reduced matinghave been reported for tup1� strains of serotype D (26).Therefore, the role of TUP1 in sexual reproduction appears tobe conserved in both serotype A and D strains.

Although we did not see any effect of tup1 deletion oncapsule formation in serotype D strains, tup1� strains in aserotype A background produced capsules that were signifi-cantly enlarged compared to those of wild-type strains. Figure2B shows the markedly increased capsule size in HL112, thetup1� strain of H99, compared to HL132, the tup1��TUP1strain, and the wild-type strain H99. Deletion of TUP1 in othergenetically unrelated serotype A strains also showed significantincreases in capsule size (data not shown). These observationsclearly associate the hypercapsular phenotype with the dele-tion of TUP1 in serotype A strains. The enlarged capsule for-mation in serotype A tup1� strains was consistent regardless ofgrowth media at both 30°C and 37°C. Among all the mediatested, RPMI induced the most pronounced difference in cap-sule size between wild-type and tup1� strains (Fig. 2B).

TUP1 regulates expression of genes involved in capsule for-mation and iron/copper homeostasis in serotype A strain H99.Given the prominent hypercapsular phenotype of serotype Atup1� strains, it is possible that TUP1 might alter the expres-sion levels of genes involved in capsule formation. Gene profileanalysis was undertaken as a preliminary screen to identify theputative genes whose expression was affected by TUP1 dele-

FIG. 1. Deletion of TUP1 in H99 does not cause density-dependentgrowth. (A) Exponentially growing cultures (OD600 of 0.5 to 1.0) werewashed, resuspended in 0.9% NaCl, and plated on SD medium. Totalsof 5 � 106 and 106 cells for LP1 (wild type) and its tup1� mutant(serotype D strains) and 5 � 105 and 5 � 102 cells for H99 (wild type)and its tup1� mutant (serotype A strains) were plated on SD mediumand incubated for 2 days at 30°C. (B) Comparison of Cqs1 sequencesin JEC21 (serotype D) and H99. The sequence of QSP1, the activepeptide purified from serotype D tup1� culture filtrate, is in bold. Twoamino acids in H99 Cqs1 which are different from those in JEC21 Cqs1are underlined.

FIG. 2. TUP1 disruption affects mating and capsule formation.(A) Strains H99 (wild type; left panel) and HL112 (tup1�; right panel)were each mixed with KN99a on V-8 juice agar, incubated for 72 h, andobserved for hyphal formation. (B) H99 (wild type [WT]), HL112(tup1�), and HL132 (TUP1-complemented strain; tup1��TUP1) weregrown on RPMI agar medium at 37°C for 2 days and examined forcapsule formation by microscopic examination of India ink slide prep-arations.

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tion. Microarray analysis was performed on the H99 and tup1�strains grown for 3 h and 6 h in RPMI liquid medium at 30°C.The expression levels of a few genes involved in capsule bio-synthesis and iron/copper homeostasis were affected by dele-tion of TUP1 in H99 (see Table S3 in the supplemental mate-rial). To confirm the expression patterns, mRNA levels ofseveral genes were examined by real-time RT-PCR. Quantita-tive PCR results showed that the transcriptional levels of threegenes involved in capsule synthesis, CAP10, CAP64, andCAS35, were about threefold higher in the tup1� strain than inH99 (Fig. 3). Conversely, the expression levels of three geneswith an annotated function involved in iron/copper homeosta-sis, CTR4, FRT1, and SIT2, were two- to ninefold lower in thetup1� strain (Fig. 3) (28). In addition, the expression of CIG1,which encodes a product believed to be an extracellular man-noprotein involved in the retention of iron at the cell surfaceand/or in the uptake of siderophore-bound iron (28), wasdownregulated by twofold in the tup1� strain. However, theexpression of CFT2, an ortholog of the Saccharomyces cerevi-siae high-affinity iron permease gene FTR1 in C. neoformans,was not affected by the deletion of TUP1 (17).

