Cloning and Expression of the Gene Which Encodes a Tube ...

INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.4.2211–2222.2001

Apr. 2001, p. 2211–2222 Vol. 69, No. 4

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Cloning and Expression of the Gene Which Encodes a Tube PrecipitinAntigen and Wall-Associated b-Glucosidase of Coccidioides immitis

CHIUNG-YU HUNG, JIEH-JUEN YU, PAUL F. LEHMANN, AND GARRY T. COLE*

Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614-5806

Received 11 September 2000/Returned for modification 5 October 2000/Accepted 10 December 2000

We report the structure and expression of the Coccidioides immitis BGL2 gene which encodes a previouslycharacterized 120-kDa glycoprotein of this fungal respiratory pathogen. The glycoprotein is recognized byimmunoglobulin M tube precipitin (TP) antibody present in sera of patients with coccidioidomycosis, a reac-tion which has been used for serodiagnosis of early coccidioidal infection. The deduced amino acid sequenceof BGL2 shows 12 potential N glycosylation sites and numerous serine-threonine-rich regions which couldfunction as sites for O glycosylation. In addition, the protein sequence includes a domain which is character-istic of family 3 glycosyl hydrolases. Earlier biochemical studies of the purified 120-kDa TP antigen revealedthat it functions as a b-glucosidase (EC 3.2.1.21). Its amino acid sequence shows high homology to several oth-er reported fungal b-glucosidases which are members of the family 3 glycosyl hydrolases. Results of previousstudies have also suggested that the 120-kDa b-glucosidase participates in wall modification during differen-tiation of the parasitic cells (spherules) of C. immitis. In this study we showed that expression of the BGL2 geneis elevated during isotropic growth of spherules and the peak of wall-associated BGL2 enzyme activity corre-lates with this same phase of parasitic cell differentiation. These data support our hypothesis that the 120-kDab-glucosidase plays a morphogenetic role in the parasitic cycle of C. immitis.

Coccidioidomycosis is a fungal respiratory disease of hu-mans caused by Coccidioides immitis. It is recognized as a re-emerging problem in regions of endemicity of the Southwest-ern United States (16). Diagnosis of early stages of C. immitisinfection is aided by a serologic test which involves detectionof patient immunoglobulin M (IgM) precipitin antibodies re-active with specific antigens of C. immitis in an immunodif-fusion-tube precipitin (ID-TP) assay (28). We have previous-ly described the isolation of a 120-kDa glycoprotein which isrecognized by precipitin antibodies present in sera of patientswith coccidioidomycosis (5, 22). The ability of this purifiedglycoprotein to bind patient IgM (TP) antibody was confirmedby both the classical TP reaction and an enzyme-linked immu-nosorbent assay (4, 22). We have also shown that the 120-kDaTP antigen is a b-glucosidase, and the active enzyme is presentin the culture medium and within the walls of young parasiticcells (presegmented spherules) (23). We have demonstratedthat the b-glucosidase can utilize isolated and boiled cell wallmaterial of C. immitis spherules as a substrate. It was suggestedthat the wall-associated enzyme may cleave structural glucansof the spherule wall and thereby contribute to wall plasticityand isotropic growth of the parasitic cells (6, 23). Such in situenzyme activity was supported by our observations that the ac-tive enzyme can be extracted from the wall of viable, preseg-mented spherules and that exposure of cultured parasitic cellsto 1-deoxynojirimycin, a specific inhibitor of glucosidases, blocksdiametric growth of the pathogen in vitro (23). Moreover, an-tibody raised against a conjugate of 1-deoxynojirimycin wasused in an immunofluorescence study to show that the inhib-itor was localized in the wall of the growth-arrested spherules.

Here we report the isolation of the BGL2 gene that encodesthe 120-kDa b-glucosidase (TP) antigen, and present results ofthe analysis of BGL2 expression during the parasitic cycle ofC. immitis.

MATERIALS AND METHODS

Fungal strain and growth conditions. C. immitis strain C735 used in this studywas originally isolated from a patient with disseminated coccidioidomycosis whoresided in Southern California. The isolate is maintained in the Medical Collegeof Ohio fungal culture collection. The saprobic phase was grown for 5 days inGYE liquid medium (1% glucose, 0.5% yeast extract) at 30°C, while the parasiticphase was grown in Converse medium for different periods of incubation aspreviously described (17).

Isolation and sequence analysis of the BGL2 genomic clone. The strategyemployed to isolate the gene that encodes the 120-kDa TP antigen was based onidentification of two conserved amino acid sequences of selected fungal b-glu-cosidases which had been deposited in the GenBank database. An amino acidsequence alignment of these proteins was performed using the MacDNASISSequence Analysis Software (version 3.5; Hitachi, San Bruno, Calif.) to identifythe conserved domains. The conserved sequences were used to design degener-ate sense and antisense primers for use in a PCR with template genomic DNAof C. immitis to amplify a fragment of the putative BGL gene. The nucleotidesequence of the sense primer deduced from the conserved, upstream peptidesequence (GRNWEGF) was 59-GGWMGDAAYTGGGARGGNTT-39 (192-fold degeneracy) (where M is A or C; D is A, G, or T; N is A, C, G, or T; R isA or G; W is A or T; and Y is C or T). The nucleotide sequence of the antisenseprimer was designed on the basis of a conserved downstream peptide sequence(ELGFQGF) which had previously been identified as part of the signature motifthat defines family 3 glycosyl hydrolases (18) (see Table 1). The nucleotidesequence of the antisense primer was 59-GAAKCCYTGRAAKCCNARYTC-39(256-fold degeneracy) (where K is G or T).

The PCR mixture (100 ml) contained 10 mM Tris-HCl (pH 8.3) plus 50 mMKCl, 1.5 mM MgCl2, a 0.2 mM concentration of each deoxynucleoside triphos-phate (dNTP), a 5 mM concentration of each primer, 50 ng of C. immitis genomicDNA, and 2.5 U of Taq DNA polymerase (Promega, Madison, Wis.). Thirty-fivecycles were conducted for amplification of the template genomic DNA. Initialdenaturation was performed at 94°C for 3 min. Each subsequent cycle consistedof a melting step (94°C for 1 min), an annealing step (50°C for 1 min), and anextension step (72°C for 1 min). Three PCR products of different molecular sizewere observed by 3.0% agarose gel electrophoresis. The mixture of PCR ampli-cons was ligated into the pZErO 2.1 cloning vector (Invitrogen, Carlsbad, Calif.)

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Medical College of Ohio, 3055 Arlington Ave.,Toledo, OH 43614-5806. Phone: (419) 383-5423. Fax: (419) 383-3002.E-mail: [email protected].

