Disruption of the Gene Which Encodes a Serodiagnostic Antigen and

INFECTION AND IMMUNITY,0019-9567/00/$04.0010

Oct. 2000, p. 5830–5838 Vol. 68, No. 10

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Disruption of the Gene Which Encodes a SerodiagnosticAntigen and Chitinase of the Human Fungal Pathogen

Coccidioides immitisUTZ REICHARD, CHIUNG-YU HUNG, PEI W. THOMAS, AND GARRY T. COLE*

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

Received 19 April 2000/Returned for modification 23 June 2000/Accepted 21 July 2000

Disruption of genes in medically important fungi has proved to be a powerful tool for evaluation of putativevirulence factors and identification of potential protein targets for novel antifungal drugs. Chitinase has beensuggested to play a pivotal role in autolysis of the parasitic cell wall of Coccidioides immitis during the asexualreproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (CTS1 and CTS2) of C.immitis have been cloned. Preliminary evidence has suggested that expression of CTS1 is markedly increasedduring endospore formation. The secreted CTS1 chitinase has also been shown to react with patient anti-Coccidioides complement-fixing (CF) antibody and is a valuable aid in the serodiagnosis of coccidioidomycosis.To examine the role of CTS1 in the morphogenesis of parasitic cells, the CTS1 gene was disrupted by a single,locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construct thatcontained a 1.1-kb fragment of the chitinase gene into the chromosomal DNA of C. immitis. Results of Southernhybridizations, immunoblot analyses of culture filtrates using both CTS1-specific murine antiserum and serumfrom a patient with confirmed coccidioidal infection, an immunodiffusion test for CF antigenicity, and sub-strate gel electrophoresis assays of chitinase activity confirmed that the CTS1 gene was disrupted andnonfunctional. This is the first report of a successful targeted gene disruption in C. immitis. However, loss ofCTS1 function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in theparental and Dcts1 strains suggested that the absence of a functional CTS1 gene can be compensated for byelevated expression of the CTS2 gene. Current investigations are focused on disruption of CTS2 in the Dcts1host to further evaluate the significance of chitinase activity in the parasitic cycle of C. immitis.

Coccidioides immitis is the causative agent of a fungal respi-ratory disease known as San Joaquin Valley fever or coccid-ioidomycosis. The fungus is considered to be a primary patho-gen of humans and dogs that is capable of establishinginfections in an immunocompetent host (16, 23). The saprobicphase of C. immitis occurs in alkaline soil of southwesterndesert regions of the United States, which extend from Cali-fornia to western Texas (26). Host infection typically occurs byinhalation of dry airborne spores (arthroconidia) that are smallenough to reach the alveoli (5). The fungus is highly virulent:BALB/c mice inoculated intranasally with as few as 100 arthro-conidia of a strain of the fungus known to be virulent die within2 to 3 weeks as a result of necrotic pulmonary lesions andrespiratory failure (6). The pathogenicity of C. immitis is fur-ther underscored by the fact that coccidioidomycosis is themost frequently diagnosed mycosis of laboratory personnelwho work with medically important fungi (13).

The saprobic and parasitic phases are considered to be hap-loid (24), although this has yet to be confirmed. C. immitis hasno known sexual state, but molecular evidence indicates thatrecombination occurs within the fungal population in nature(2). Morphogenesis of the asexual, parasitic phase of C. immitisis unlike that of any of the other human fungal pathogens (5).The parasitic cycle can be reproduced in vitro (21) and isinitiated by conversion of the cylindrical arthroconidium into amultinucleate round cell (spherule). The latter undergoes iso-tropic growth and segmentation and ultimately gives rise to a

multiplicity of endospores. The high fecundity of the parasiticphase of C. immitis may contribute to the ability of the patho-gen to overcome innate cellular immune defenses of the host,particularly when a large inoculum of arthroconidia has beeninhaled. Morphogenetic factors that control pivotal events ofendosporulation represent potential anti-Coccidioides drugtargets.

Our knowledge of the molecular biology of C. immitis is stillin its infancy, largely because few laboratories have focusedtheir efforts on this microorganism. A major emphasis in C.immitis gene cloning studies has been antigen identification,expression, and characterization. One immunoreactive macro-molecule that has been the focus of multiple investigations isthe complement fixation (CF) antigen, which has been usedclinically for decades in the serodiagnosis of coccidioidal in-fections (27). The molecular size of the native CF antigen,estimated under reducing conditions by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE), is 48 kDa(42). Biochemical characterization of the purified 48-kDapolypeptide revealed that the antigen functions as a chitinase(15) and is secreted by C. immitis in both the saprobic andparasitic phases. It was suggested that the active chitinaseassociates with the segmentation wall of parasitic cells duringearly endosporulation (3) and participates in an essential pro-cess of cell wall modification as endospores undergo differen-tiation and subsequent release from the maternal spherule (4).The gene which encodes the CF antigen (CTS1) has beencloned (29), and temporal expression of the chitinase gene wasevaluated in vitro during parasitic cell development (P. W.Thomas and G. T. Cole, Abstr. 99th Gen. Meet. Am. Soc.Microbiol. 1999, abstr. F-52, p. 306, 1999). The results of thelatter study suggested that the maximum level of expression of

* 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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the CTS1 gene occurs during the endosporulation phase of C.immitis. In this paper, we report the first targeted disruption ofa C. immitis gene and further evaluate whether the CTS1chitinase plays a morphogenetic role in the parasitic cycle ofthe pathogen.

MATERIALS AND METHODS

Strains, media, and growth conditions. To obtain arthroconidia, C. immitisstrain C735 in the saprobic phase was grown on GYE agar (1% glucose, 0.5%yeast extract, 1.5% agar) at 30°C for 3 to 4 weeks. For DNA extraction, parentaland transformant strains in the saprobic phase were grown in GYE liquid me-dium at 30°C without or with hygromycin (75 mg/ml; Sigma, St. Louis, Mo.). Forparasitic-phase growth, the same strains were grown in Converse medium aspreviously described (21).

