Isolation, Characterization, and Localization of a Capsule ... · Isolation, Characterization, and...

JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Sept. 1999, p. 5636–5643 Vol. 181, No. 18

Isolation, Characterization, and Localization of a Capsule-AssociatedGene, CAP10, of Cryptococcus neoformans

Y. C. CHANG AND K. J. KWON-CHUNG*

Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, Maryland 20892

Received 29 April 1999/Accepted 14 June 1999

Cryptococcus neoformans is a pathogenic fungus which most commonly affects the central nervous system andcauses fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellularpolysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation andcharacterization of a novel gene, CAP10, which is required for capsule formation. Complementation of theacapsular cap10 mutant produced an encapsulated strain and the deletion of CAP10 from a wild strain resultedin an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa,which is similar to the size predicted from sequence analysis. When CAP10 was fused with a hybrid greenfluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reportergene construct, we found that CAP10 was expressed at high levels in late-stationary-phase cells. In addition,we found that the expression levels of CAP10 are modulated by the transcriptional factor STE12a. Deletion ofSTE12a downregulated the expression levels of CAP10 while overexpression of STE12a upregulated theexpression levels of CAP10. Animal model studies indicate that deletion of the CAP10 gene results in the lossof virulence, and complementation of the acapsular phenotype of cap10 restores virulence. Thus, CAP10 isrequired for capsule formation and virulence.

Cryptococcus neoformans is a pathogenic fungus which mostcommonly affects the central nervous system and causes fatalmeningoencephalitis in AIDS patients (20). This fungus pro-duces a thick extracellular polysaccharide capsule which is awell-recognized virulence factor (15, 22). The predominantcapsular polysaccharide of C. neoformans is glucuronoxylo-mannan (GXM), which consists of an O-acetylated, a-1,3-man-nose backbone with xylosyl and glucuronosyl side chains. Theextent of O-acetylation and xylosyl substitution varies withserotype. The biochemical pathway for synthesis of the poly-saccharide capsule, however, is not clear.

Several acapsular mutants have been isolated by classic mu-tational approaches (3, 27). Molecular cloning by directcomplementation of acapsular mutants has resulted in the iso-lation of three genes, CAP59, CAP60, and CAP64, which arerequired for capsule formation (2–4). While these genes arenot essential for growth in vitro, each has been shown to berequired for virulence in the murine model. DNA sequenceanalysis was not indicative of their biochemical function exceptthat Cap59p and Cap60p contained a putative transmembranedomain and shared sequence similarity at the center of theircoding regions. Functional analysis indicated that the trans-membrane domain of Cap59p was required for its ability tocomplement the cap59 acapsular mutant (2). Immunogoldelectron microscopy revealed the location of Cap60p to bearound the nuclear membrane (3).

Difficulties in using histochemical methods to localize geneproducts in C. neoformans (3, 29) have been encountered. Thegreen fluorescent protein (GFP) from the bioluminescent jel-lyfish Aequorea victoriae (24) has emerged as a useful marker instudying protein localization in a variety of organisms. Theformation of fluorophore appears to be cell autonomous be-

sides the requirement for molecular oxygen (9, 18, 25). Directvisualization of gene expression in individual cells is thereforepossible without distortion caused by fixation, sectioning, andstaining.

Although the molecular mechanisms of regulation in capsulesynthesis are not clear, it has been shown that a homolog of theGPA1 gene encoding the G-protein alpha subunit in the signaltransduction pathway influences capsule production in re-sponse to iron limitation (1). Recently, another gene involvedin the pheromone response signal transduction cascade,STE12a, was also found to modulate the expression of severalcapsule-associated genes, including CAP59, CAP60, andCAP64 (5). The STE12a gene of C. neoformans shares se-quence similarity with the Saccharomyces cerevisiae STE12gene and its homologs, but STE12a exists only in MATa strainsof C. neoformans (31). The STE12a gene is required for hap-loid fruiting on filamentous agar but not for mating. Experi-mental infections in the murine model suggested that theSTE12a gene is important for virulence in C. neoformans (5).

We isolated and characterized a novel capsular gene,CAP10, which may also encode a protein containing a trans-membrane domain. CAP10 is required for capsule formationand its deletion abolishes the ability of the fungus to cause fatalinfection in mice. Cellular location of the CAP10 gene productwas determined by tagging the Cap10p with a new hybrid GFPspecific for C. neoformans. In addition, the Escherichia colib-glucuronidase (GUS) gene was used as a reporter to monitorthe levels of expression of CAP10 during different stages ofgrowth. The importance of STE12a in regulating CAP10 ex-pression was also demonstrated.