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by the TA cloning method (2). The clones were subsequently screened by PCRusing primers derived from nucleotide sequences in the multiple cloning site ofthe vector (2). Selected clones were sequenced using the ThermoSequenaseradiolabeled terminator cycle sequencing kit (Amersham, Cleveland, Ohio). Aclone which contained a 423-bp insert was selected on the basis of homology ofits translated sequence to the reported sequence of a secreted b-glucosidase ofHistoplasma capsulatum (11, 13). This same glycosyl hydrolase of H. capsulatumhas been identified as a seroreactive antigen (H antigen) and is used in thediagnosis of histoplasmosis (11). H. capsulatum has been shown to be a closerelative of C. immitis (27). The 423-bp PCR product was purified, labeled with[a-32P]dCTP (.3,000 Ci/mmol; ICN, Costa Mesa, Calif.) using a MultiprimerDNA labeling system (Amersham), and used to screen a genomic library ofC. immitis C735 which has been reported (32). Positive phages were selected andamplified, and DNA was extracted for restriction enzyme digestion and Southernhybridization using the 423-bp PCR product as previously described (33). Thisresulted in detection of a 4.9-kb XbaI genomic fragment of the BGL2 genethat was subcloned into pZErO 2.1 and sequenced as described above. TheMacDNASIS software package was used for sequence analysis.

RACE and sequence analysis of BGL2 cDNA. The rapid amplification ofcDNA ends (RACE) procedure (17) was used to resolve the location of thenucleotide termination of the 59 untranslated region (UTR) as well as thepoly(A) addition sites of the BGL2 gene. In brief, total RNA was first isolatedfrom mycelia of C. immitis as described (17). Reverse transcription (RT)-PCRwas conducted as reported by Ausubel et al. (2). High fidelity Taq polymerase(SuperMix High Fidelity DNA Taq polymerase; Gibco BRL, Grand Island, N.Y.)was used for the PCR. The two gene-specific primers used for 59 RACE were asfollows: 59-CCTTTTATCGTTTCAGCG-39 (BG 2.4 [nucleotides {nt} 2300 to2317] [see Fig. 2B]), and 59-ACAAGCTTCTTGCATCCA-39 (BG 2.10 [nt 1927to 1944]). For 39 RACE, one of the two primers used was the synthesized oligo-d(T)17-adapter construct described by Frohman (15). The amplified RT-PCRproduct was obtained using the BGL2 gene-specific primer, 59-TGGTGTCAGCATCCTCAA-39 (BG 2.15 [nt 3662 to 3679]) and the oligo(dT) construct. ThePCR conditions were the same as those used for amplification of the genomicfragment described above. The RACE products were separated by 1.5% agarosegel electrophoresis, ligated into the pZErO 2.1 vector, and subjected to nucleo-tide sequence analysis as described above.

To amplify the remainder of the cDNA fragment of BGL2, two primers weresynthesized on the basis of the sequences of the RACE products. The nucleotidesequences of the sense and antisense primers were 59-GAAAGATCTGGCCTTCTCACCTCCATA (BG 2.16 [nt 1734 to 1751]) and 59-ATGTCGACCCTACGAAGACGGGGCTAGAG (BG 2.18; nt 4485 to 4505), which contained engi-neered BglII and SalI restriction sites, respectively (nucleotides in boldface type).Thirty-five cycles were performed to amplify the RT-PCR product. The PCRconditions were the same as described above, except that the extension step wasconducted at 72°C for 3 min. The 2.5-kb RT-PCR product was digested withBglII and SalI, separated by 1% agarose gel electrophoresis, isolated, and sub-cloned into the BamHI/SalI site of pET28b (Novagen, Madison, Wis.) to yieldthe pET28b-BGL2 plasmid construct (17). The plasmid insert was sequenced asdescribed above.

The NCBI-BLAST and PSI-BLAST programs were used to search for proteinsequences in the GenBank and SWISS-PROT databases with similarity to thetranslated sequence of the C. immitis BGL2 gene (1). The PROSITE databasewas used to identify motifs and signature sequences in BGL2 with homology toreported proteins (20), the PSORT database was used for for prediction ofprotein localization sites (24), and the CLUSTALW program was used to per-form sequence alignments (19).

Southern hybridization. Intact chromosomal DNA of C. immitis was preparedby the agarose-spheroplast procedure (25) and subjected to contour-clampedhomogeneous electric field (CHEF) gel electrophoresis under the conditionspreviously described (34). The separated chromosomal DNA was transferred toa Zeta-probe GT Blotting Membrane (Bio-Rad, Hercules, Calif.) and hybridizedwith the radiolabeled, 423-bp PCR product which was described above. In ad-dition, aliquots of equal amounts of total genomic DNA of C. immitis wereseparately digested with the restriction endonucleases XbaI, PstI, or KpnI andanalyzed by Southern hybridization with the same 423-bp probe. Hybridizationwas conducted at low stringency as described by Yu et al. (33).

Purification of the 120-kDa TP antigen for amino acid sequence analysis. The120-kDa glycoprotein was isolated from the mycelial filtrate-lysate (F-L) prep-aration of C. immitis as described previously (4). In brief, the F-L preparationwas first subjected to concanavalin A (ConA) affinity chromatography. TheConA-bound fraction was eluted from the column and subjected to sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducingconditions (22). The Coomassie brilliant blue (Sigma)-stained 120-kDa band was

excised, and the glycoprotein was electroeluted from the gel, dialyzed againstwater, and concentrated by drying under vacuum. The purified antigen wasreconstituted in phosphate-buffered saline (pH 7.2) to a concentration of 0.1 mgof protein/ml and tested for patient TP antibody reactivity in the ID-TP assay aspreviously reported (4).

Enzymatic digestion of the purified 120-kDa glycoprotein was conducted byuse of the Protein Finger-printing System kit (Promega). The Lys-C digest wassubjected to SDS-PAGE (10% polyacrylamide), and the separated fragmentswere electrotransferred to an Immobilon-P membrane (Millipore, Bedford,Mass.). Selected peptide bands visualized with Coomassie stain were excised andsubjected to N-terminal sequence analysis in an Applied Biosystems model 477Agas phase sequencer by standard procedures (26).

Expression of BGL2 by Escherichia coli. The 2.5-kb cDNA fragment of theBGL2 gene, which encodes a predicted 90.6-kDa protein (amino acids [aa] 23 to858 [see Fig. 2B]), was amplified by PCR and subcloned into the pET28b vectoras described above. The pET28b-BGL2 plasmid construct encodes a recombi-nant protein that contained a polyhistidine (His) tag at its N terminus derivedfrom the vector. The stop condon in the plasmid construct was derived from theBGL2 gene insert. The pET28b-BGL2 construct was used to transform E. colistrain BL21(DE3) as described (17). Growth of the transformed cells, inductionof expression, purification, and internal amino acid sequence analysis of therecombinant protein (rBGL2) were conducted as previously reported (17). C-terminal amino acid sequence analysis of the purified rBGL2 was conducted toconfirm that a C-terminally truncated fragment of the recombinant protein wasexpressed by E. coli. The rBGL2 was isolated by nickel-affinity chromatographyas previously described (33). C-terminal sequence analysis of the rBGL2 wasdetermined with a Perkin-Elmer Applied Biosystems model 428 amino acidanalyzer and conducted by the Macromolecular Structure Facility, MichiganState University, East Lansing, using a standard procedure (3).