Isolation of a BamHI genomic fragment which contains the CTS1 gene. An8-kb genomic fragment was obtained by screening a genomic library of C. immitis(38) with a radioisotope-labeled 515-bp PCR product derived from amplicationof a fragment of the CTS1 gene as described by Pishko et al. (29). The 8-kbgenomic fragment was subcloned into pBluescript SK1 and restriction mappedusing the known sequence of the CTS1 gene (29) and a standard digestionprotocol (34).

Construction of a transformation plasmid (pDcts1) for disruption of the CTS1gene. The pDcts1 plasmid was designed as a gene disruption vector and wasconstructed using pAN7.1 (32) and a 1.1-kb internal fragment of the CTS1 geneamplified by PCR (bp 68 to 1177 of the reported CTS1 gene [29]). To facilitatecloning, BglII and BstEII sites were added to the 59 end of the upstream anddownstream primers, respectively (primers A and B in Table 1). These same tworestriction sites are present in pAN7.1 upstream from the functional GPD pro-moter sequence (32). Both pAN7.1 and the 1.1-kb CTS1 PCR product weredigested with BglII and BstEII. The CTS1 PCR product and the larger fragmentof digested pAN7.1 (6.5 kb) were purified by agarose gel separation and extrac-tion using a QIAEX II Gel Extraction Kit (Qiagen, Chatsworth, Calif.). The twofragments were then ligated, and the product was used to transform Escherichiacoli strain DH5a. The pDcts1 plasmid isolated from E. coli was used for subse-quent gene-targeted disruption experiments. Prior to transformation, the plas-mid was linearized with ClaI and then purified by phenol-chloroform extractionand ethanol precipitation in accordance with standard protocols (34).

Transformation procedure. Transformation of C. immitis was performed usinga modified protocol which has been successfully employed for Aspergillus nidu-lans and Aspergillus fumigatus (28, 33). Arthroconidia from 8 to 10 agar plateswith dense sporulating mycelia were harvested by flooding each plate with 5 mlof GYE medium plus 0.1% Tween 80 (Sigma), followed by gentle disruption ofthe mycelia with a flamed wire inoculation loop. Suspensions of arthroconidiafrom each plate were pooled in a 50-ml tube, vortexed vigorously, and used toinoculate a 1-liter flask that contained 150 ml of GYE medium. In a typicalexperiment, the flask contained approximately 1.0 3 106 to 3.0 3 106 arthro-conidia per ml of GYE medium. The cell suspension was incubated in a gyratoryshaker (100 rpm, 30°C, 12 h) to obtain germinated arthroconidia, resulting ingerm tubes that were approximately two to five times the length of the conidia.Germ tube formation was monitored microscopically. The germlings were har-vested by centrifugation (2,000 3 g, 4°C, 10 min) and transferred to a sterile,transparent 50-ml polypropylene tube. The germinated arthroconidia were re-suspended in 20 ml of osmotic medium (OM; 10 mM Na-phosphate buffer [pH5.8], 1.2 M MgSO4), centrifuged (2000 3 g, 4°C, 10 min), and again resuspendedin 20 ml of OM. This washing step was repeated twice and followed by suspen-

sion of the germinated conidia in 5 ml of OM contained in the 50-ml polypro-pylene tube.

To obtain protoplasts of C. immitis, the germlings were exposed to a mixtureof fungal-wall-targeted enzymes. A stock solution of lytic enzymes from Tricho-derma harzianum (Sigma; catalog no. L2265) was first prepared at a concentra-tion of 25 mg/ml of OM and sterilized by filtration. Four milliliters of thissolution was initially added to the fungal cell suspension in 5 ml of OM, whichwas then incubated on ice for 5 min. After the addition of 1 ml of a filter-sterilized solution of bovine serum albumin (Sigma; 3 mg/ml of OM), the samplewas incubated in a shaker incubator (60 rpm, 30°C, 90 min) and protoplastformation was monitored by phase-contrast light microcopy. The sample in the50-ml centrifuge tube was then carefully overlaid with 10 ml of trapping buffer(TB; 100 mM morpholinepropanesulfonic acid [MOPS]-NaOH buffer [pH 6.5],0.6 M sorbitol), followed by centrifugation using a swing-arm rotor (3,900 3 g,4°C, 15 min). The protoplasts were visible as a cloudy band at the interphase ofthe OM and the TB. The dense pellet consisted primarily of cell debris. Theprotoplast-containing interphase was carefully aspirated, transferred to a new50-ml tube, and mixed with 9 parts of MOPS-sorbitol (MS) buffer (10 mMMOPS-NaOH buffer [pH 6.5], 1 M sorbitol). The protoplasts were pelleted bycentrifugation (1,000 3 g, 15 min, 4°C) and washed two times with 20 ml of MSbuffer plus calcium chloride (MSC buffer; MS plus 20 mM CaCl2) using thecentrifugation conditions described above. The protoplast pellet was then resus-pended in 150 ml of MSC buffer and stored on ice, and the number of protoplastsper milliliter was determined by light microscopy using a hemocytometercounter. Typically, the final concentration of protoplasts ranged from 5.0 3 107

to 2.0 3 108/ml. Prior to transformation, the concentration of protoplasts wasadjusted to 5.0 3 107/ml using MSC buffer.