MATERIALS AND METHODS

Strains and media. The cDNA library was constructed by J. C. Edman frommRNA of log-phase cells. Table 1 summarizes the strains used in this study. C.neoformans var. neoformans strains B-3501 (MATa) and B-3502 (MATa) havebeen described previously (19). B-4500 is a wild-type congenic strain of B-4476(21). B-4500FO2 is a ura5 auxotroph and LP1 is an ade2 ura5 strain derived fromB-4500 (3). Strain cap10F2 is an F2 progeny obtained from a cross between the

* Corresponding author. Mailing address: Building 10, Room11C304, National Institutes of Health, Bethesda, MD 20892. Phone:(301) 496-1602. Fax: (301) 402-1003. E-mail: [email protected].

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acapsular mutant, cap10C (3), and a MATa strain. Strain cap10F2FO is a ura5auxotroph of cap10F2 and was isolated according to the methods describedpreviously (23). TYCC245F1FO is a ura5 auxotroph of Dste12a and TYCC259 isa ura5 auxotroph containing GAL7(p)::STE12a (5). YEPD contains 1% yeastextract, 2% Bacto Peptone, and 2% dextrose. Minimal medium (YNB) contains6.7 g of yeast nitrogen base without amino acids (Difco) and 20 g of glucose perliter. 5-Fluoroorotic acid (5-FOA) medium contains 6.7 g of yeast nitrogen base(Difco), 1 g of 5-FOA, 50 mg of uracil, and 20 g of glucose per liter.

Transformation of C. neoformans. The electroporation method described byEdman and Kwon-Chung (13) was used to transform C. neoformans. TYCC133is a stable encapsulated transformant of cap10F2FO containing pYCC133 andCIP3 is a stable acapsular transformant of cap10F2FO containing pCIP3. Thestable transformants were uracil prototrophs obtained after three transfers onYEPD medium.

Preparation and analysis of nucleic acid and proteins. Genomic DNA isola-tion and analysis were as described previously (2). Random hexamer priming wasused to label the DNA probes to specific activities of .108 dpm/mg (14). TotalRNA was isolated by using the FastRNA kit (Bio 101, Vista, Calif.) andpoly(A)1 RNA was isolated by using the Oligotex mRNA kit (Qiagen, Valencia,Calif.). Northern blot analysis was performed as described previously (6). Fol-lowing each hybridization, the blot was exposed to PhosphorImager Screen andthe CAP10 specific signal, normalized to that of the actin gene, was quantifiedwith ImageQuant 1.1 (Molecular Dynamics). DNA sequencing was performed bythe dideoxy-mediated chain-termination method using a Sequenase version 2.0kit (U.S. Biochemicals, Cleveland, Ohio). Programs of the University of Wis-consin Genetics Computer Group (Madison, Wis.) were used for analysis ofnucleic acid sequences (11).

Total protein isolation, polyacrylamide gel electrophoresis, and Western blotanalyses were as described previously (3). The membrane was incubated withanti-hemagglutinin (HA) monoclonal antibody (BAbCO, Richmond, Calif.) fol-lowed by secondary antibody obtained from the Western-Star chemilumines-cence detection system (Tropix, Bedford, Mass.) and was used as suggested bythe manufacturers.

Construction of plasmids. Table 2 summarizes the plasmids used in this study.The URA5-containing plasmid, pCIP3, was obtained from J. C. Edman. TheBamHI-EcoRI fragment of pYCC76 (3), which contained the functional ADE2gene, was cloned into the BamHI-EcoRI site of pBC(KS1) to yield pYCC123.To recover free plasmids from C. neoformans, genomic DNA from encapsulatedtransformants was digested with NotI, ligated, and transformed into E. coli.Plasmid pYCC125 was one of several plasmids recovered from E. coli which wereable to complement the mutation of cap10F2FO. Plasmids pYCC130, pYCC131,and pYCC132 were subclones of pYCC125 in pCIP3 (Fig. 1A).