Production of antiserum against rBGL2. The chromatographically isolatedrBGL2 was subjected to SDS-PAGE and electroeluted from the gel as previouslydescribed (26), and the purified recombinant protein was used to immunizeBALB/c mice (6 weeks old) for production of specific antiserum as reported (21).The antiserum was used for examination of BGL2 protein production during invitro growth of C. immitis by immunoblot analysis as described below. Preim-mune mouse serum was used as a control.

RT-PCR evaluation of BGL2 gene expression during the parasitic cycle. Semi-quantitative analysis of BGL2 mRNA levels in different morphogenetic stages ofthe parasitic cycle was conducted essentially as described by Guevara-Olvera etal. (17). First-generation parasitic cells of C. immitis derived from arthroconidiawere grown in Converse medium and harvested by centrifugation (1,500 3 g for10 min) after 16, 24, 36, 72, 84, 96, 120, and 132 h of incubation on a gyratoryshaker under conditions previously reported (17). The first generation of para-sitic cells isolated at these various times after inoculation with arthroconidia wasfairly well synchronized in development (7). Mature, ruptured spherules whichhad released their endospores (i.e., 132 h postinoculation) were isolated, washedonce with Converse medium, and used as the inoculum for second-generationcultures. The cells were grown in fresh Converse medium for 48 h and thenharvested as described above. Cells harvested at each incubation time wereseparated into three aliquots; one was used for light-microscopic analysis of thedegree of synchrony of cell development, one was used immediately for RNAextraction as described below, and the rest of the cells were quick-frozen in1.5-ml microcentrifuge tubes and then stored at 270°C until processed forprotein extraction. For light microscopy, the cell types isolated at each incubationtime were scored to characterize their stage of development. At least 200 cellswere examined in each aliquot. The cell types of the first generation were clas-sified as follows: (i) swollen, cylindrical spherule initials that were ,5 mm in di-ameter; (ii) swollen, cylindrical spherule initials that were $5 but ,10 mm indiameter; (iii) spherules that were $10 mm but ,20 mm in diameter; (iv) non-segmented spherules that were .20 mm in diameter; (v) segmented spherules;(vi) early endosporulating (,50%) spherules; (vii) early endosporulating (.50%)spherules; and (viii) mature, ruptured spherules ($90%) showing released en-dospores. The second-generation spherules were scored as nonsegmented par-asitic cells that were 15 to 20 mm in diameter. To evaluate stages of segmenta-tion, wall formation, and early endospore differentiation which occur withinintact spherules, aliquots of parasitic cells from 72- to 120-h cultures were chem-ically fixed and sectioned as previously described (30). Thick sections (approxi-mately 1 mm) were stained with wheat germ agglutinin (WGA) conjugated withfluorescein isothiocyanate (FITC) (Sigma) and examined by fluorescence mi-croscopy. The WGA-FITC conjugate stained the chitin in the spherule andendospore wall.

Total RNA was isolated from each of the cell types described above and usedfor RT-PCR analysis of levels of BGL2 expression. Total RNA was also isolated

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from C. immitis mycelia grown in liquid GYE culture medium for 120 h at30°C. Since the 120-kDa b-glucosidase was originally purified from 5-day myce-lial cultures (23), the total RNA isolated from the saprobic phase served as apositive control for the RT-PCR. Total RNA was extracted from 100 mg of freshparasitic cells or mycelial pellet using the Plant RNeasy mini kit (Qiagen, Chats-worth, Calif.) as previously described (17). The crude extract was digested withRQ1 RNase-free DNase (Promega), and the RNA was purified using the RNAclean-up protocol (Plant RNeasy mini kit; Qiagen). The purity and quantity ofRNA were monitored by UV absorbance. A ratio of optical density at 260 nm tothat at 280 nm that was .1.9 was obtained for each preparation.

Gene expression was examined by comparison of the level of mRNA whichencodes BGL2 to that which encodes the glyceraldehyde-3-phosphate dehydro-genase (GAPDH) gene of C. immitis (GenBank accession no. AF288134). Thelatter is expressed constitutively in C. immitis (J.-J. Yu, C.-Y. Hung, P. W.Thomas, and G. T. Cole, Abstr. 99th Gen. Meet. Am. Soc. Microbiol. 1999, abstr.F-52, p. 305, 1999). The RT-PCR protocol employed was essentially the same aspreviously reported (17). In brief, the cDNA was synthesized in a 50-ml solutioncontaining 50 mM Tris-HCl (pH 8.3) plus 75 mM KCl, 3 mM MgCl2, 10 mMdithiothreitol (DTT), 0.5 mM concentration of each dNTP, a 200 mM concen-tration of oligo PCR d(T)17-adapter primer, 5 mg of C. immitis total RNA, and400 U of SuperScript II RNase H2 reverse transcriptase (Gibco BRL). Thereaction mixture was incubated at 42°C for 50 min and then shifted to 70°C for10 min to denature the reverse transcriptase. The PCR mixture contained 1 ml ofthe cDNA, which was separated as aliquots diluted in Milli-Q (Millipore) water(1:1, 1:2, 1:4, 1:8, 1:16, 1:32, or 1:50). Each aliquot was mixed with 10 mMTris-HCl (pH 8.3), 50 mM KCl, 1.5 mM Mg Cl2, a 0.2 mM concentration of eachdNTP, a 5 mM concentration of each primer, and 1 U of Taq DNA polymerase(Sigma) in a total volume of 25 ml. PCR primer pairs synthesized for C. immitisBGL2 and GAPDH were each designed to span at least one intron. Inclusion ofthe intron allowed us to distinguish cDNA from genomic DNA amplicons basedupon their different sizes after separation by agarose gel electrophoresis. In orderto further rule out contamination of the RNA preparations with genomic DNA,controls included samples of the C. immitis RNA preparation that had not been

reverse transcribed but were subjected to PCR and examined by agarose gelelectrophoresis as described above. For GAPDH, the sequences of the PCRprimers were the same as reported by Guevara-Olvera et al. (17). For BGL2, theprimer sequences were as follows: sense, 59-GAAACGATAAAAGGAATCCAGGATGCT-39 (BG2.7 [nt 2304 to 2330]), and antisense, 59-GCTGTTGTTGATTTGGTTATATGAACA-39 (BG2.8 [nt 2577 to 2603]). The primers yieldedsingle RT-PCR products for GAPDH and BGL2 which were 246 and 243 bp,respectively. The PCR conditions were the same as described above, except thatthe annealing temperatures were 56°C for GAPDH and 60°C for BGL2. Thirty-five cycles were used to amplify the BGL2 and GAPDH genes. The PCR productswere subjected to agarose gel electrophoresis (2.0%) and the amplified cDNAbands were visualized by staining with ethidium bromide (EtBr). The intensityof the EtBr stain for each band was determined by UV transillumination anddensitometric analysis using the Bio-Rad Gel Documentation 1000 system andMulti-Analyst software program (Bio-Rad). The intensities of the bands, whichrepresent amount of BGL2 and GAPDH amplicons for each dilution of cDNAtemplate, were recorded. The relative amounts of BGL2 and GAPDH cDNAdetermined for each stage of parasitic cell development were calculated as thedilution factor that yielded the same band intensity as that of the mycelial BGL2amplicon at a 1:50 dilution.