For transformation, 2 mg of ClaI-digested plasmid (pDcts1) DNA in a totalvolume of 5 ml of sterile distilled H2O was added to 100 ml of the protoplastsuspension (approximately 5 3 106 protoplasts) in a 1.5-ml microcentrifuge tubeand thoroughly mixed with 30 ml of 60% polyethylene glycol 3350 (PEG; Sigma)prepared in MSC buffer. After 30 min of incubation on ice, another 900 ml of60% PEG was mixed with the sample, followed by incubation for 30 min at roomtemperature. The protoplasts were then pelleted by centrifugation (5,000 3 g,4°C, 10 min), and the PEG solution was carefully removed by aspiration. Theprotoplasts, which had presumably taken up the DNA at this stage, were resus-pended in 500 ml of MSC buffer transferred in different volumes (10, 25, 50, and100 ml) to sterile 2-ml polypropylene tubes. Sterile GYE soft agar (1% glucose,0.5% yeast extract, 0.7% agar, 1 M sucrose) was prepared as a stock and heldmolten at 45 to 50°C. An aliquot (1.6 ml) of this soft agar was added to each tube,mixed with the protoplasts, and immediately poured onto prewarmed (37°C)petri plates (100 by 15 mm) which already contained 16 ml of GYE agar plus 1M sucrose as an osmotic stabilizer. The plates were incubated at 30°C for 20 hand then overlaid with another 2.4 ml of GYE soft agar containing 1.5 mg ofhygromycin. The final concentration of hygromycin after diffusion into the plateswas estimated to be 75 mg/ml of agar. The plates were incubated at 30°C for anadditional 6 days. Typically, saprobic-phase colonies of C. immitis which wereresistant to hygromycin were visible after 4 days. Putative transformants wereisolated with the tip of a 3-ml disposable pipette (March Biomedical Products,Rochester, N.Y.) and transferred to separate, fresh GYE agar plates whichcontained 75 mg of hygromycin/ml. For each transformation experiment, a neg-ative control of C. immitis protoplasts treated as described above but in theabsence of transforming DNA was prepared.

Isolation of homokaryons. Since the protoplasts of C. immitis prepared forthese and earlier investigations (24) were shown to be multinuclear, it wasreasonable to assume that at least some of the transformants produced would beheterokaryons. To obtain homokaryons, the isolated transformants were grown

TABLE 1. PCR primers used to screen for disruptants and amplify selected probes for Southern hybridization

Primera Nucleotide sequence Sequencederivationb

A 59-ATTAGATCTTGCCCAATTCTTATCCAGTCC-39 CTS1B 59-ATTGGTCACCTTGAAGCTAGTGCCGATCCC-39 CTS1C 59-TTTATGCTTCCGGCTCGTATG-39 pAN7.1D 59-CCCATTCCATTCTTCGTAATG-39 CTS1E 59-CGACGACAGATTCCATCTCAC-39 CTS1 UTR (59)F 59-CTCCAGTTCTTCTCGGCGTTC-39 pAN7.1 pGPDG 59-CAAGATCGACGACAGATTCC-39 CTS1 UTR (59)H 59-AAGTTCGGTAACCTGAGCGC-39 CTS1I 59-TGCTTTGCCCGGTGTATGAAACC-39 pAN7.1 pGPDJ 59-AAGGGATGGGAAGGATGGAGTATGG-39 pAN7.1 pGPDK 59-TGGGACTACAAAGACATGCCG-39 CTS1L 59-GCCAATACAACAAAGGTCGGC-39 CTS1 UTR (39)

a Primers A and B, which include engineered BglII and BstEII restriction sites, respectively (underlined), were used to amplify a 1.1-kb internal fragment of the CTS1gene (29); primers C to L were used to amplify fragments of pAN7.1 or CTS1, as indicated in Fig. 1 and described in the text.

b Nucleotide sequences of primers are based on reported DNA sequences of pAN7.1 (GenBank accession no. Z32698) (32) and CTS1 (GenBank accession no.L41663) (29).

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on GYE agar plates containing 100 mg of hygromycin/ml for 10 days, followed bythree repeated streak-outs from single colonies of sporulating cultures. The finalisolated colonies were transferred to separate hygromycin-containing GYE agarplates as described above. Transformants obtained by this procedure were thensubjected to PCR, Southern, and immunoblot analyses.

PCR screening procedures. To obtain DNA for PCR screening, approximatelytwo inoculating loops of fungal mycelia were isolated from plate cultures of eachof the putative transformants or the parental strain and transferred separately to1.5-ml microcentrifuge tubes containing 100 mg of glass beads (0.45 to 0.55 mmin diameter) and 250 ml of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA).The fungal cells were homogenized using a Mini-Beadbeater (Biospec, Bartles-ville, Okla.) at 3,000 rpm for 60 s, incubated at 100°C for 5 min, and thencentrifuged (16,000 3 g, 5 min). The DNA present in the supernatant wasextracted with phenol-chloroform and precipitated with ethanol in accordancewith standard protocols (34). The transformants were screened for the desiredintegration event by PCR using primers which amplified a specific 1.5-kb productonly if the plasmid construct integrated into the C. immitis chromosomal DNAby homologous recombination. For this purpose, the upstream primer (primer Cin Table 1) was located within the pAN7.1 vector sequence (see Fig. 1), whereasthe downstream primer (primer D in Table 1) was located in the 59 region of theCTS1 gene, which was not present in the pDcts1 construct. A second primer pair(E and F in Table 1 and Fig. 1) was used to confirm homologous integrationbased on the same rationale. PCR amplification of genomic DNA isolated fromputative transformants in the latter case yielded a predicted amplicon of 1.4 kb.Initial screening for homokaryons among the putative transformants was accom-plished by PCR using a primer pair (G and H in Table 1 and Fig. 1) which yieldeda 1.3-kb product only if the nondisrupted CTS1 gene was present in the templateDNA. Transformant template DNA which displayed the two PCR products (1.5and 1.4 kb) specific for the desired homologous integration event and the ab-sence of the 1.3-kb PCR product provided initial evidence for the isolation of ahomokaryotic disruptant.

Southern hybridization and selection of probes. Southern hybridizations wereperformed in accordance with standard procedures (34) using restriction en-zyme-digested genomic DNAs of parental and transformant strains. Probes werederived by PCR amplification using the specific primers described below. Tem-plate DNA included either pBluescript SK1, which contained the full-lengthCTS1 gene, or pAN7.1 plus the 1.1-kb insert of the CTS1 gene. The PCR-derivedprobes were conjugated with digoxigenin-11-dUTP (Boehringer, Mannheim,Germany) during the amplification reaction, and digoxigenin was detected withspecific peroxidase-labeled antibody as recommended by the supplier. Insertionof the HPH gene present in pDcts1 into the C. immitis genome as a singleintegration event was confirmed by Southern hybridization. The probe was a392-bp PCR product (Pb1) amplified with primers I and J (Table 1 and Fig. 1),

which were derived from the GPD promoter sequence. Confirmation of disrup-tion of the CTS1 gene was performed by Southern hybridization using a 584-bpPCR product (Pb2). This was amplified with primers K and L (Table 1 and Fig.1), which had been selected from sequences in the 59 region of the CTS1 geneand were not present in the pDcts1 construct. A hypothetical restriction map wasconstructed, assuming homologous integration of pDcts1, to predict the size ofhybridized fragments in the Southern blot analysis. Restriction enzymes werechosen on the basis of this map and used to digest parental and transformantDNA preparations prior to hybridization with Pb1 or Pb2.