To construct a partial library, genomic DNA of B-4500 was digested with XbaIand fractionated on a 1.0% agarose gel. The region from 1.5 to 2.5 kb was gelisolated and cloned into pBluescript vector. The library was screened with the1.2-kb BamHI-NotI fragment of pYCC125. One of the positive clones, pYCC147,containing a 1.6-kb insert was isolated. The deletion construct, pYCC150 (Fig.2A), was constructed as follows. The 1.2-kb MscI-XbaI fragment of pYCC133(Fig. 1A) was replaced with the 3.0-kb EcoRI-XbaI fragment of the ADE2 genefrom pYCC123 to give pYCC149. The 1.2-kb NsiI-NotI fragment of pYCC147was cloned into the BamHI-NotI site of pYCC149 to give pYCC150. The 59 rapidamplification of cDNA ends (RACE) method was performed in accordance withthe protocol accompanying the Marathon cDNA amplification kit (Clontech,Palo Alto, Calif.).

The HA epitope (YPYDYPDYA) was inserted in frame at the carboxylterminus of Cap10p by PCR amplification of pYCC147 as described previously

(3). The resulting plasmid (pYCC151) was sequenced to confirm that no errorshad been introduced during amplification. The 39 end of CAP10 in pYCC133 wasreplaced with the tagged fragment in pYCC151 to generate pYCC152. GETP1containing three tandem copies of HA was developed by M. Tyers and B.Futcher. The BstXI-XbaI fragment of GETP1 was cloned into pYCC151 to givepYCC199 and the 39 end of CAP10 in pYCC133 was replaced with the three-HA-tagged fragment in pYCC199 to generate pYCC203.

To create the CAP10(p)::GUS fusion, an NdeI site was created at the first ATGsite of the CAP10 coding region by PCR and the resulting promoter was clonedinto the NdeI site created at the first ATG of the GUS gene. Plasmid pYCC330contained the CAP10(p)::GUS reporter gene construct in pPM8 which containedURA5, telomeres, and a 1.08-kb STAB sequence (a gift from P. Mondon). Thetelomeres increase the transformation frequency (12) and the 1.08-kb STABsequence confers stability to the episomal plasmid (28). Plasmid pYCC331 con-tained a promoterless GUS gene in pPM8.

Construction of the GFP-containing plasmid, pYCC352, was briefly as follows.The first 77 amino acids of GFP, which includes the chromophore region of GFP,were from the 0.6-kb SalI-NdeI fragment of pYGFP3 developed for Candidaspecies (7). The rest of the GFP was from the 0.5-kb NdeI-SacI fragment of pBIN35S-mgfp5-ER (a gift from J. Haseloff), in which a cryptic intron between the127th and 155th amino acids was removed from a thermotolerant mutant ofGFP. This hybrid GFP was inserted into the C terminus of Cap10p and the entirefusion construct was placed in the plasmid pCIP3 to yield pYCC352.

GUS activity assay. To monitor the GUS activity in different growing stages,transformants of B-4500FOF2 containing pYCC330 were inoculated in 10 ml ofminimal medium containing 2% raffinose in 50-ml Falcon tubes and incubated at30°C with shaking at 200 rpm for 24 h. One milliliter of this culture was thendiluted in 9 ml of fresh minimal medium containing 2% glucose and reincubatedas described above. Cells were harvested at 5, 10, 15, 25, 45, and 75 h aftertransfer and GUS activity was assayed as previously described (30). GUS activitywas expressed as picomoles of 4-methylumbelliferone produced per minute per200 mg of protein. To study the effect of overexpression of STE12a on CAP10expression, TYCC259 was transformed with pYCC330. For galactose induction,cells were inoculated in 10 ml of minimal medium containing 2% raffinose in a50-ml Falcon tube and incubated at 30°C with shaking at 200 rpm for 24 h. Onemilliliter of the culture was inoculated into 9 ml of fresh minimal mediumcontaining either 2% glucose or 2% galactose as a carbon source. Cells wereharvested after an additional 20-h incubation. To assay GUS activity in culturesof stationary phase, B-4500FO2 and TYCC245F1FO were transformed withpYCC330 separately. Cells were grown in 10 ml of minimal medium containing2% raffinose for 24 h and 1 ml of this culture was diluted into 9 ml of freshminimal medium containing 2% glucose. This culture was then grown for anadditional 45 h under the same conditions. Cells were harvested and GUSactivity was assayed. Six independent transformants from each strain were as-sayed for GUS activity.

Protein localization. The immunofluorescence and immunogold localizationmethods were as described previously (3). GFP was visualized by using anAxiovert 100TV microscope (Carl Zeiss, Jena, Germany), and images wererecorded using a C5810 color chilled 3CCD camera (Hamamatsu Corporation,Bridgewater, N.J.) and processed using Adobe Photoshop.