Evaluation of 120-kDa glycoprotein production by immunoblot analysis. De-tection of the 120-kDa glycoprotein in total homogenates of the 5-day mycelialmat and homogenates of cells from the same stages of parasitic cell developmentas described above were conducted by SDS-PAGE followed by immunoblotanalysis using the murine antiserum raised against the rBGL2. Aliquots of eachfungal cell isolate were mechanically disrupted with glass beads (50-mm diame-ter) in a Mini-Beadbeater (Biospec, Bartlesville, Okla.). Total protein of eachcell preparation was extracted with 50 mM sodium acetate (NaAc) buffer (pH5.5) containing octyl-b-D-glucopyranoside (1% [vol/vol]; Calbiochem, La Jolla,Calif.), 100 mM NaCl, 6 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride, L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone (50 mg/ml), leupeptin (1 mg/ml),and pepstatin (1 mg/ml); (Sigma). The protein concentration of each sample wasdetermined using the Detergent Compatible Protein Assay kit (Bio-Rad), and

FIG. 1. (A) Alignment of amino acid sequences of two conserved regions of fungal b-glucosidase reported in GenBank for Aspergillus aculeatus(Aa), H. capsulatum (Hc), Saccharomycopsis fibuligera (Sf), and Pichia anomala (Pa). (B) PCR products (a, b, and c) amplified from C. immitisgenomic DNA using degenerate primers designed on the basis of conserved amino acid sequences in panel A. (C) Translated sequences of PCRproducts shown in panel B. The translated primer sequences are indicated (#). An asterisk indicates amino acid identity; a dot indicates aconserved substitution (see Table 2).

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FIG. 2. Restriction map of 4.9-kb l phage insert digested with XbaI and subcloned into pZErO (A), and nucleotide sequence of C. immitisBGL2 gene and deduced amino acid sequence (B). The 423-bp probe in panel A is derived from PCR product (a) in Fig. 1. The two underlinedamino acid sequences represent matched peptide sequence of the Lys-C-digested, native 120-kDa glycoprotein. The amino acid sequence inboldface type (aa 559 to 577) matched the peptide sequence of Lys-C-digested recombinant BGL2. The boxed sequence represents the 18-aasignature motif of family 3 glycosyl hydrolases. The aspartic acid residue within this sequence (boldface type) is the putative active site. Residuescontained within square brackets are putative N glycosylation sites. The arrow between aa 18 and 19 indicates a putative cleavage site of the signalpeptide. The double-underlined nucleotide sequences indicate conserved 59-39 sequences of introns. The gene-specific primers used for RACE andRT-PCR are indicated. The putative CAAT box (boldface type), 59 end of the UTR (c), stop codon (asterisk), and putative poly(A) addition sites(●) are also indicated.



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adjusted so that equal amounts of protein were applied to each lane of theSDS-PAGE gel. The protein preparations (approximately 80 mg each) wereseparated by SDS-PAGE (10% polyacrylamide), and the reducing gels werestained with Coomassie brilliant blue. The immunoblot procedure was conductedas previously described (26), except that secondary antibody conjugated withhorseradish peroxidase and a chemiluminescent substrate were used (ECL West-ern blot analysis system; Amersham, Arlington Heights, Ill.).

Evaluation of b-glucosidase activity. The same set of equilibrated proteinextracts as described above were also used to detect b-glycosyl-hydrolase activityby substrate gel electrophoresis (8). Approximately 40 mg of total protein of eachcell homogenate were mixed with 63 sample buffer (0.35 M Tris-HCl [pH 6.8],1% SDS, 10% glycerol, 0.002% bromophenol blue, and 0.6 M dithiothreitol),incubated at 37°C for 5 min, and separated by SDS-PAGE (10% polyacryl-amide). The gel was washed three times with 50 mM NaAc buffer (pH 5.5)containing 0.1% Triton X-100 (37°C; 10 min each wash) to remove SDS and thenincubated with 10 ml of a solution of 4-methyl-umbelliferyl-b-D-glucoside (Sig-ma) in the same NaAc buffer (0.1 mg/ml) for 30 min at 37°C. Fluorescent bandsindicative of b-glucosidase activity were viewed under UV light, and their inten-sities were determined by image analysis as described above.

Nucleotide sequence accession number. The GenBank accession number forthe C. immitis BGL2 gene is AF022893, and that for the BGL2 protein isAAF21242.

RESULTS

Selection of putative BGL2 amplicon. Four fungal b-gluco-sidase sequences obtained from the GenBank database werealigned, and two conserved regions were identified (Fig. 1A).The b-glucosidase sequences were selected on the basis thatthe native proteins were reported to have molecular sizes inthe range of 80 to 144 kDa. Degenerate oligonucleotide prim-ers designed on the basis of these sequences were used forPCR amplification with genomic template DNA. Three EtBr-stained amplicons visible in the agarose gel (Fig. 1B) were iso-lated, purified, cloned, and subjected to nucleotide sequenceanalysis. The sizes of the PCR products (amplicons a, b, and c),as determined by sequence analysis, were 423, 409, and 351 bp,respectively. Each amplicon was translated to yield the openreading frames shown in Fig. 1C. The translated PCR primersequences are shown at the N and C termini. Alignment of thesequences, excluding the translated primer regions, was con-

TABLE 1. Alignment of 18-aa signature sequence which defines fungal family 3 glycosyl hydrolases

Fungus Accession no.a EC no.b Amino acid sequencec Sequence range (total aa)d

Coccidioides immitis BGL2 AAF21242 3.2.1.21 LLKGELGFQGFIMSDWQA 268–283 (858)Coccidioides immitis BGL1 AAB67972 3.2.1.21 ILKDELGFQGFVMTDWYA 275–292 (870)Histoplasma capsulatum H Antigen AAA86880 NDe LLKAELGFQGFIMSDWQA 267–284 (863)Aspergillus niger BGL1 CAB75696 3.2.1.21 LLKAELGFQGFVMSDWAA 266–283 (860)Aspergillus aculeatus BGL BAA10968 3.2.1.21 LLKAELGFQGFVMSDWAA 266–283 (860)Saccharomycopsis fibuligera BGL1 AAA34314 3.2.1.21 LLKEELGFQGFVVSDWGA 281–298 (876)Pichia anomala BGL CAA26662 3.2.1.21 LLKEELGFQGFVMTDWGA 285–302 (825)Pichia capsulata BGLN AAA91297 ND LLKSELGFQGFVVSDWGG 269–286 (763)Geaumannomyces graminis avenacinase I AAB09777 3.2.1.21 LLKTELGFQGFVVSDWAA 265–282 (793)Kluyveromyces marxianus BGL CAA29353 3.2.1.21 ILRDEWKWDGMLMSDWFG 211–228 (845)

a Accession number for protein sequences in GenBank.b Enzyme nomenclature based on recommendations of the International Union of Biochemistry (18).c Conserved residues indicated in boldface type.d Positions of 18-aa sequence in total number of amino acids (in parentheses).e ND, not determined.