Since TUP1 affected the expression of genes involved in ironand copper uptake/homeostasis, growth of the tup1� strain wastested on both iron-chelated medium (LIM�EDDA) and iron-replete medium (LIM�Fe). Interestingly, growth of the tup1�strain was reduced on LIM�EDDA medium compared to thatof the wild-type strain, and iron repletion (LIM�Fe) restoredgrowth of the tup1� strain (Fig. 4A). Furthermore, on LIMmedium treated with a chelator to remove copper(LIM�BCS�EDDA), the growth difference between the wild-type and tup1� strains was even more drastic (Fig. 4B, rightpanel). These results indicated that TUP1 regulates the utili-zation of iron and copper, which corroborates the expression

data for several iron/copper homeostasis genes affected by thedeletion of TUP1 in H99. Melanin production is one of theknown virulence factors in C. neoformans, and iron/copperhomeostasis can affect melanin production (14). Melanin pro-

FIG. 3. TUP1 positively regulates iron-related genes and negativelyregulates capsule-related genes. Total RNA was isolated from wild-type (H99) and tup1� strains. Transcriptional changes in several geneswere determined by real-time RT-PCR. Data were normalized withactin levels and expressed as the relative amount in the tup1� straincompared to that in H99. Genes and their annotated functions arelisted.

FIG. 4. Pleiotropic effects of TUP1 deletion. H99, wild type;HL112, tup1� mutant; HL132, tup1��TUP1 complemented strain.(A) Iron conditions affect growth of the tup1� strain. Serially dilutedyeast cells from the indicated strains were spotted on SD medium,iron-chelated medium (LIM-EDDA), or iron-replete medium (LIM-Fe) and incubated at 30°C for 3 days. (B) The tup1� strain showsadditive growth defects on iron- and copper-limited medium. Seriallydiluted yeast cells were spotted on YES, LIM-Fe, or LIM-BCS-EDDA(1 mM BCS, copper chelator; 0.056 mM EDDA, iron chelator) andincubated at 30°C for 3 days. (C) Melanin production is affected in thetup1� strain. Serially diluted yeast cells were spotted onto norepineph-rine-containing medium. The plates were then incubated for 2 days at30°C in the dark. CuSO4 was added at 10 �M to the norepinephrine-containing medium (right panel). (D) Iron concentrations affect cap-sule size in the tup1� strain. Cells were grown on RPMI (left panel),LIM (middle panel), or LIM-Fe (right panel) for 2 days at 30°C, andcapsule formation was examined. (E) The tup1� strain is sensitive toSDS and fluconazole. Serially diluted yeast cells were spotted on YPD,YPD plus 0.01% SDS, or YPD plus 8 �g/ml fluconazole (FLC) andincubated at 30°C for 2 days.

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duction in the tup1� strains, examined on norepinephrine-containing medium, was observed to be notably reduced com-pared to that in the wild-type and complemented strains (Fig.4C, left panel). Laccase is a key enzyme for melanin biosyn-thesis in C. neoformans. Since it requires four bound copperions (43, 44) and copper is known to suppress the defect inmelanin formation caused by mutation in genes involved inmetal ion homeostasis (48, 49), the effect of Cu2� on themelanin phenotype of the tup1� strain was studied. Melaninproduction was apparently restored in the tup1� strain bysupplementing the growth medium with 10 �M CuSO4. Theseresults suggest that the tup1� strain is defective in copperhomeostasis affecting melanin production (Fig. 4C, rightpanel).

An insufficient iron concentration in the growth environ-ment is known to induce large capsules in C. neoformans (15).Although the tup1� strain already showed an enlarged capsulein RPMI medium, it was interesting to determine if iron levelsstill influence the capsule size. The capsule size was measuredafter growing cells on RPMI, LIM, and LIM�Fe agar plates(Fig. 4D). In concordance with previous studies, strain H99produced larger capsules in LIM (2.51 � 0.71 �m; n � 33)than in RPMI (1.87 � 0.39 �m; n � 25) and iron-repletemedium (LIM�Fe) (0.64 � 0.22 �m; n � 24) (Fig. 4D, upperpanels). In the tup1� strain, cells grown on RPMI were alreadyhypercapsulated compared to H99 cells, but the capsules be-came even larger when cultured on LIM. The capsule size wassignificantly reduced upon addition of Fe to LIM (RPMI, 5.34�m � 1.21 [n � 14]; LIM, 6.14 �m � 2.98, [n � 26], andLIM�Fe, 2.47 �m � 0.62 [n � 49]) (Fig. 4D, middle panels).The TUP1-complemented strain (HL132) behaved similarly tothe wild-type strain in all growth conditions (Fig. 4D, bottompanels). Thus, deletion of TUP1 results in the formation of anenlarged capsule under non-iron-limiting conditions, whichcan be altered further in tup1� cells by iron levels in theenvironment.