Virulence study. Female BALB/c mice (body weight, 20 g) were injected viathe lateral tail vein with each yeast strain as described previously (2) and mor-tality was monitored.

TABLE 1. List of strains relevant to this study

Strain Genotype Source orreference

B-3501 MATa 19B-3502 MATa 19B-4500 MATa 21B-4476 MATa 21B-4500FO2 MATa ura5 3cap10C MATa cap10 This studycap10F2 MATa cap10 This studycap10F2FO MATa cap10 ura5 This studyCIP3 MATa ura5 cap10 pCIP3(URA5) This studyLP1 MATa ura5 ade2 3TYCC133 MATa ura5 cap10

pYCC133(URA5 CAP10)This study

TYCC150 MATa ura5 ade2 Dcap10::ADE2 This studyTYCC259 MATa ura5 ade2 ADE2::GAL7

(p)::STE12a5

TYCC245F1FO MATa ura5 Dste12::ADE2 5

TABLE 2. List of plasmids relevant to this study

Plasmid Description Source

pBIN 35S-mgfp5-ER GFP designed for plant J. HaseloffpCIP3 URA5 in pBluescript J. EdmanpYGFP3 GFP designed for C. albicans B. CormackpYCC123 ADE2 in pBC KS This studypYCC125 CAP10 subclone; see Fig. 1 This studypYCC130 CAP10 subclone; see Fig. 1 This studypYCC131 CAP10 subclone; see Fig. 1 This studypYCC132 CAP10 subclone; see Fig. 1 This studypYCC133 CAP10 subclone; see Fig. 1 This studypYCC147 1.6-kb XbaI fragment at 39 end

of CAP10This study

pYCC150 Deletion construct of CAP10;see Fig. 2A

This study

pYCC152 HA epitope-tagged CAP10 This studypYCC203 Three-HA epitope-tagged

CAP10This study

pYCC330 CAP10(p)::GUS in pPM8 This studypYCC331 Promoterless GUS in pPM8 This studypYCC352 Hybrid GFP-tagged CAP10 This study

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Nucleotide sequence accession number. The GenBank nucleotide accessionnumber for CAP10 is AF144574.

RESULTS

Cloning of the CAP10 gene. We have generated 16 acapsularstrains by mutagenesis (3). Among the 16 acapsular strains,four were complemented by CAP59, four were complementedby CAP60, and three were complemented by CAP64 (3). Fromthe remaining five strains, one (cap10C) was randomly chosenin an attempt to complement its acapsular phenotype with alibrary of genomic DNA from B-4500. Several encapsulatedtransformants of cap10F2FO were obtained after enrichingwith a two-polymer aqueous-phase system (2). The plasmidresponsible for the complementation was recovered and trans-formed into E. coli. The plasmid, pYCC125, containing a5.0-kb insert, complemented the acapsular mutation ofcap10F2FO but not any other acapsular mutants in our collec-tion (Fig. 1A). The 5-kb insert was subcloned to minimize theregion required for complementation. Plasmid pYCC133 con-

taining a 3.3-kb insert was the smallest clone obtained (Fig.1A). We designated the newly isolated gene CAP10.

Sequence analysis and genomic structure of CAP10. DNAsequences of the genomic and cDNA clones of CAP10 weredetermined. Because the full-length cDNA was absent in ourcDNA library, the 59 portion of the cDNA was obtained by the59 RACE method. Comparisons of the genomic and cDNAsequences revealed the presence of three introns in CAP10.The presence of multiple introns in genes is a commonly ob-served feature in C. neoformans. Unlike the other three CAPgenes (2–4), no other transcript was detected in close proximityto the CAP10 locus when pYCC125 was used as a probe. TheCAP10 gene encodes a putative protein containing 640 aminoacids with a calculated molecular mass of 73 kDa. Databasesearches did not reveal any gene sharing significant similaritywith CAP10. However, a putative type II transmembrane re-gion was detected close to the N terminus.

Southern blot analysis of CAP10 suggested the existence ofa single copy of the CAP10 gene in the genome of B-4500 (Fig.1B). Two of the previously isolated capsule-related genes,CAP59 and CAP60, are both on chromosome I and CAP64 ison chromosome III. To determine the chromosomal locationof CAP10, chromosomal DNA was separated by contour-clamped homogeneous electric field (CHEF) electrophoresisand the resulting blot was hybridized with a probe ofpYCC133. The result showed that CAP10 was on a chromo-some which is different from the other three capsule-relatedgenes (Fig. 1C).