Recombinant CTS1 production and generation of anti-CTS1 polyclonal anti-body. A 1.2-kb PCR product which encodes part of the CTS1 protein (aminoacids 1 to 411 [29]) was amplified using template cDNA obtained by reversetranscription of total RNA of C. immitis. The RNA was isolated from mycelial-phase C. immitis grown in GYE liquid medium at 30°C for 6 days. High-fidelityTaq DNA polymerase (PCR SuperMix High Fidelity DNA Taq polymerase;Gibco BRL, Grand Island, N.Y.) was used for reverse transcription (RT)-PCR.Isolation of total RNA and the RT reaction were performed as previouslydescribed (9). PCR amplification of the CTS1 cDNA was conducted using asense primer (nucleotides [nt] 427 to 447) derived from the genomic sequence ofCTS1 (29). The sense primer included the start codon (AUG) and contained anengineered NdeI restriction site constructed as previously described (9). Theantisense primer (nt 1973 to 1953) was also designed on the basis of the CTS1genomic sequence. The PCR product was isolated as previously described (40)and digested with NdeI and SstI to yield a 1.2-kb cDNA fragment. The SstIrestriction site (nt 1973) was previously determined to be upstream from the stopcodon of the CTS1 gene (29). The cDNA fragment was subcloned into the E. coliexpression vector pET28b (Novagen, Madison, Wis.) by ligation at the NdeI andHindIII restriction sites as previously reported (41). Confirmation of the pre-dicted nucleotide sequence of the pET28b-CTS1 construct was performed bystandard sequence analysis as previously described (9). The plasmid constructwas used to transform E. coli strain BL21 (DE3) (Novagen), and expression wasinduced in the presence of isopropyl-b-D-thiogalactopyranoside (IPTG) as pre-viously reported (40). The recombinant CTS1 (rCTS1) expressed by E. colicontained 20 amino acids at its N terminus and 13 amino acids at its C terminuswhich represented vector-translated peptides. The predicted molecular size ofthe rCTS1 fusion protein was 49.3 kDa. The rCTS1 was purified from homoge-nates of the IPTG-induced bacterial cells by nickel affinity chromatography. Thecolumn eluate was separated by SDS-PAGE and electrotransferred to an Im-mobilon P membrane (Millipore Corp., Bedford, Mass.), and the 49-kDa rCTS1was excised for amino acid sequence analysis as previously described (9).

The recombinant protein was also isolated by electroelution from a copper-stained (Bio-Rad) SDS-PAGE gel separation of the nickel affinity chromato-graphic fraction and destained in half-strength SDS-PAGE running buffer (25

FIG. 1. Plasmid construct and predicted outcome of pDcts1 integration event. A 1.1-kb internal fragment of CTS1 was cloned into the pAN7.1 plasmid. Homologousrecombination of the plasmid construct with CTS1 by a single crossover event results in the generation of two incomplete copies of the C. immitis gene (labeled cts1*)separated by the linearized sequence of pAN7.1. The primers used for amplification of the parental CTS1 gene are G and H. The primers used to screen for theincomplete cts1 genes are C and D (39 fragment) and E and F (59 fragment). The probes used for Southern hybridization are Pb1 and Pb2. UTR, untranslated region.

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mM Tris, 192 mM glycine, 0.1% SDS). Approximately 10 mg of purified proteinwas used to immunize each of five female BALB/c mice (6 weeks old). Theimmunization protocol was the same as previously described (12).

Immunoblot analyses. Saprobic-phase C. immitis was grown in modified GYEmedium containing 0.1% glucose, 1% yeast extract, and 1% chitin in suspension(purified powder from crab shells; Sigma). Chitin was added to enhance secre-tion of the native CTS1 protein. After 5 days of incubation on a gyratory shaker(120 rpm, 30°C), the culture supernatant was harvested under aseptic conditionsby filtration through a Whatman no. 1 filter. Residual fungal debris was removedfrom the filtrate by centrifugation (15,000 3 g, 20 min, 4°C), and proteins wereconcentrated 200-fold with a Centriprep YM-10 filtration device in accordancewith the protocol of the manufacturer (Millipore). The concentrated proteinswere subjected to SDS-PAGE, and immunoblots were prepared essentially aspreviously described (12) using either mouse or human serum. The murineanti-rCTS1 serum described above and serum from a patient with confirmedcoccidioidomycosis were diluted 1:500 in phosphate buffer (pH 7.4). Preimmunemouse serum and normal human serum were used as controls.

Immunoblot analysis of native proteins of the mycelial culture filtrate whichwere separated by nondenaturing PAGE was also conducted to identify theenzymatically active CTS1 polypeptide band in substrate gels as described belowfor the chitinase assay. After electrophoresis, the nondenaturing gel was incu-bated in SDS running buffer for 30 min before electrotransfer of proteins to theImmobilon membrane. Native CTS1 was identified using the specific murineantibody described above.

ID-CF assay. The immunodiffusion (ID) assay was performed by a methodpreviously described (17) for detection of the CF antigen (CF-Ag). The referenceC. immitis CF-Ag and goat anti-CF reference serum were obtained from Me-ridian Diagnostics (Cincinnati, Ohio).