TUP1 deletion affects cell wall integrity and susceptibility tofluconazole. Cir1 is another cryptococcal transcriptional regu-lator involved in iron homeostasis. CIR1 (CNAG_04864) isimportant for capsule formation and negatively regulates lac-case expression in H99 (18). In addition, it has been shown thatcir1 mutants are sensitive to sodium dodecyl sulfate (SDS) andthe azole drug fluconazole, suggesting that Cir1 is involved incell wall integrity and membrane functions (18). Since capsuleand melanin production were affected in the tup1� strain, theeffect of TUP1 deletion relative to changes in CIR1 expressionwas examined. Quantitative RT-PCR results showed CIR1 ex-pression to be mildly affected in the tup1� strain compared toH99, as the relative expression level was only 1.36- � 0.05-foldhigher in the tup1� strain. Furthermore, expression of ironpermease genes, such as CFT1 and CFT2, which are regulatedby CIR1, was not affected by the deletion of TUP1 according toour microarray data (see Table S3 in the supplemental mate-rial). These data suggested that TUP1 does not regulate ironhomeostasis through the CIR1 regulatory circuit. However,growth of the tup1� strain was significantly hampered in thepresence of 0.01% SDS, and the tup1� strain displayed in-creased sensitivity to fluconazole (Fig. 4E). These data sug-gested that TUP1 is also involved in cell wall integrity andmembrane functions.

TUP1 deletion reduces virulence. Since deletion of TUP1in H99 resulted in both positive and negative effects withrespect to the three major C. neoformans virulence factors,which include growth at 37°C and formation of melanin andcapsule, its effect on virulence was investigated. Groups of10 mice were challenged with different yeast stains via tailvein injection. Figure 5A shows that all mice challenged withthe wild-type or the complemented strain succumbed toinfection by 9 days postinjection, while it took 20 days for allmice infected with the tup1� strain to succumb (P 0.001compared to wild-type-infected mice), indicating that dele-

FIG. 5. Virulence study. (A) Survival of mice injected with H99(wild type), HL112 (tup1� mutant) and HL132 (tup1��TUP1 com-plemented strain). Groups of 10 female BALB/c mice were injected viathe lateral tail vein with 106 viable yeast cells, and mortality wasmonitored. (B) Fungal load in the mouse brain. Three mice per yeaststrain were sacrificed at several intervals after injection as indicated.The brains were homogenized and plated onto YEPD agar. Colonieswere counted after 2 days of incubation (DAI) at 30°C. Error barsindicate standard deviations. (C) Brain smear showing cells of the H99and tup1� strains at 3, 9, and 14 days postinfection as indicated. Braintissue of mice injected with H99 or tup1� strains was smeared on amicroscopic slide and examined under a microscope with a Nomarskiinterference condenser.

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tion of TUP1 causes attenuation of virulence in C. neofor-mans.

To examine the pathobiological differences in mice infectedwith the wild-type or tup1� strain, the brain fungal burden andcapsule size were determined at different stages of infection.Significant differences in the number of CFU were observedfor the H99 and tup1� strains (5.93 � 103 versus 5.3 � 102 perbrain) as early as 2 days after injection (Fig. 5B). The numberof CFU, however, increased exponentially and differed evenmore at 6 days after injection. These data suggest that TUP1 isimportant for growth in vivo, although the tup1� strain onlyshowed a marginal reduction in growth at 37°C in vitro. An-other noteworthy observation was that fungal burden analyzedon the day of death in a mouse injected with the tup1� strain(1.25 � 107 at day 13) was 80-fold lower than that in a mouseinjected with H99 (8.35 � 108 at day 9) (Fig. 5B). It is possiblethat the larger capsule size in the tup1� strain in vitro (Fig. 2A)might have contributed to such a difference. Surprisingly, thecapsule size of the yeast cells in the brain smear from miceinfected with tup1� strain was similar to that for mice infectedwith H99 (Fig. 5C). These findings clearly indicate that Tup1plays an important role in the pathobiology of C. neoformans.