The importance of CAP10 in capsule formation and viru-lence. A positive-negative selection method was used to deleteCAP10 from a wild-type strain (2). This method required adouble crossover at the flanking region of the gene. However,the largest clone, pYCC125, which complemented the cap10mutation contained only 90 bp beyond the stop codon ofCAP10 (Fig. 1A). To construct the CAP10 deletion construct,a longer 39 flanking region of CAP10 was obtained by screeningan XbaI-digested partial genomic library. The deletion con-struct, pYCC150, which contained 1.1 kb flanking both 59 and39 regions of CAP10 is shown in Fig. 2A. This plasmid was usedto transform an ade2 ura5 strain, LP1, and the yeast cells were

FIG. 1. Genomic structure of CAP10. (A) Restriction map of CAP10. Over-lapping subclones of pYCC125 are depicted. Plasmids were transformed into theacapsular strain cap10F2FO. The capsular phenotypes of the resulting transfor-mants were scored as indicated. Arrow represents transcriptional direction ofCAP10 and triangles represent introns. Filled box represents the coding region ofCAP10. B, BamHI; Bg, BglII; D, HindIII; M, MscI; N, NsiI; Xb, XbaI. (B)Southern blot analysis. Genomic DNA of B-4500 was digested with differentrestriction enzymes and fractionated on an 0.8% agarose gel. The DNA blot washybridized with the 3.3-kb fragment of pYCC133. (C) Chromosomal location ofCAP10. The chromosomal DNA was separated by CHEF gel electrophoresis andstained with ethidium bromide (I). The gel-separated chromosomal DNA wastransferred to a nylon membrane and hybridized with the CAP10 probe (II).B-3501 (MATa) and B-3502 (MATa) are wild-type strains.

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plated on 5-FOA plates. The DNA of acapsular transformantswas isolated and analyzed by Southern blotting. Figure 2Bshows that the .12-kb band in B-4500 was replaced by an8.0-kb and a 2.0-kb band in TYCC150, which indicated thereplacement of the wild-type CAP10 with the deletion con-struct. The replacement event was further supported by thelack of hybridization signal in TYCC150 when the blot washybridized with a probe of the deleted DNA fragment ofCAP10 (Fig. 2BII). These results indicated that the acapsularphenotype of the transformant was a result of the CAP10disruption.

Previous studies have shown that each of the other threecapsule-associated genes in C. neoformans is required to pro-duce fatal infections in mice. Two sets of animal experimentswere conducted to study the importance of CAP10 in virulence.First, four strains of C. neoformans were used to infect groupsof mice, including a stable Cap1 transformant of cap10F2FO(TYCC133), an acapsular transformant of cap10F2FO con-taining vector only (CIP3), an acapsular mutant (cap10F2),and a wild-type congenic strain (B-4500). Both TYCC133 andB-4500 produced fatal infection in all eight mice within 65 days(Fig. 3A). In contrast, CIP3 and the acapsular mutant(cap10F2) remained healthy when the experiment was termi-

nated at 100 days postinfection. The mortality rate in miceinfected with TYCC133 was higher than that in mice infectedwith B-4500. It was due to the slightly larger size of inoculantin mice receiving TYCC133. However, the size of inoculumamong mice that received TYCC133, CIP3, and cap10F2 wassimilar.

In the second set of experiments, virulence of an acapsularstrain produced by deletion of CAP10 (TYCC150) and a con-genic encapsulated strain (B-4500FO2) was compared.B-4500FO2 produced fatal infection in all eight mice within 60days whereas mice injected with TYCC150 remained healthyover 100 days (Fig. 3B). The slightly faster killing byB-4500FO2 compared to B-4500 may have been due to differ-ences between batches of mice used in these experiments (Fig.3A and B). These results corroborated the hypothesis thatcapsule is required for the virulence of C. neoformans.