Chitinase assay. Approximately 108 arthroconidia isolated from plate culturesof the parental strain and the strain with the CTS1 gene disrupted (Dcts1) wereinoculated separately into 500-ml flasks which contained 100 ml of GYE mediumplus chitin as described above. The cultures were grown with constant shaking(100 rpm) at 30°C for 5 to 6 days. The mycelia were removed by filtration (0.2-mmpore size). The culture filtrate was dialyzed overnight against sterile distilledwater (3,500 molecular-weight [MW] cutoff; BioDesign Dialysis Inc., Carmel,N.Y.) and then concentrated 10-fold against PEG (Sigma; 15,000 to 20,000average MW). Each sample was then further concentrated to a final volume of1 ml using a centrifugal filter unit (Ultrafree-4; Millipore; MW cutoff of 10,000).The protein concentration of each sample was determined by the Bio-Rad assay.The same amount of protein (20 mg) for each sample was separated by nonde-naturing PAGE (8% polyacrylamide without SDS and reducing agent). Substrategel electrophoresis was used to detect chitinase activity by the method reportedby Tronsmo and Harman (36). Briefly, after electrophoresis, the gel was washedthree times (10 min, room temperature) in substrate buffer containing 50 mMNaPO4 (pH 6.1). The washed gel was immediately transferred to the substratesolution containing 0.1 mM 4-methylumbelliferyl-(b-N-N1-N11-triacetylchitotri-ose) and incubated for 10 min at 37°C. The gel was irradiated with UV light tovisualize the bands of chitinase activity. The same gel was either stained withCoomassie brilliant blue to detect protein bands or subjected to immunoblotanalysis as described above.

Evaluation of genetic stability, virulence, and morphogenesis of the Dcts1mutant. Arthroconidia (103 cells) obtained from GYE plate cultures (30 days at30°C) of the parental and Dcts1 strains were used to inoculate BALB/c mice (twomice each; females, 8 weeks old) by the intraperitoneal (i.p.) route. Two weeksafter challenge, the spleen and lungs of each mouse were separately homoge-nized, dilution plated on GYE agar plus chloramphenicol (50 mg/ml; Sigma)butlacking hygromycin, and incubated for 3 days (30°C). Dilution plating was per-formed to determine the relative numbers of CFU derived from organ homog-enates of the two groups of mice. Equal numbers of CFU from the parental-strain- and Dcts1 strain-challenged mice (3 3 103) were transferred to separate,fresh GYE agar plates in the absence of hygromycin and incubated for 2 weeksfor arthroconidium production. Conidia from the two sets of cultures wereseparately pooled and used to inoculate liquid GYE medium lacking hygromy-cin. After 3 days of incubation (30°C) on a gyratory shaker, separate pools oftotal genomic DNA from the parental and Dcts1 mutant strains were extracted,subjected to restriction enzyme digestion, and examined by Southern hybridiza-tion using the 584-bp probe (Pb2) as described above.

Arthroconidia (200) obtained from GYE agar plate cultures of the mouse-passaged parental and Dcts1 strains were used to challenge BALB/c mice (fe-males, 25 g) by the i.p. route as described above. Survival plots for the two C.immitis strains were compared and analyzed by Kaplan-Meier statistics. Histo-logical sections of mouse tissue infected with the parental or Dcts1 strain werecompared by light microscopy for morphological features of the parasitic cells.

RESULTS

Transformation efficiency and identification of transfor-mants. The transformation plasmid construct pDcts1 was de-signed to integrate into C. immitis chromosomal DNA by asingle crossover event involving the homologous fragment of

the CTS1 gene as shown in Fig. 1. Transformation of 107

protoplasts with 2 mg of ClaI-digested pDcts1 plasmid DNAtypically yielded 30 to 50 hygromycin-resistant colonies. Afterinitial regeneration of mycelia from protoplasts plated on GYEsoft agar plus hygromycin (75 mg/ml), colonies were isolatedand transferred to GYE agar plates containing hygromycin at100 mg/ml. Surviving colonies were then transferred to freshplates, reisolated, and transferred again in an attempt to obtainhomokaryons. The transformants were screened for disruptionof the CTS1 gene by PCR as described above. Figure 2 showsthe results of a typical transformation experiment. Templategenomic DNA from 48 hygromycin-resistant isolates wasscreened by PCR using two sets of primers (C plus D and Gplus H; Table 1). The use of one pair of primers (C plus D)resulted in the amplification of a 1.5-kb fragment of the pDcts1construct if it integrated into the CTS1 gene. The use of theother primer pair (G plus H) resulted in amplification of the1.3-kb parental CTS1 gene. Four of the 48 transformants werethus identified as candidates which had apparently undergonethe desired homologous integration. Results of PCR amplifi-cation of template DNAs from three of these putative trans-formants (D10.2, D2, and D3) are compared to that of theparental strain (P) in Fig. 2. The transformants, but not theparental strain, revealed a 1.5-kb amplicon if primers C and Dwere used. Primers G and H amplified the 1.3-kb CTS1 geneproduct in the presence of parental genomic DNA only. Ab-sence of the amplified full-length CTS1 gene in chromosomalDNA preparations of the transformants suggested that theywere homokaryons. One candidate transformant (D10.2), which isreferred to below as the Dcts1 strain, was selected for furtheranalysis.

Confirmation of CTS1 gene disruption by Southern hybrid-ization. To confirm that integration of pDcts1 into the C. im-mitis genome had occurred at a single, homologous site andthat the transformant was homokaryotic, genomic DNA of theDcts1 strain was extracted, digested with selected restrictionenzymes, and hybridized with one of two nucleotide probes. Arestriction map was constructed for the 8.0-kb genomic frag-ment which included the encoding region of the CTS1 geneplus the 59 and 39 flanking regions (Fig. 3A). A restriction mapwas also constructed for the hypothetical form of this fragmentwith pDcts1 integrated into the CTS1 gene (Fig. 3B). Hybrid-

FIG. 2. Results of PCR screening of genomic DNAs of the parental (P)strain and three putative transformant (D10.2, D2, and D3) strains. Two separateprimer pairs were used (G and H or C and D; Fig. 1 and Table 1) to amplifyeither the 1.3-kb parental CTS1 gene or the 1.5-kb 39 incomplete cts1 fragmentplus part of the plasmid construct, respectively. std., standards.