DISCUSSION

This study investigated the function of TUP1 in C. neofor-mans serotype A strains, including H99, and presents anotherexample of serotype-specific difference in gene regulation.TUP1 plays a conserved role with respect to growth and matingbut distinctly different roles in strains of serotypes A and D.Serotype A-specific phenotypes of tup1� strains include a lackof density-dependent growth, an enlarged capsule size, re-duced melanin production, and a defect in iron/copper ho-meostasis.

The prominent capsule size in the tup1� strain under non-inducing conditions indicated the important role of TUP1 incapsule formation. Many environmental factors have beenshown to influence the size of the capsule in C. neoformans.Low concentrations of glucose and iron, high concentrations ofcarbon dioxide, and the presence of serum components havebeen shown to enhance capsule formation (15). However, lim-ited information is available concerning the regulation of cap-sule formation. The Gpa1-cAMP-protein kinase A (PKA) sig-naling pathway has been thoroughly studied in regard to itseffect on capsule production. Both pka1� and gpa1� strainsexhibited a marked defect in capsule production, while a pkr1�strain overproduced the capsule (10). Several observations in-dicate that TUP1 affects capsule production independent of thecAMP-PKA signaling pathway. First, our preliminary microar-ray data did not show any significant change in the gene ex-pression of cAMP-PKA pathway components such as CAC1(adenyl cyclase), PKA1, and PKR1 (data not shown). Second,the addition of cAMP to growth media did not alter the hy-percapsular phenotype of the tup1� strain (data not shown).Third, the hypercapsular phenotype of pkr1� was observed notonly in vitro but also in vivo, resulting in hypervirulence, whilethe tup1� strain exhibited reduced virulence and yet its capsulesize in vivo was comparable to that of the wild-type strain.Another signaling pathway involved in capsule formation is theHOG pathway. HOG1 negatively regulates synthesis of capsule

and melanin in the serotype A strain H99 but not in theserotype D strain JEC21 (1). Deletion of TUP1 or HOG1 hasa similar effect on capsule production, and their serotype-specific regulatory function offers the possibility that Tup1 andHog1 might share downstream regulatory targets either in aparallel signaling pathway or by direct interaction. The HOG1pathway in S. cerevisiae is activated by osmotic stress and mod-ulates diverse osmo-adaptive gene expression through the re-cruitment of the general transcription repressor complexTup1-Ssn6 and the sequence-specific DNA-binding proteinSko1 (36). Since the tup1� strains also show sensitivity to 2 mMH2O2 as observed with the hog1� strains (1) (data not shown),this possibility was considered. However, the hog1� strain de-rived from H99 exhibited enhanced melanization and temper-ature sensitivity at 40°C, neither of which is shared in the tup1�strains. Furthermore, the H2O2-sensitive phenotype of thetup1� strain was rescued by addition of copper or iron to themedium, suggesting that the low intracellular iron/copper con-tent and not a defect in the HOG pathway resulted in the H2O2

sensitivity (data not shown). Therefore, the involvement ofTUP1 in regulating capsule formation does not share all thecommon characteristics with the aforementioned knownpathways.

Given that TUP1 affects capsule production independentlyof previously known regulatory pathways, we tried to identifythe targets of TUP1. Preliminary microarray and RT-PCR ex-periments revealed that several genes involved in iron/copperhomeostasis were downregulated in tup1� strains. The require-ment of iron/copper in the growth of the tup1� strain andrestoration of melanin production by copper in the tup1�strain further support the importance of TUP1 in iron/copperhomeostasis in serotype A strain H99. A role of TUP1 in ironmetabolism was first suggested by the identification of theferric reductase gene, RBT2, as one of the genes repressed byTUP1 in C. albicans (2). Subsequent study showed that ferricreductase activity in �tup1/�tup1 cells was constitutively ele-vated and iron-dependent transcriptional alteration of C. albi-cans FTR1 and FTR2 mRNAs was abrogated in �tup1/�tup1mutant (21). Furthermore, examples of the physical interactionbetween a corepressor and an iron-sensing factor controllingthe expression of iron uptake genes have been shown in Schizo-saccharomyces pombe (50). S. pombe fep1� encodes a GATAtranscription factor that represses the expression of iron trans-port genes in response to elevated iron levels. Using yeasttwo-hybrid analysis, it has been shown that Tup11, a Tup1homolog of S. pombe, and Fep1 physically interact with eachother (50). Whether C. neoformans Tup1 directly interacts withthe proteins involved in iron homeostasis is yet to be deter-mined.