The influence of different stages of growth on the expressionof CAP10. The GUS reporter gene has been successfully usedto study the expression of several genes in C. neoformans (5,30). We constructed a plasmid, pYCC330, in which the codingregion of GUS was placed under the control of the CAP10promoter. This construct was transformed into B4500FO2.GUS activity was measured by using protein extracts fromtransformants of different stages of growth (see Materials andMethods). Noticeable GUS activity was observed from over-night cultures using raffinose as a carbon source (Fig. 4). Whencultures were transferred from raffinose to glucose medium,GUS activity decreased initially and its activity stayed at lowlevels for 25 h. However, GUS activity increased significantlyafter prolonged incubation in glucose medium (.45 h). At 3days after transfer, GUS activity increased about sixfold com-pared to the activity of 5-h cultures. These results indicatedthat the expression of the CAP10(p)::GUS was influenced bydifferent growth stages.

CAP10 expression is regulated by STE12a. Because STE12aof C. neoformans regulates the expression of several virulence-associated genes (5), it was of interest to test whether STE12aregulates the expression of CAP10. Poly(A)1 RNA was iso-lated from 45-h glucose-grown cultures of the ste12a dis-ruptant and the wild-type strain. The mRNA levels of CAP10were 1.7-fold higher in the wild-type strain than in the ste12adisruptant (Fig. 5A). To test whether the disruption of STE12aaffects the CAP10(p)::GUS reporter activity, pYCC330 wastransformed into an STE12a strain (B-4500FO2) and ste12adisruptant (TYCC245F1FO). GUS activity was determinedfrom the same stationary-phase culture of both sets of trans-formants. A significant decrease in GUS activity was observedin transformants of the ste12a disruptant compared to trans-formants of the wild-type strain (Fig. 5B). Furthermore, tostudy the effect of overexpression of STE12a on CAP10expression, pYCC330 was transformed into TYCC259.TYCC259 contains a GAL7(p)::STE12a construct, which canoverexpress STE12a when galactose is used as a sole carbonsource. GUS activity was measured from protein extracts ofglucose- and galactose-grown cultures. GUS activity in galac-tose-grown culture was slightly higher than that in glucose-grown culture (Fig. 5C). Because the GUS activity in the trans-formants containing just the vector was also higher ingalactose-grown cells than in glucose-grown cells (Fig. 5C), itwas possible that the observed differences in GUS activity werecaused by differences in culture medium and were not inducedby overexpression of STE12a. To evaluate this possibility, GUSactivity from transformants of a wild-type strain (B-4500FO)containing pYCC330 grown in both culture media was deter-mined. No significant differences in GUS activity were ob-served between glucose- and galactose-grown cultures for

FIG. 2. Deletion of the CAP10 gene. (A) CAP10 locus and gene replacementvector. B, BamHI; Bg, BglII; M, MscI; N, NsiI; S, SmaI; X, XhoI; Xb, XbaI. (B)Southern blot analysis. Genomic DNAs were prepared from the wild-type strainB-4500 and an acapsular strain, TYCC150. DNAs were digested with XhoI andseparated in a 0.8% agarose gel. The blot was hybridized with a probe of the6.0-kb SmaI-NsiI fragment of pYCC150 (I) or the 1.7-kb MscI-NsiI fragmentwhich was deleted from pYCC150 (II). Arrows at 2.0 and 0.8 kb (B-4500; I)indicate the faint signals corresponding to ADE2 hybridization signals.



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transformants of B-4500FO containing pYCC330 (glucose,8.00 6 1.56, versus galactose, 7.37 6 2.16). Thus, the differ-ences of GUS activity in transformants of TYCC259 werecaused by overexpression of STE12a, which induced theCAP10(p)::GUS reporter gene activity. Therefore, the ob-served changes of GUS activity in transformants containing thevector may be due to the existence of a cryptic promoter in thevector. These data indicated that STE12a modulates the ex-pression of CAP10.

Localization of Cap10p. To understand the function ofCAP10, we attempted to determine the cellular location of theCAP10 gene product by peptide epitope-tagging methods.Nine amino acids of the influenza virus HA protein were in-

serted into the C terminus of CAP10, and the resulting con-struct was transformed into a cap10 strain. The resulting trans-formants produced capsule, indicating that insertion of the HAtag at the C terminus of Cap10p did not interfere with itsfunction. Total proteins were extracted from these capsule-containing transformants and analyzed by Western blottingusing an anti-HA antibody. The size of the protein detected byWestern blotting corroborated the predicted molecular weight(Fig. 6I). Immunofluorescence and immunoelectron micros-copy were used to determine the cellular location of Cap10p.However, we did not obtain satisfactory results due to technicaldifficulties, such as suboptimal levels of fluorescence and poorpreservation of organelles in immunogold electron microscopystudies (data not shown). Similar negative results were ob-tained even when three tandem copies of HA were used to tagCap10p.