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ducted using the CLUSTALW program, and the sequencesshowed 62 to 75% homology to each other and 45 to 58%homology to other nonfungal b-glucosidase sequences in theGenBank database. These results suggested that the threePCR products translate three distinct glycosyl hydrolases ofC. immitis. In fact, the deduced amino acid sequence of am-plicon b showed complete identity to BGL1, a cytosolic b-glu-cosidase of C. immitis which has been reported (J.-J. Yu andS. L. Smithson, Abstr. 96th Gen. Meet. Am. Soc. Microbiol.1996, abstr. F-57, p. 83, 1996) and deposited in the GenBankdatabase (accession no. AAB67972). BGL1 was not examinedfurther in this study. The sequences of amplicons a and c werealigned with the sequence of a previously reported 144-kDaserodiagnostic antigen (H antigen) of H. capsulatum which wassuggested to function as a b-glucosidase (11, 13). The homol-ogies of sequences a and c to this putative b-glucosidase were92 and 69%, respectively. Earlier studies in our laboratory havesuggested a close phylogenetic relationship between C. immitisand H. capsulatum (27). For example, 72% amino acid se-quence identity was revealed between the heat shock proteins(HSP60) of these two pathogens (31). On the basis of theabove structural and functional homology data, we tentativelyidentified sequence a as a fragment of the gene which encodesthe 120-kDa b-glucosidase of C. immitis, and we refer to thisgene as BGL2. This 423-bp PCR product was used as a probeto screen the genomic library. The sequence of amplicon c wasdesignated as a fragment of the BGL3 gene, which is furtherexamined in temporal expression studies in this work.

Isolation and structure of the BGL2 gene. The random hex-amer primer-labeled 423-bp PCR product was used to screen aC. immitis genomic library constructed in lFIXII (32). A clonewhich hybridized with the probe was isolated, digested with XbaIto yield a 4.9-kb fragment, subcloned into pZErO 2.1, and sub-jected to DNA sequence analysis. The restriction map of the4.9-kb fragment of the phage insert is shown in Fig. 2A. The de-duced open reading frame of the cDNA sequence, which wasobtained by 59-39 RACE as described above, matched the ge-nomic sequence and confirmed that the gene contained five in-trons (Fig. 2B). A putative CAAT box was located 224 bpupstream of the 59 end of the UTR (nt 1359). The latter wasidentified by 59 RACE. No discernible TATA box was found.Two putative poly(A) tail addition sites were identified at nt4712 and nt 4850.

The translated BGL2 gene contains 858 aa and, in the ab-sence of any posttranslational modification, has a predictedmolecular mass of 92.8 kDa and a pI of 5.0. Sequence analysisperformed by PSORT (24) showed that 18 aa at the N termi-nus have the characteristic of a signal peptide with a putativecleavage site between A18 and E19. The predicted molecularsize of the mature BGL2 protein is 90.9 kDa. The translatedprotein showed 12 potential N glycosylation sites, multiple S/T-rich regions which could function as O glycosylation sites, aglycosyl-hydrolase family 3 signature motif at aa 266 to 283(18), and a predicted active-site residue at D280 (10). The 18-aasequence of the signature motif of BGL2 is very similar to thatreported for all other fungal family 3 glycosyl hydrolases cur-rently deposited in the GenBank database, with the exceptionof Kluyveromyces marxianus (Table 1). The predicted full-length sequence of the C. immitis BGL2 protein was comparedto reported amino acid sequences of fungal family 3 glycosylhydrolases in the database. The highest identity (74.3%) wasshown between BGL2 and the H antigen of H. capsulatum(Table 2).

Southern hybridization. Southern hybridization of chromo-somal DNA separated by CHEF gel electrophoresis was con-ducted using the same random hexamer primer-labeled 423-bpPCR amplicon as described above. The Southern blot showedthat the BGL2 gene is located on chromosome II (Fig. 3A).Total genomic DNA preparations of C. immitis were separate-ly digested with XbaI, PstI, and KpnI; subjected to agarose gelelectrophoresis; and hybridized with the same labeled 423-bpprobe (Fig. 3B). The sizes of the three single bands were pre-dicted by the restriction map of the 4.9-kb BGL2 sequence(Fig. 2A). The Southern hybridization data indicate that BGL2is a single-copy gene.

Purification and amino acid sequence analysis of 120-kDaTP antigen. The dialyzed F-L preparation of the mycelialphase of C. immitis was bound to ConA, eluted, and separatedby SDS-PAGE, and the 120-kDa glycoprotein was isolatedfrom the gel by electroelution (Fig. 4A). Antigenic activityof the isolated glycoprotein was confirmed by the ID-TP assay

FIG. 3. EtBr-stained CHEF electrophoresis gel of C. immitis strainC735 with Southern blot (S.B.) of separated chromosomal DNA (A),and Southern blot of restriction enzyme-digested genomic DNA (B).Abbreviations: Chrom., chromosome; Std., standard.

TABLE 2. Summary of calculated values for conserved amino acidsimilarities and identities between C. immitis BGL2 and

b-glucosidase sequences of other fungi

Sequence compareda Similarity (%)b Identity (%)

Histoplasma capsulatum H-antigen 85.2 74.3Aspergillus aculeatus BGL1 79.3 66.6Aspergillus niger BGL1 79.2 66.2Saccharomycopsis fibuligera BGL1 58.8 42.9Coccidioides immitis BGL1 54.3 40.2Pichia capsulata BGLN 52.4 37.8Pichia anomala BGL 52.0 34.8Geaumannomyces graminis avenacinase I 43.0 39.1Kluyveromyces marxianus BGL 37.1 18.5

a The amino acid sequences of the b-glucosidases of the indicated fungi wereobtained from GenBank. The protein accession numbers are listed in Table 1.

b Based on CLUSTALW alignment of amino acid sequences, taking intoaccount conserved substitutions of residues (19).



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(Fig. 4B). The purified TP antigen was subjected to Lys-Cdigestion, and two of the proteolytic fragments with molecularsizes of 55 and 60 kDa (Fig. 4C) were subjected to N-terminalamino acid sequence analysis. The sequence of the 60-kDafragment (LTAVIGEDAGPNL) matched aa 416 to 428 of thetranslated BGL2 sequence (Fig. 2B), while the sequence of the50-kDa fragment (EWAFSPPYY) was identical to aa 21 to 29.