The importance of copper homeostasis in C. neoformans issuggested by the copper dependency of two well-known viru-lence factors, the Cu/Zn superoxide dismutase (7) and laccase,a key enzyme in melanin synthesis (37, 43). Both enzymesrequire copper as a cofactor for their function. Deletion of C.neoformans LAC1, encoding the laccase, or mutation in thecopper-binding site of the gene resulted in a significant reduc-tion in virulence (37, 43). Copper also induces laccase tran-scription in wild-type cells and can restore laccase activity invph1� mutants (48). In addition, the close relationship be-tween copper and iron homeostasis has been reviewed (16).

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For example, copper homeostasis also affects iron, since Fet3,the high-affinity iron transporter, requires the incorporation offour copper ions for the function (16). Thus, ineffective copperloading of Fet3 due to a defect in copper homeostasis can alsolead to lower intercellular levels of iron, which affects capsuleand melanin production. In fact, mutation in CCC2 (encodinga copper transporter) or ATX1 (encoding a copper chaperone)resulted in large capsules under iron-replete conditions andimpaired growth under iron-limiting condition (40). Althoughour microarray data did not show significant changes in CCC2and ATX1 gene expression (see Table S3 in the supplementalmaterial), reduced expression of CTR4 (encoding coppertransporter 4) in the tup1� strain and additive growth defectsof the tup1� strain in iron/copper-chelated media lend addi-tional support for the regulatory role of TUP1 in both iron andcopper homeostasis.

Another intriguing result of our study is that Tup1 appearsto function as both a repressor and an activator in C. neofor-mans. In contrast to the prevailing view of Tup1 as a globalrepressor, our results showed that many genes were also down-regulated in the absence of TUP1, suggesting that Tup1 func-tions as an activator for the expression of those genes. Ananalogy is seen with Hap1 in S. cerevisiae, which was originallyidentified as a heme-dependent transcriptional activator butwas reported to function also as transcriptional repressor, de-pending on oxygen levels (11). Mammalian nuclear hormonereceptors also are examples of factors that can act both posi-tively and negatively through the recruitment of coactivatorsand corepressor complexes, respectively (45). Conversely, it isalso possible that the downregulated genes in the tup1� strainare due to the indirect effect of Tup1. For instance, Tup1 couldinteract with a negative regulator, and inactivation of Tup1could lead to the activation of a negative regulator, which inturn would cause the observed downregulation of genes in thetup1� strain. Additional experiments are required to identifythe direct target(s) of Tup1 and possible interacting partners,if any, to understand the mechanism of Tup1 regulation in C.neoformans.

In C. albicans, disruption of TUP1 causes an inability toswitch between yeast and filament forms and results in consti-tutive filamentous growth, which presumably is the reason whythe tup1� strain is avirulent (2, 3). Previously, virulence studiescould not be carried out properly with tup1� strains in a sero-type D background because of their inability to grow at low celldensities, which hindered the precise determination of inocu-lum size based on CFU (26, 27). Here, we showed that deletionof TUP1 in H99 affected virulence. Since the TUP1 deletiondisplayed pleiotropic effects, it is likely that the reduced viru-lence of the tup1� strain resulted from the combination ofthese effects and is possibly related to iron/copper homeostasis.Given the global regulatory role of TUP1 in fungi and themanifestation of different phenotypes in serotype A and Dtup1� strains, an in-depth analysis of TUP1 function wouldoffer a valuable tool toward understanding the divergence ofgene regulation in C. neoformans.

ACKNOWLEDGMENT

This study was supported by funds from the intramural program ofthe National Institute of Allergy and Infectious Diseases, NIH.

REFERENCES

1. Bahn, Y. S., K. Kojima, G. M. Cox, and J. Heitman. 2005. Specialization ofthe HOG pathway and its impact on differentiation and virulence of Cryp-tococcus neoformans. Mol. Biol. Cell 16:2285–2300.

2. Braun, B. R., W. S. Head, M. X. Wang, and A. D. Johnson. 2000. Identifi-cation and characterization of TUP1-regulated genes in Candida albicans.Genetics 156:31–44.