A modified GFP, yGFP3, has been engineered and success-fully expressed in Candida albicans (7). Two modificationshave been introduced in the coding region of GFP in yGFP3.First, several mutations surrounding the chromophore of GFPwere introduced in the amino acid sequence between residues64 and 72. These alterations increased the fluorescent intensityof GFP 100-fold compared to the wild-type GFP construct (8).Secondly, many codons were modified for optimal translationin C. albicans. The yGFP3-tagged Cap10p, however, failed toproduce satisfactory fluorescence of GFP in C. neoformans.Recently, a different version of GFP, mgfp5, in which a crypticintron present between amino acids 127 and 155 was removedfrom a thermotolerant mutant of GFP was successfully ex-pressed in a plant system (17, 26). Experiments suggested thatit is possible to increase fluorescence intensity by further mod-ification of the chromophore region of this thermotolerantmutant GFP (26). In order to express GFP successfully in C.neoformans, we constructed a hybrid protein. The first 77amino acids of the hybrid GFP, which contains the chro-mophore region, were derived from yGFP3 and the remainderwere derived from the C-terminal portion of mgfp5. This hy-brid GFP was inserted at the C terminus of Cap10p and the

FIG. 3. Virulence test. Groups of eight mice each were injected with about106 viable cells and monitored for 100 days to determine mortality. (A) B-4500,a wild-type strain; TYCC133, a stable Cap1 transformant of cap10F2FO; CIP3,a stable Cap2 transformant of cap10F2FO harboring only the vector sequence;cap10F2, a cap10 mutant. (B) B-4500FO2, a CAP10 ura5 auxotroph; TYCC150,a ura5 auxotrophic cap10 disruptant.

FIG. 4. GUS activity of cultures from different growth stages. The GUSreporter construct, pYCC330, was transformed into the wild-type strainB-4500FO2. Protein extracts from three independent transformants were iso-lated at different time points and the GUS activity was determined. Error barsrepresent the sample standard deviations.



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resulting plasmid, pYCC352, was transformed into the cap10disruptant, TYCC150. The resulting transformants producedabundant capsule. When these encapsulated transformantswere viewed by fluorescence microscopy, green fluorescentsignals appeared as patches within the cytoplasm (Fig. 6IIB).The green fluorescence could be detected only in transfor-mants containing hybrid GFP and not in the controls.

DISCUSSION

Cloning and characterization of CAP10 revealed that thegene contains three introns and encodes a novel protein.CAP10 was not contiguous with another transcript in closeproximity. This observation was different from those obtainedwith the other three CAP genes which are tightly linked withother genes: CAP59 with L27, CAP60 with CEL1, and CAP64with PRE1 (2–4). Animal studies demonstrated that cap10mutants constructed by deletion or mutagenesis were unable toproduce fatal infection in mice, as demonstrated with otheracapsular strains of cap59, cap60, and cap64 (2–4). Comple-mentation of the cap10 mutation restored capsule and viru-lence. Thus, CAP10 is the fourth characterized gene requiredfor capsule formation and virulence in C. neoformans.

The GFP-tagged Cap10p appeared as patches within thecytoplasm of yeast cells. Because of the presence of a putativetype II transmembrane region close to the N terminus, wespeculate that Cap10p may be associated with certain types oforganelles, although insertion of GFP may have affected thelocation. We used HA epitope-tagging and immunoelectronmicroscopy to further define the location of Cap10p withoutsatisfactory results. Similar difficulties have been encounteredusing histochemical methods to localize gene products in C.neoformans (3, 29). Raising high-titer antibodies againstCap10p may increase the sensitivity of detecting the proteinand reveal the definite location of Cap10p.