Expression of rBGL2 and antibody production. To expressthe BGL2 gene, the PCR-generated cDNA was subcloned intopET28b to yield the pET28b-BGL2 construct. The predictedmolecular size of the recombinant protein was 94 kDa (includ-ing the vector-encoded peptide which contained the His tag atthe N terminus). SDS-PAGE was used to analyze cell lysatesobtained from E. coli strain BL21(DE3) which had been trans-formed with the plasmid construct and induced with IPTG(isopropyl-b-D-thiogalactopyranoside). An unpredicted 66-kDa band was detected in the lysate of IPTG-induced bacteria(Fig. 5A). The cDNA insert of the pET28b-BGL2 constructwas sequenced and confirmed to contain an open reading framethat was identical to the sequence in Fig. 2B. The 66-kDa proteinwas isolated by nickel-affinity chromatography, separated by SDS-PAGE, and purified by electroelution (Fig. 5A). The protein wasdigested with Lys-C, separated by high-pressure liquid chroma-tography (17), and subjected to N-terminal amino acid sequenceanalysis, which yielded the following: WYDHPNVTAILWAGLPGQE. The sequence was identical to the predicted amino acidsequence of the BGL2 gene (aa 559 to 577) (Fig. 2B). C-terminalsequence analysis of the rBGL2, isolated by Ni-affinity chroma-tography, revealed that the last three residues were WAA. Thissequence matches aa 600 to 602 of the translated sequence of theBGL2 gene (Fig. 2B). The predicted molecular size of rBGL2,taking this C terminus into account, is 66.1 kDa. This predictedsize is the same as the SDS-PAGE estimate of the molecular size

of the recombinant protein. It appears that the transformed bac-teria produced a C-terminally truncated form of the rBGL2.

The purified rBGL2 was used to immunize mice for produc-tion of polyclonal antibody. The antiserum recognized the 66-kDa recombinant protein in the bacterial lysate (Fig. 5A) aswell as the native 120-kDa glycoprotein in the crude, deter-gent-extracted mycelial homogenate (Fig. 5B). The 120-kDaglycoprotein was isolated from the mycelial homogenate asreported (23) and confirmed to have b-glucosidase activity(data not shown). This same antiserum was subsequently usedin the immunoblot assay of BGL2 in cell homogenates ofC. immitis obtained from different stages of the parasitic cycle.

Isolation of C. immitis cell types for studies of BGL2 expres-sion, BGL2 production, and enzyme activity. Figure 6A to Nshow light micrographs of the mycelia (Fig. 6A, 5-day culture),parasitic cell types isolated from first-generation culturesgrown for 16 to 132 h (Fig. 6B to M), and a second-generationculture grown for 48 h (Fig. 6N). Inserts (Fig. 6F, H, J, and L)show thick sections of representative spherules isolated from72-, 84-, 96-, and 120-h cultures which were stained with theWGA-FITC chitin-specific, lectin conjugate. The sectionedcells show stages of development of the segmentation wallcomplex (Fig. 6F and H) and early stages of endospore differ-entiation (Fig. 6J and L) within the intact spherules. The iso-tropic growth phase of the first-generation parasitic cells priorto segmentation is represented by Fig. 6B to E. Early differ-entiation of endospore initials (Fig. 6I and J) is signaled byisotropic growth of cells contained within the maternal spher-ule. Some swelling of the latter occurs at this stage as growthof the endospores occurs. Continued isotropic growth of theendospores (Fig. 6K and L) leads to rupture of the first gen-eration spherules (Fig. 6M) and maturation of second-gener-ation parasitic cells (Fig. 6N). The homogenate of each of

FIG. 4. (A) SDS-PAGE gel separation of ConA-bound fraction of mycelial filtrate plus lysate preparation (Con A-Bd. Fr.), and gel-electroeluted (EE) 120-kDa glycoprotein. Std., standard. (B) ID-TP assay of immunoreactivity of purified 120-kDa glycoprotein. (C) SDS-PAGEgel separation of Lys-C-digested 120-kDa glycoprotein.



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these developmental stages was used to monitor BGL2 geneexpression, 120-kDa glycoprotein production, and b-glucosi-dase activity. The similar morphology of the parasitic cells ateach progressive stage of differentiation shown in Fig. 6B to Nsuggests the near-synchronous state of the first- and second-generation cultures grown in vitro.

RT-PCR analysis of BGL2 expression during the parasiticcycle. A diagrammatic representation of first- and early sec-ond-generation parasitic cell development is shown in Fig. 7A.Each developmental stage is designated by the culture time(i.e., hours postinoculation), which was used to identify the cellhomogenates examined in Fig. 7B to D and Fig. 8. The EtBr-

stained bands in Fig. 7B represent PCR products of BGL2cDNA amplification using diluted template cDNA (1:1 to 1:32).The cDNA templates were derived from RT of separate RNApreparations obtained from selected developmental stages ofthe parasitic cycle. The intensity of each gel band examined bydensitometric analysis was compared to that of the mycelialBGL2 amplicon. The latter was generated by PCR amplifica-tion of mycelial cDNA template that was diluted by a factor of1:50. The relative amounts of BGL2 and GAPDH cDNA atselected developmental stages of the parasitic cycle are shownin two separate analyses of gene expression (i.e., experiments 1and 2 in Fig. 7C and D). The relative amounts of cDNA werecalculated as described in Materials and Methods. As indi-cated, expression of the GAPDH gene was constitutive duringthe parasitic cycle. In contrast, expression of the BGL2 gene infirst-generation cultures was elevated during the isotropicgrowth phase of spherules (16 to 36 h postinoculation) butdecreased sharply once this diametric expansion was arrestedand the cells began to undergo segmentation (72 h postinocu-lation in the first generation). As endospore differentiation wasinitiated (;96 h postinoculation) and the cells began a secondphase of isotropic growth, the expression level of BGL2 roseagain. At 48 h after transfer of the released endospores to freshculture medium, a sharp decrease in expression of BGL2 cor-related with the near completion of isotropic growth of thesecond-generation spherules.

BGL2 protein production. The relative intensity of the Coo-massie blue-stained protein bands of cell homogenates shownin Fig. 8 indicates that equal amounts of protein were appliedto the respective lanes of the two SDS-PAGE gels. Detectionof the BGL2 glycoprotein in each lane was accomplished byimmunoblot analysis using the murine, polyclonal anti-rBGL2antiserum. The results of this assay support the interpretationof the RT-PCR data. The 120-kDa glycoprotein was detectedin cell homogenates during the isotropic growth phases of boththe first-generation spherules (16, 24, and 36 h) and second-generation endospores (96, 120, and 132 h), but was not de-tectable once the parasitic cells ceased diametric growth andbegan to undergo segmentation. Detection of the 120-kDaglycoprotein in the immunoblot of the mycelial homogenateserved as a positive control.

b-Glucosidase activity. The results of substrate gel electro-phoretic analysis of the same cell homogenates as describedabove are shown in Fig. 8. The parasitic cell homogenate prep-arations were first separated by SDS-PAGE under reducingconditions, and the gels were then washed to renature theproteins and remove the SDS. After incubation of the gel with4-methyl-umbelliferyl-b-D-glucoside substrate, fluorescentbands were visible which corresponded to the 120-kDa b-glu-cosidase activity. The results suggest that enzyme activity re-mains high during the isotropic growth phase of both the first-generation spherules (16, 24, and 36 h) and endospores (96,120, and 132 h) but decreases sharply once diametric growth isarrested (72 h). Results of the RT-PCR semiquantitative anal-ysis of BGL2 expression at 72 and 84 h in the first generationshowed the absence and then slight increase in BGL2 mRNA,respectively (Fig. 7B to D). The immunoblot assay demon-strated the presence of minute amounts of BGL2 protein atthese same developmental stages. However, substrate gel anal-ysis of BGL2 enzyme activity appears to be more sensitive than