3. Braun, B. R., and A. D. Johnson. 1997. Control of filament formation inCandida albicans by the transcriptional repressor TUP1. Science 277:105–109.

4. Chang, Y. C., and K. J. Kwon-Chung. 1994. Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol.Cell. Biol. 14:4912–4919.

5. Chang, Y. C., B. L. Wickes, G. F. Miller, L. A. Penoyer, and K. J. Kwon-Chung. 2000. Cryptococcus neoformans STE12� regulates virulence but is notessential for mating. J. Exp. Med. 191:871–882.

6. Chen, J., A. Varma, M. R. Diaz, A. P. Litvintseva, K. K. Wollenberg, andK. J. Kwon-Chung. 2008. Cryptococcus neoformans strains and infection inapparently immunocompetent patients, China. Emerg. Infect. Dis. 14:755–762.

7. Cox, G. M., T. S. Harrison, H. C. McDade, C. P. Taborda, G. Heinrich, A.Casadevall, and J. R. Perfect. 2003. Superoxide dismutase influences thevirulence of Cryptococcus neoformans by affecting growth within macro-phages. Infect. Immun. 71:173–180.

8. Cruz, M. C., R. A. Sia, M. Olson, G. M. Cox, and J. Heitman. 2000. Com-parison of the roles of calcineurin in physiology and virulence in serotype Dand serotype A strains of Cryptococcus neoformans. Infect. Immun. 68:982–985.

9. DeRisi, J. L., V. R. Iyer, and P. O. Brown. 1997. Exploring the metabolic andgenetic control of gene expression on a genomic scale. Science 278:680–686.

10. D’Souza, C. A., and J. Heitman. 2001. Conserved cAMP signaling cascadesregulate fungal development and virulence. FEMS Microbiol. Rev. 25:349–364.

11. Hickman, M. J., and F. Winston. 2007. Heme levels switch the function ofHapI of Saccharomyces cerevisiae between transcriptional activator and tran-scriptional repressor. Mol. Cell. Biol. 27:7414–7424.

12. Hicks, J., R. A. Lockington, J. Strauss, D. Dieringer, C. P. Kubicek, J. Kelly,and N. Keller. 2001. RcoA has pleiotropic effects on Aspergillus nidulanscellular development. Mol. Microbiol. 39:1482–1493.

13. Hicks, J. K., C. A. D’Souza, G. M. Cox, and J. Heitman. 2004. CyclicAMP-dependent protein kinase catalytic subunits have divergent roles invirulence factor production in two varieties of the fungal pathogen Crypto-coccus neoformans. Eukaryot. Cell 3:14–26.

14. Jacobson, E. S. 2000. Pathogenic roles for fungal melanins. Clin. Microbiol.Rev. 13:708–717.

15. Janbon, G. 2004. Cryptococcus neoformans capsule biosynthesis and regula-tion. FEMS Yeast Res. 4:765–771.

16. Jung, W. H., and J. W. Kronstad. 2008. Iron and fungal pathogenesis: a casestudy with Cryptococcus neoformans. Cell. Microbiol. 10:277–284.

17. Jung, W. H., A. Sham, T. Lian, A. Singh, D. J. Kosman, and J. W. Kronstad.2008. Iron source preference and regulation of iron uptake in Cryptococcusneoformans. PLoS Pathog. 4:e45.

18. Jung, W. H., A. Sham, R. White, and J. W. Kronstad. 2006. Iron regulationof the major virulence factors in the AIDS-associated pathogen Cryptococcusneoformans. PLoS Biol. 4:e410.

19. Keleher, C. A., M. J. Redd, J. Schultz, M. Carlson, and A. D. Johnson. 1992.Ssn6-Tup1 is a general repressor of transcription in yeast. Cell 68:709–719.

20. Keller, S. M., and B. L. Wickes. 2003. Abstr. 15th Congr. Int. Soc. Hum.Anim. Mycol., San Antonio, TX, abstr. O17.

21. Knight, S. A., E. Lesuisse, R. Stearman, R. D. Klausner, and A. Dancis. 2002.Reductive iron uptake by Candida albicans: role of copper, iron and theTUP1 regulator. Microbiology 148:29–40.