We have previously used several versions of modified GFP,including yGFP3, to tag Cap10p, but without success. TheyGFP3 was used as a reporter by fusing it to different promot-ers of C. neoformans (10). It is not clear why the CAP10-yGFP3fusion construct failed to produce strong fluorescence. Theonly GFP construct that yielded satisfactory results was a hy-brid GFP, pYCC352. This hybrid protein contained a portionof yGFP3 engineered for C. albicans at the N terminus and aportion of GFP designed for the plant system at the C termi-nus. The success of expressing this hybrid GFP in C. neofor-mans may be due to combinative effects: the removal of thecryptic intron from a thermotolerant GFP mutant (17, 26) andintroducing modified chromophore region to increase the flu-orescence intensity (7, 8). This hybrid GFP was also success-fully used to localize the Cap60p (data not shown) which, byimmunogold electron microscopy, has been localized to thenuclear membrane (3). Several factors influenced the results ofour hybrid GFP expression. When the promoter of CAP10 inthe GFP fusion construct was replaced with a strong inducible

FIG. 5. STE12a modulates CAP10 expression. (A) Northern blot analysis.Poly(A)1 RNA isolated from the wild-type strain (B-4500) and the ste12a dis-ruptant (TYCC245F1) was fractionated in a 2.2 M formaldehyde–1% agarose geland transferred to a nylon membrane. The blot was hybridized with a probe ofCAP10 cDNA and a probe of actin cDNA. The phosphorimaging results wereused to determine the relative signal intensities. (B) The effect of the deletion ofSTE12a on GUS reporter activity. (C) The effect of STE12a overexpression onGUS reporter activity. CAP10 denotes the transformants containing pYCC330and vector denotes the transformants containing the promoterless GUS(pYCC331). Data are averages of GUS activity from six independent transfor-mants. Error bars represent the standard deviations.



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GAL7 promoter of C. neoformans (30), the resulting constructwas able to complement the acapsular mutation of TYCC150on galactose medium. However, no GFP signal was detected inthese encapsulated transformants from galactose-grown cul-ture (data not shown). Thus, overexpression of fusion GFPshowed an adverse effect on the GFP fluorescence signal. Phys-iological conditions of yeast cells also affected the level of GFPsignals. GFP fluorescence was reliably detected only when cul-tures were grown on agar for no more than 24 h. When oldercultures were used, not only did the GFP signals fade but alsomany yeast cells showed copious autofluorescence. Therefore,it may be important to have appropriate expression levels ofthe fusion construct for detection of GFP signals in C. neofor-mans. The expression levels of the fusion construct, however,appeared to have no effect on its function to complement theacapsular mutation.

Using the GUS gene as a reporter system, we found thatCAP10 expression is influenced by different stages of growth;the CAP10 gene was expressed at much higher levels duringthe late stationary phase. It appears that there is a basal levelof expression of CAP10 in young cultures and the expression ofCAP10 increases when the nutrient of the medium is depleted.These data appear to corroborate the observations that yeastcells produce abundant capsule in late stationary phase (16),although it is not clear how this process is regulated. Interest-ingly, GUS activity and accumulation of CAP10 mRNA de-creased in a strain containing a deletion of a well-conservedtranscriptional factor, STE12a. In addition, the CAP10(p)::GUS reporter activity was induced by overexpression ofSTE12a. Thus, CAP10 expression is modulated by STE12a.Since STE12a is present only in MATa cells, it would be ofinterest to know what transcriptional factor(s) controls theexpression of CAP10 in MATa strains. STE12a is a globalregulator, which also controls the expression of several genes

involved in virulence, such as capsule and phenol oxidase pro-duction (5). Although four capsule-associated genes have beenisolated, the regulation of expression of these genes is not welldefined. Further investigation on the mechanisms of regulatingage-dependent CAP10 expression and how STE12a modulatesCAP10 expression may lead to further understanding of theregulation of capsule synthesis.

ACKNOWLEDGMENTS

We thank L. Penoyer for technical assistance, A. Varma for a criticalreading of the manuscript, B. Cormack and J. Haseloff for GFP plas-mids, and J. Hanover and H. Edskes for help with fluorescence mi-croscopy.

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FIG. 6. Detection and localization of Cap10p. (I) Immunoblot analysis. Yeast cells were grown in YNB and total protein extracts were analyzed by sodium dodecylsulfate–8% polyacrylamide gel electrophoresis, incubated with anti-HA antibody, and analyzed using the Western-Star chemiluminescence detection system. Lane 1,a wild-type strain, B-4500; lane 2, an encapsulated transformant of TYCC150 containing pYCC152. (II) Localization of Cap10p-GFP fusion protein. Yeast cells weregrown on YNB medium for no more than 24 h at 30°C. Panels A and B show the transformants of TYCC150 containing Cap10p-GFP fusion construct, pYCC352. PanelsC and D show the transformants of B-4500FO2 containing pCIP3. Shown are phase-contrast micrographs (A and C) and fluorescence micrographs (B and D).



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