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ization of PstI- or BamHI-digested genomic DNA of the Dcts1strain with a probe derived from the GPD promoter region ofpDcts1 (Pb1) revealed single 4.0- and 6.6-kb bands, respectively(Fig. 3C). The size of each band was predicted by the restric-tion map in Fig. 3B. The Pb1 probe failed to hybridize with thesame restriction enzyme digests of genomic DNA derived fromthe parental strain. These results provide evidence that homol-ogous integration had taken place at a single locus in thechromosomal DNA. The Dcts1 and parental strain DNA prep-arations were also digested with ApaI, XhoI, XbaI, or PstI andsubjected to Southern hybridization to further test if integra-tion had occurred at the homologous CTS1 site and whetherthe selected transformant was homokaryotic (Fig. 3C). South-ern hybridization in this case was performed with a probe(Pb2) derived from the 39 end of the parental CTS1 gene, aregion outside the pDcts1 construct (Fig. 3A and B). The re-sults of Southern hybridization of Pb2 with the Dcts1 (T) andparental (P) DNA digests showed single bands which wereeach of predicted size. These data provide additional evidencethat homologous integration had occurred at a single locus,resulted in disruption of the CTS1 gene, and yielded the ho-mokaryotic Dcts1 transformant.

Specificity of anti-rCTS1 serum and immunoblot analysesof Dcts1 culture filtrate. The 1.2-kb cDNA which encodes partof the CTS1 protein was subcloned into pET28b and expressedby E. coli. As a control, the same E. coli strain was separately

transformed with the pET28b plasmid which lacked the C.immitis gene insert. SDS-PAGE separations of total cytosolicpreparations derived from bacterial cells transformed withpET28b-CTS1 and induced or not induced with IPTG duringgrowth are shown in Fig. 4. A prominent Coomassie brilliantblue-stained band with a molecular size of approximately 49kDa was visible in the cytosolic fraction of induced cells. Theresult of purification of rCTS1 by nickel affinity chromatogra-phy is also shown in Fig. 4. The gel-isolated protein was elec-trotransferred to an Immobilon P membrane, stained, excised,and digested with Lys-C in preparation for amino acid se-quence analysis. The Lys-C digest was then fractionated byhigh-performance liquid chromatography (9), and the selectedpeptide which was purified to homogeneity was subjected toN-terminal sequence analysis. The sequence obtained was(K)FLLTIASPAGPQNY, which exactly matches the reportedsequence of the translated CTS1 gene (amino acids 205 to 219[29]). The SDS-PAGE-electroeluted rCTS1 was used to im-munize mice, and the antiserum was evaluated for specificityby immunoblot analysis (Fig. 4). A single, 49-kDa band wasidentified in the cytosolic fraction of the IPTG-induced bacte-rial transformant. Preimmune mouse serum failed to recognizethe rCTS1 protein (data not shown).

The same rCTS1-specific antiserum was used in immuno-blots to compare expression of the native antigen in reducedSDS-PAGE separations of culture filtrate concentrates derivedfrom the saprobic-phase Dcts1 and parental strains (Fig. 5).The difference between the molecular size of the native anti-gen estimated by SDS-PAGE (i.e., 48 kDa) and the predictedsize of the mature protein encoded by the CTS1 gene (45.5kDa [29]) is probably due to glycosylation of the former. Im-munoblot analysis revealed that the antiserum showed no re-activity with the filtrate preparation of the Dcts1 strain butrecognized a 48-kDa band in the culture filtrate of the parentalstrain. A commercial preparation of the CF-Ag used for sero-diagnosis of patients with coccidioidal infection was also sep-arated by SDS-PAGE and used as a positive control (MeridianDiagnostics). A distinct 48-kDa band was detected by the anti-rCTS1 serum in this preparation (Fig. 5).

Mycelial culture filtrates of the parental and Dcts1 strainswere also separated by SDS-PAGE and subjected to immuno-blot analysis using serum from a patient with confirmed C.

FIG. 3. Restriction map of the 8-kb BamHI genomic fragment which con-tains the CTS1 gene (A), predicted map of the corresponding genomic fragmentafter homologous integration of the pDcts1 plasmid (B), and results of Southernhybridization of genomic DNA using either the Pb1 probe (left) with restrictionenzyme digests of the Dcts1 transformant (T) strain or the Pb2 probe (right) withdigests of both the parental (P) and Dcts1 (T) strains (C). The size of each bandin panel C correlates with the predicted size in panels A and B, which providesevidence for homologous integration. The single bands revealed by hybridizationof both Pb1 and Pb2 with genomic digests of the Dcts1 transformant indicate thatintegration occurred at a single chromosomal locus. Std., standards.

FIG. 4. SDS-PAGE and corresponding immunoblot (Ib.) analysis of expres-sion of C. immitis rCTS1 by E. coli strain BL21 (DE3) transformed with pET28b-CTS1. The 49-kDa recombinant protein was visualized only in lysates derivedfrom transformed, IPTG-induced bacterial cells grown in vitro. The purifiedrCTS1 in lane 4, obtained by nickel affinity column chromatography, was used toimmunize mice for production of antisera. The specificity of the antisera wasconfirmed by immunoblot assay of the lysate of transformed bacteria. Std.,standards.



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immitis infection (Fig. 5). The patient had a CF antibody titerof .1:64. The immunoblot of the filtrate preparation derivedfrom the parental strain suggested the presence of a 48-kDaband, which was confirmed in a separate immunoblot of thesame preparation using the specific anti-rCTS1 murine serum.On the other hand, the patient serum failed to detect a 48-kDaband in the culture filtrate separation of the Dcts1 strain.

Loss of CF-Ag from culture filtrates of the Dcts1 transfor-mant. The ID-CF assay was used to compare the immunore-activities of culture filtrates derived from the parental andDcts1 strains (Fig. 6). Fusion of the precipitin produced by thereference anti-CF serum and CF-Ag reaction with the preci-pitin produced between the wells which contained the refer-ence antiserum and culture filtrate of the parental strain con-firmed the presence of the CF-Ag in the filtrate preparation.The same volume and protein concentration of culture filtratederived from the Dcts1 strain and the parental strain were

added to separate wells of the ID plate (T and P, respectively,in Fig. 6). No precipitin band was formed between the T andreference wells, suggesting loss of the CF-Ag from the trans-formed strain. Saline was added to a single well (S) as a neg-ative control.