22. Kuwayama, H., S. Obara, T. Morio, M. Katoh, H. Urushihara, and Y.Tanaka. 2002. PCR-mediated generation of a gene disruption constructwithout the use of DNA ligase and plasmid vectors. Nucleic Acids Res.30:E2.

23. Kwon-Chung, K. J., and J. E. Bennett. 1992. Medical mycology. Lea &Febiger, Philadelphia, PA.

24. Kwon-Chung, K. J., J. E. Bennett, and J. C. Rhodes. 1982. Taxonomicstudies on Filobasidiella species and their anamorphs. Antonie van Leeu-wenhoek 48:25–38.

25. Lee, H., C. M. Bien, A. L. Hughes, P. J. Espenshade, K. J. Kwon-Chung, andY. C. Chang. 2007. Cobalt chloride, a hypoxia-mimicking agent, targets sterolsynthesis in the pathogenic fungus Cryptococcus neoformans. Mol. Microbiol.65:1018–1033.

26. Lee, H., Y. C. Chang, and K. J. Kwon-Chung. 2005. TUP1 disruption revealsbiological differences between MATa and MAT� strains of Cryptococcusneoformans. Mol. Microbiol. 55:1222–1232.

27. Lee, H., Y. C. Chang, G. Nardone, and K. J. Kwon-Chung. 2007. TUP1disruption in Cryptococcus neoformans uncovers a peptide-mediated density-

VOL. 8, 2009 Tup1 REGULATION IN CRYPTOCOCCUS NEOFORMANS 1907

on July 13, 2020 by guesthttp://ec.asm

.org/D

ownloaded from

dependent growth phenomenon that mimics quorum sensing. Mol. Micro-biol. 64:591–601.

28. Lian, T., M. I. Simmer, C. A. D’Souza, B. R. Steen, S. D. Zuyderduyn, S. J.Jones, M. A. Marra, and J. W. Kronstad. 2005. Iron-regulated transcriptionand capsule formation in the fungal pathogen Cryptococcus neoformans. Mol.Microbiol. 55:1452–1472.

29. Loftus, B. J., E. Fung, P. Roncaglia, D. Rowley, P. Amedeo, D. Bruno, J.Vamathevan, M. Miranda, I. J. Anderson, J. A. Fraser, J. E. Allen, I. E.Bosdet, M. R. Brent, R. Chiu, T. L. Doering, M. J. Donlin, C. A. D’Souza,D. S. Fox, V. Grinberg, J. Fu, M. Fukushima, B. J. Haas, J. C. Huang, G.Janbon, S. J. Jones, H. L. Koo, M. I. Krzywinski, J. K. Kwon-Chung, K. B.Lengeler, R. Maiti, M. A. Marra, R. E. Marra, C. A. Mathewson, T. G.Mitchell, M. Pertea, F. R. Riggs, S. L. Salzberg, J. E. Schein, A. Shvartsbeyn,H. Shin, M. Shumway, C. A. Specht, B. B. Suh, A. Tenney, T. R. Utterback,B. L. Wickes, J. R. Wortman, N. H. Wye, J. W. Kronstad, J. K. Lodge,J. Heitman, R. W. Davis, C. M. Fraser, and R. W. Hyman. 2005. The genomeof the basidiomycetous yeast and human pathogen Cryptococcus neoformans.Science 307:1321–1324.

30. Meyer, W., A. Castaneda, S. Jackson, M. Huynh, and E. Castaneda. 2003.Molecular typing of IberoAmerican Cryptococcus neoformans isolates.Emerg. Infect. Dis. 9:189–195.

31. Moreno, S., A. Klar, and P. Nurse. 1991. Molecular genetic analysis of fissionyeast Schizosaccharomyces pombe. Methods Enzymol. 194:795–823.

32. Murad, A. M., C. d’Enfert, C. Gaillardin, H. Tournu, F. Tekaia, D. Talibi, D.Marechal, V. Marchais, J. Cottin, and A. J. Brown. 2001. Transcript profilingin Candida albicans reveals new cellular functions for the transcriptionalrepressors CaTup1, CaMig1 and CaNrg1. Mol. Microbiol. 42:981–993.

33. Nielsen, K., G. M. Cox, P. Wang, D. L. Toffaletti, J. R. Perfect, and J. Heit-man. 2003. Sexual cycle of Cryptococcus neoformans var. grubii and virulenceof congenic a and � isolates. Infect. Immun. 71:4831–4841.