FIG. 5. (A) SDS-PAGE and immunoblot analysis of E. coli-ex-pressed rBGL2. Shown are standards (Std.), separations of lysates oftransformed bacteria grown in the presence (1) or absence (2) ofIPTG, nickel-affinity-isolated rBGL2 (Ni-Bd), purified rBGL2 ob-tained by gel electroelution (EE), and an immunoblot (Ib.) of thelysate of transformed E. coli using murine anti-rBGL2 antibody. (B)SDS-PAGE separation of detergent-extracted mycelial homogenate(My. Hm.) and corresponding immunoblot (Ib.) of native glycoproteinusing anti-rBGL2 antibody.



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the immunoblot technique. We suggest that the absence ofBGL2 mRNA at 72 h but detection of a low level of BGL2enzyme activity at this same developmental stage (Fig. 8) is dueto presence of a small amount of residual enzyme from theearlier developmental stage. On the other hand, the slightlyelevated BGL2 enzyme activity at 84 h revealed by the sub-strate gel correlates with the slight increase in BGL2 mRNA atthis same developmental stage (Fig. 7B to D). Similarly, thelow level of BGL2 enzyme activity suggested by the fluorescentband, which represents the second-generation spherules at48 h, correlates with the slightly elevated level of BGL2 mRNA(Fig. 7B to D) and BGL2 protein detected by immunoblotanalysis of this same developmental stage (Fig. 8).

The second-generation cells in Fig. 6N (corresponds to laneslabeled 48 h in Fig. 7 and 8) had not initiated segmenta-tion and, therefore, had not completed their isotropic growthphase. The substrate gel revealed two additional fluorescentbands with estimated molecular sizes of 45 and 50 kDa. Incontrast to the 120-kDa b-glucosidase, the highest activity ofthese enzymes apparently correlated with phases of spherule

segmentation (72 and 84 h) and early endosporulation (96 h) inthe first generation of the parasitic cycle. To test whether theBGL3 gene fragment (Fig. 1C) possibly encodes either the 45-or 50-kDa glycosyl hydrolase, RT-PCR analysis of BGL3 ex-pression was conducted using gene-specific primers. The re-sults indicated that BGL3 is constitutively expressed during theparasitic cycle (data not shown). The anti-rBGL2 mouse se-rum, which was raised against approximately two-thirds of themature BGL2 protein, including the putative active site, didnot recognize the 45- or 50-kDa bands in the immunoblot.These data suggest that the 45- and 50-kDa proteins were notdegradation products of BGL2 and may represent novel gly-cosyl hydrolases of C. immitis.

DISCUSSION

The secreted 120-kDa glycoprotein of C. immitis has beenshown to be both a serodiagnostic antigen and a b-glucan-degrading enzyme (23). In this study, we have cloned andcharacterized the gene which encodes this TP antigen and

FIG. 6. Light micrographs of 5-day mycelia of C. immitis (A), and developmental stages of first-generation spherules (B to M) and second-generation spherules (N). Inserts (F, H, J, L) show WGA-FITC-stained sections of spherules at stages which correspond to parasitic cells shownin panels E, G, I, and K, respectively. Developmental stages (B, C, D, E, G, I, K, and M) are derived from parasitic-phase cultures inoculated witharthroconidia and incubated for 16, 24, 36, 72, 84, 96, 120, and 132 h, respectively. Second-generation spherules in panel N were derived fromendosporulating spherules (M) which were incubated in fresh medium for 48 h. (A and F) Bars represent 20 mm.



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wall-associated b-glucosidase. On the basis of analysis of thetranslated BGL2 gene sequence, the predicted molecular sizeof the mature protein is 90.9 kDa. It is argued, therefore, thatthe native 120-kDa glycoprotein is highly glycosylated and thecarbohydrate moiety contributes approximately 25% of its mo-lecular weight. In fact, the translated amino acid sequence ofthe BGL2 gene reveals several potential sites for both N and Oglycosylation. The recombinant BGL2 protein expressed by E.coli was not recognized by the reference patient antibody whichhad been used to detect the purified, native TP antigen in theID-TP assay. This result is consistent with our earlier findingthat 3-O-methyl-D-mannose residues added to BGL2 by post-translational modification are largely responsible for the reac-tivity of IgM precipitin antibodies with this TP antigen (5).

Our earlier studies had also confirmed that the purified

120-kDa glycoprotein is a b-glucosidase (23). Of the 70 recog-nized families of glycosyl hydrolases (http://afmb.cnrs-mrs.fr/;pedro/CAZY/ghf_3.html) (9), the translated BGL2 se-quence is closely related to family 3, which currently accom-modates both prokayotic and eukaryotic enzymes with a broadrange of substrate specifities (e.g., EC 3.2.1.21, -.37, -.52, -.55,-.57, -.58, and -.74). This family is characterized by an aspartateresidue at the active site (10) and a specific peptide signaturemotif (9). Our sequence analysis showed that the location ofthe conserved 18-aa signature motif within the BGL2 sequenceis very similar to that of all other reported fungal family 3glycosyl hydrolases except for K. marxianus. However, the lat-ter does contain the putative aspartate active site and fouradditional conserved residues within the signature motif ofother members of this family.

FIG. 7. Diagrammatic representation of the parasitic cycle of C. immitis (A) and RT-PCR analysis of BGL2 and GAPDH expression (B to D).(A to D) Developmental stages of first- and second-generation parasitic cells are identified by incubation time after inoculation of the parasitic-phase cultures. (B) The EtBr-stained gels show BGL2 amplicons produced by RT-PCR as described in Materials and Methods. The dilution factorsfor the parasitic and mycelium phase-derived template cDNAs are indicated. The intensity of the gel bands was determined by densitometricanalysis. (C and D) The relative amounts of BGL2 and GAPDH cDNAs are plotted as the dilution factor of the template cDNA that yielded thesame EtBr-stained gel band intensity as that of the mycelial BGL2 amplicon using a cDNA template dilution of 1:50. The data presented inExperiment 1 (C) were derived from densitometric analysis of the gels shown in panel B.