Loss of CTS1 chitinase activity by the Dcts1 transformant.Approximately equal amounts of protein derived from 5- and6-day-old culture filtrates of the parental and Dcts1 strainswere separated electrophoretically under nondenaturing con-ditions and subjected to substrate gel analysis (Fig. 7). Fourfluorescent bands, each indicative of chitinase activity, werevisible in the filtrate of the parental strain, with the strongestenzyme activity clearly associated with one of the upper bandsin the 6-day culture filtrate. The culture filtrate of the trans-formant, on the other hand, showed only three fluorescentbands in the substrate gel. The prominent band present in thefiltrate of the parental strain was absent from both the 5-dayand 6-day culture filtrates of the Dcts1 strain. Immunoblotanalysis of the nondenaturing gel separations of the culturefiltrates was conducted using the rCTS1-specific antiserum.The antiserum recognized the prominent band in both the5-day and 6-day filtrates of the parental strain, which identifiesit as the CTS1 chitinase. No bands were recognized by thespecific antiserum in filtrate preparations of the Dcts1 strain.

The uppermost band in the two Dcts1 filtrate preparations inFig. 7 is enhanced compared to the parental strain, while thetwo lower bands showed little difference in intensity in all fourfiltrate preparations. Evidence has been obtained, based onseparate immunoblot analysis, that the uppermost band rep-resents the CTS2 chitinase (unpublished data). The amino acidsequence identity between C. immitis CTS1 and CTS2 is only12.7% (29), suggesting that anti-CTS1 and -CTS2 would notcross-react with the reciprocal chitinases. The two lower bandsin the culture filtrates of the two strains were not recognized byantisera raised against CTS1 or CTS2 and, therefore, mayrepresent an additional chitinase(s).

Mitotic stability of the Dcts1 transformant after animal pas-sage and serial transfer in vitro. Since the Dcts1 strain wasproduced by disruption rather than replacement of the CTS1gene (Fig. 3B), it is possible that the integrated plasmid couldbe excised and result in reversion of the mutant to the parentalgenotype. For this reason, we examined the stability of themutation after animal passage, reisolation, and sequential cul-ture transfer of the Dcts1 strain to GYE agar plates and liquid

FIG. 5. SDS-PAGE and corresponding immunoblot (Ib.) analysis of expres-sion of the native 48-kDa CTS1 in culture filtrates of the Dcts1 transformant andparental (P) strains. The murine antiserum raised against the purified rCTS1recognized the native CTS1 only in the filtrate of the parental strain (left). Acommercial preparation of the CTS1 CF-Ag was included in the immunoblotanalysis as a positive control. Also shown are SDS-PAGE and a correspondingimmunoblot (Ib.) analysis of culture filtrates (parental [P] strain versus the Dcts1transformant) reacted with serum from a CF antibody-positive patient withconfirmed coccidioidal infection (right). The patient antibody (Pt.Ab) recog-nized a 48-kDa band only in the filtrate of the parental strain. Antiserum raisedagainst purified CTS1 (anti-rCTS1) confirmed the presence of native CTS 1 inthe parental strain culture filtrate only.

FIG. 6. Comparison of immunoreactivity by ID of culture filtrates derivedfrom the parental (P) and Dcts1 transformant (T) strains. The reference wellscontained the CF-Ag and goat anti-CF serum. One well contained saline (S) andwas used as a negative control.

FIG. 7. Nonreducing (nr) PAGE separation, substrate (Subst.) gel electro-phoresis, and immunoblot (Ib.) analysis of filtrates derived from 5-day (5d) and6-day (6d) mycelial cultures of the parental (P) strain and the Dcts1 transformant.Two reported chitinases (CTS1 and CTS2 [29]) of C. immitis were identified inthe substrate gel by immunoblot analysis (only CTS1 identification using anti-rCTS1 serum is shown). Note the apparent increased CTS2 activity in the en-zyme assay of the Dcts1 filtrate. Additional low-MW bands in the substrate gelhave not been identified.



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GYE medium, both in the absence of the selective pressure ofhygromycin. Genomic DNA digests of the parental strain andthe serially transferred Dcts1 strain were subjected to Southernhybridization with the Pb2 probe as previously described (Fig.8). As in Fig. 3C, the blot was designed to detect both integra-tion of the pDcts1 construct at the single homologous locus andthe presence of the intact parental CTS1 gene. The sizes of thesingle bands of the Dcts1 transformant and the parental strainwere identical to those reported for the original Dcts1 trans-formant examined in Fig. 3C. No reversion to the wild-typeCTS1 gene was detected.

Comparative analyses of virulence and morphogenesis ofthe parental and Dcts1 strains. Two groups of 10 BALB/c miceeach were challenged by the i.p. route with arthroconidia iso-lated from GYE agar plate cultures of either the parental orthe Dcts1 strain. Survival plots for the two groups of mice areshown in Fig. 9A. The mean survival times of mice infectedwith the parental and mutant strains were 14 and 16.5 days,respectively. These values are not significantly different, basedon a Kaplan-Meier survival analysis, which suggests that theparental and transformant strains demonstrate comparablelevels of virulence. Histological sections of abscesses associatedwith mesenteric tissues from mice infected with each stain werecompared at 12 days after challenge (Fig. 9B and C). Normalspherule segmentation and endosporulation appeared to bepresent in mice infected with either the parental strain (Fig.9B) or the Dcts1 transformant (Fig. 9C).