34. Nyhus, K. J., and E. S. Jacobson. 1999. Genetic and physiologic character-ization of ferric/cupric reductase constitutive mutants of Cryptococcus neo-formans. Infect. Immun. 67:2357–2365.

35. Perfect, J. R., S. D. Lang, and D. T. Durack. 1980. Chronic cryptococcalmeningitis: a new experimental model in rabbits. Am. J. Pathol. 101:177–194.

36. Proft, M., A. Pascual-Ahuir, E. de Nadal, J. Arino, R. Serrano, and F. Posas.2001. Regulation of the Sko1 transcriptional repressor by the Hog1 MAPkinase in response to osmotic stress. EMBO J. 20:1123–1133.

37. Salas, S. D., J. E. Bennett, K. J. Kwon-Chung, J. R. Perfect, and P. R.Williamson. 1996. Effect of the laccase gene CNLAC1, on virulence ofCryptococcus neoformans. J. Exp. Med. 184:377–386.

38. Todd, R. B., J. R. Greenhalgh, M. J. Hynes, and A. Andrianopoulos. 2003.TupA, the Penicillium marneffei Tup1p homologue, represses both yeast andspore development. Mol. Microbiol. 48:85–94.

39. Toffaletti, D. L., T. H. Rude, S. A. Johnston, D. T. Durack, and J. R. Perfect.1993. Gene transfer in Cryptococcus neoformans by use of biolistic delivery ofDNA. J. Bacteriol. 175:1405–1411.

40. Walton, F. J., A. Idnurm, and J. Heitman. 2005. Novel gene functionsrequired for melanization of the human pathogen Cryptococcus neoformans.Mol. Microbiol. 57:1381–1396.

41. Wang, P., C. B. Nichols, K. B. Lengeler, M. E. Cardenas, G. M. Cox, J. R.Perfect, and J. Heitman. 2002. Mating-type-specific and nonspecific PAKkinases play shared and divergent roles in Cryptococcus neoformans. Eu-karyot. Cell 1:257–272.

42. Williams, F. E., and R. J. Trumbly. 1990. Characterization of TUP1, amediator of glucose repression in Saccharomyces cerevisiae. Mol. Cell. Biol.10:6500–6511.

43. Williamson, P. R. 1994. Biochemical and molecular characterization of thediphenol oxidase of Cryptococcus neoformans: identification as a laccase. J.Bacteriol. 176:656–664.

44. Williamson, P. R. 1997. Laccase and melanin in the pathogenesis of Cryp-tococcus neoformans. Front. Biosci. 2:e99–e107.

45. Xu, L., C. K. Glass, and M. G. Rosenfeld. 1999. Coactivator and corepressorcomplexes in nuclear receptor function. Curr. Opin. Genet. Dev. 9:140–147.

46. Yamashiro, C. T., D. J. Ebbole, B. U. Lee, R. E. Brown, C. Bourland, L.Madi, and C. Yanofsky. 1996. Characterization of rco-1 of Neurospora crassa,a pleiotropic gene affecting growth and development that encodes a homologof Tup1 of Saccharomyces cerevisiae. Mol. Cell. Biol. 16:6218–6228.

47. Yue, C., L. M. Cavallo, J. A. Alspaugh, P. Wang, G. M. Cox, J. R. Perfect, andJ. Heitman. 1999. The STE12� homolog is required for haploid filamenta-tion but largely dispensable for mating and virulence in Cryptococcus neo-formans. Genetics 153:1601–1615.

48. Zhu, X., J. Gibbons, S. Zhang, and P. R. Williamson. 2003. Copper-medi-ated reversal of defective laccase in a �vph1 avirulent mutant of Cryptococ-cus neoformans. Mol. Microbiol. 47:1007–1014.

49. Zhu, X., and P. R. Williamson. 2003. A CLC-type chloride channel gene isrequired for laccase activity and virulence in Cryptococcus neoformans. Mol.Microbiol. 50:1271–1281.

50. Znaidi, S., B. Pelletier, Y. Mukai, and S. Labbe. 2004. The Schizosaccharo-myces pombe corepressor Tup11 interacts with the iron-responsive transcrip-tion factor Fep1. J. Biol. Chem. 279:9462–9474.

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