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The translated sequence of the C. immitis BGL2 geneshowed 74% identity and 85% similarity to that of the serodi-agnostic H antigen of H. capsulatum (11). Based on amino acidsequence homology of the H antigen to reported extracellularb-glucosidases of other fungi (11), and results of functionalanalysis of the recombinant protein (13), it has been reportedthat the H. capsulatum antigen is a secreted glycosyl hydrolase.We have shown that the 120-kDa b-glucosidase of C. immitis isboth associated with the cell wall of presegmented spherulesand secreted into the culture medium (4, 23). However, animportant additional observation is that the concentration ofthe C. immitis macromolecule in the culture filtrate fluctuatesduring the parasitic cycle. The peaks of concentration of thesecreted 120-kDa b-glucosidase in the medium correspondedto stages of endospore release, and the lowest concentrationscorresponded to phases of isotropic growth (23). We previ-ously showed that the active enzyme could be extracted fromviable, intact, presegmented spherules by incubation of thecells with 1% octyl-b-D-thioglucoside (23). After isolation ofthe active b-glucosidase by this method the spherules remainedviable, suggesting that the enzyme was derived from the spher-

ule wall. Furthermore, exposure of spherule initials (Fig. 6C)to an inhibitor of the 120-kDa b-glucosidase resulted in the ar-rest of isotropic growth of the parasitic cells. These data sug-gested that the active enzyme is associated with the spherulewall during its isotropic growth phase and may perform amorphogenetic role during the parasitic cycle.

Our ability to further evaluate the function of the 120-kDab-glucosidase was enhanced by the isolation of the BGL2 geneand our success in achieving near synchrony of parasitic celldevelopment in liquid culture. The parasitic cycle can be sep-arated into three fairly distinct morphogenetic phases: isotro-pic growth, segmentation, and endosporulation (17). An im-portant developmental feature relevant to this study is thatinitiation of isotropic growth of endospores (i.e., cells whichdifferentiate into new generations of spherules) occurs whilethe cells are still within the maternal spherule. The results ofanalyses of temporal expression of the BGL2 gene, productionof the BGL2 protein, and activity of the BGL2 enzyme suggestthat the peaks of 120-kDa b-glucosidase activity in detergentextracts of spherule homogenates correlate with the isotropicgrowth phases of first- and second-generation parasitic cells.

FIG. 8. SDS-PAGE separation of Coomassie blue-stained detergent extracts of total parasitic cell and mycelial homogenates and results of bothimmunoblot analysis and substrate gel electrophoresis (SUBST. GEL) of these same total protein preparations. The developmental stages of theparasitic cycle are represented by incubation time (h) after inoculation of cultures (first generation, 16 to 132 h; second generation, 48 h). LaneM, homogenate of 5-day mycelial culture. Three bands are identified in the substrate gel with estimated molecular sizes of 120, 50, and 45 kDa.



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The first morphological change that is observed after arthro-conidia are incubated in Converse medium is cell swelling andmost likely involves water uptake and concomitant increase ininternal cell pressure, new wall biosynthesis, and wall loosening(12). The last of these events may be at least partly accom-plished by BGL2. The 120-kDa b-glucosidase is capable ofdigesting boiled, b-mercaptoethanol-treated and washed, pre-segmented spherule wall material (23). Quantitative and qual-itative analysis of the C. immitis b-glucosidase activity in thepresence of laminarin (Km, 1.03 mM) indicated that the en-zyme can utilize b-1,3-linked polyglucans as a substrate to re-lease monomeric and polymeric fragments (23). The 120-kDab-glucosidase is also capable of efficient digestion of the syn-thetic p-nitrophenol-b-D-glucopyranoside substrate, which ischaracteristic of b-1,3-exoglucosidases (14) rather than endo-glucosidases as previously suggested (23). Fungal wall-associ-ated exo- and endo-b-glucosidases have been proposed to playa role in morphogenesis (14). However, apparently not all suchglycosyl hydrolases participate in cell wall modification. Theability of the C. immitis b-glucosidase to digest its own wallcontrasts with activity of the exoG-II wall-associated hydrolaseof Aspergillus fumigatus, which showed very limited activity inthe presence of cell wall b-1,3-glucans (14). Although defini-tive proof of function of the C. immitis BGL2 is not yet avail-able, the recent development of a transformation system forC. immitis (29) now permits us to evaluate the phenotype of aBGL2 knockout strain. Nevertheless, we have provided evi-dence for a morphogenetic role of BGL2 in isotropic growthof parasitic cells based on results of immunolocalization, bio-chemical analyses, in vitro inhibition studies, and temporalevaluations of gene, glycoprotein, and enzyme expression innear-synchronized parasitic-phase cultures of C. immitis.

ACKNOWLEDGMENTS

We are grateful to K. R. Seshan for technical assistance in culturepreparation and morphological examinations.

This investigation was supported by Public Health Service grant AI19149 and the National Institute of Allergy and Infections Diseases.

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24. Nakai, K., and M. Kanehisa. 1992. A knowledge base for predicting proteinlocalization sites in eukaryotic cells. Genomics 14:897–911.

25. Pan, S., and G. T. Cole. 1992. Electrophoretic karyotypes of clinical isolatesof Coccidioides immitis. Infect. Immun. 60:4872–4880.

26. Pan, S., and G. T. Cole. 1995. Molecular and biochemical characterization ofa Coccidioides immitis-specific antigen. Infect. Immun. 63:3994–4002.

27. Pan, S., L. Sigler, and G. T. Cole. 1994. Evidence for a phylogenetic con-nection between Coccidioides immitis and Uncinocarpus reesii (Onygen-aceae). Microbiology 140:1481–1494.

28. Pappagianis, D., and B. L. Zimmer. 1990. Serology of coccidioidomycosis.Clin. Microbiol. Rev. 3:247–268.

29. Reichard, U., C.-Y. Hung, P. W. Thomas, and G. T. Cole. 2000. Disruptionof the gene which encodes a serodiagnostic antigen and chitinase of thehuman fungal pathogen Coccidioides immitis. Infect. Immun. 68:5830–5838.

30. Seshan, K. R., and G. T. Cole. 1994. Structural studies in Coccidioidesimmitis, p. 265–273. In B. Maresca and G. S. Kobayashi (ed.), Molecularbiology of pathogenic fungi. Telos Press, New York, N.Y.

31. Thomas, P. W., E. E. Wyckoff, E. J. Pishko, J.-J. Yu, T. N. Kirkland, andG. T. Cole. 1997. The hsp60 gene of the human pathogenic fungus Coccid-ioides immitis encodes a T-cell reactive protein. Gene 199:83–91.

32. Wyckoff, E. E., E. J. Pishko, T. N. Kirkland, and G. T. Cole. 1995. Cloningand expression of a gene encoding a T-cell reactive protein from Coccid-ioides immitis: homology to 4-hydroxyphenylpyruvate dioxygenase and themammalian F antigen. Gene 161:107–111.

33. Yu, J.-J., S. L. Smithson, P. W. Thomas, T. N. Kirkland, and G. T. Cole.1997. Isolation and characterization of the urease gene (URE) from thepathogenic fungus Coccidioides immitis. Gene 198:387–391.

34. Yu, J.-J., L. Zheng, P. W. Thomas, P. J. Szaniszlo, and G. T. Cole. 1999.Isolation and confirmation of function of the Coccidioides immitis URA5(orotate phosphoribosyl transferase) gene. Gene 226:233–242.

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