DISCUSSION

Disruption of genes in fungal pathogens, such as Cryptococ-cus neoformans, Candida albicans, and two close relatives of C.immitis, Histoplasma capsulatum and Blastomyces dermatitidis(25), has proved to be a powerful genetic tool for the identi-fication of virulence factors and potential antifungal drug tar-gets (19). Factors which contribute to the asexual reproductivecapacity of these microbes in vitro and in vivo are relevant toour understanding of the pathogenicity of medically importantfungi. Disruption of a gene that encodes an endochitinase ofSaccharomyces cerevisiae resulted in a defect in separation ofthe daughter from the mother yeast cell at the culmination ofthe budding process (18). The authors have suggested that thechitin binding region of the enzyme functions in localizing it tothe cell wall chitin, where it hydrolyzes chitin fibers that jointhe daughter and mother cells. We have suggested that certainfungal cell wall-associated hydrolases, including chitinases and

b-glucanases, can digest structural components of the chitin-glucan wall of C. immitis and thereby play key roles in themorphogenesis of this systemic pathogen (4, 17). Endosporu-lation in C. immitis is apparently accompanied by digestion ofa network of cross walls (segmentation apparatus) that com-partmentalizes the cytoplasm of the spherule in preparationfor endospore differentiation (5). Chitin is a major componentof the segmentation apparatus (5, 10). A secreted 48-kDachitinase, which has been shown to be an immunodominant,serodiagnostic complement fixation antigen (CF-Ag) of C. im-mitis (14), was suggested to play a pivotal role in the digestionof the segmentation wall during endospore formation (3, 15).The CF-Ag–chitinase has been cloned (CTS1 [29]), and therecombinant protein expressed in E. coli has been crystallized.The latter undertaking was motivated by speculation thatCTS1 is a rational molecular target for a chitinase inhibitor-derived antibiotic (11). The present study was conducted tofurther test these hypotheses.

Gene disruption in medically important fungi has progressedat a considerably slower pace than the development of gene

FIG. 8. Southern hybridization of the Pb2 probe with restriction enzyme-digested genomic DNAs obtained from the parental (P) and Dcts1 (T) strainsafter mouse passage, reisolation, and serial transfer in vitro in the absence ofhygromycin. The sizes of the single bands identified in the Dcts1 strain areidentical to those shown in Fig. 3C, which provides evidence for mitotic stabilityof the single-crossover integration of the pDcts1 construct. Std., standards.

FIG. 9. (A) Survival plot of mice challenged i.p. with either the parentalstrain or the Dcts1 transformant. The mean survival times of the two groups ofmice were not significantly different. (B and C) Histological sections of C.immitis-infected tissue from mice at 12 days post-i.p. challenge with the parentalstrain (B) or the Dcts1 strain (C). Typical segmented spherules (SS) and endos-porulating spherules (ES) are shown in both preparations. Bar, 20 mm.



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knockout strategies in S. cerevisiae (30). In fact, transformationsystems among the human fungal pathogens have so far beendeveloped only for C. albicans, C. neoformans, A. fumigatus,Wangiella dermatitidis, H. capsulatum, B. dermatitidis, and C.immitis (20). With the exception of A. fumigatus (8), transfor-mation of filamentous fungal pathogens by use of a protoplastmethod developed for A. nidulans has not been productive.This has necessitated the use of alternative approaches, includ-ing electroporation, biolistic transformation (20), and DNAtransfer from Agrobacterium to recipient cells (1, 7). There aremajor problems with these methods of transformation of hu-man fungal pathogens. Chromosomal integration of a geneknockout construct at a single site and its homologous recom-bination that typically results in disruption of the targeted geneeither have not yet been demonstrated (1, 39) or are rare (37).However, here a procedure was developed for transformationof C. immitis which was based on one that had been usedsuccessfully to generate mutants of A. fumigatus (28). Proto-plasts of C. immitis were prepared from germinated arthro-conidia, and the uptake of exogenous DNA was promoted inthe presence of PEG-CaCl2. The pAN7.1 plasmid construct thatwas introduced into the recipient cells contained the E. coli-derived hygromycin resistance gene (HPH), whose expressionis under the control of 59 and 39 signal elements of A. nidulans(32). Protoplast regeneration was conducted in an osmoticallystabilized selective medium that contained hygromycin at a con-centration which is known to inhibit the growth of the wild-type strain of C. immitis. Transformation efficiency was 24 hy-gromycin-resistant transformants/mg of transforming DNA. Thisis comparable to the efficiency reported for Aspergillus spp. (8).Approximately 10% of the hygromycin-resistant colonies (i.e.,two to three transformants per microgram of DNA) revealedsingle, chromosomal locus-specific integration of the plasmidconstruct that resulted from homologous recombination.

Our transformation strategy involved disruption of the wild-type CTS1 gene by a single crossover event with a 1.1-kbinternal fragment of CTS1 carried by pAN7.1. We predictedthat the plasmid construct would integrate into the chromo-somal DNA by homologous recombination at the site of theCTS1 gene. We linearized the plasmid construct as shown inFig. 1, based on the assumption that uptake of exogenousDNA across the protoplast membrane would be more efficientin this configuration than in the native, circular plasmid form.In fact, transformation efficiencies in this study using the twoforms of the plasmid construct were not significantly different(data not shown). The result of homologous integration of thepDcts1 plasmid was predicted to be the generation of twoincomplete copies of the CTS1 gene separated by the pAN7.1vector. We further predicted that the two mutated forms of theCTS1 gene would be nonfunctional, since one is truncated andlacks 369 bp at the 39 end of the open reading frame, as well asthe poly(A) tail, while the other lacks the promoter plus 69 bpof the 59 coding region (29). This same transformation strategyhas been used successfully for disruption of genes in A. fumiga-tus (8). However, potential problems associated with this genedisruption approach have been identified, including the possi-bilities that a truncated peptide is still functional and that thegene disruption construct is unstable under nonselective con-ditions. Both problems can be resolved by the use of a genereplacement approach (8) in which a selectable marker is in-serted within a targeted gene and an essential part of thecoding region of that gene is simultaneously deleted (33, 35).We have recently compared the transformation efficiency ofthe single-crossover gene disruption strategy to the gene re-placement approach in knockout experiments with a C. immitisgene that encodes a parasitic cell surface glycoprotein (unpub-

lished data). The transformation efficiency for generation ofmutants which occurred by single-locus, homologous recombi-nation was 2.6-fold higher using the gene replacement methodthan using the gene disruption approach. It is not knownwhether this improvement in transformation efficiency appliesto other genes of C. immitis.

In spite of these potential reservations about the use of thegene disruption strategy, the CTS1 gene was successfully dis-rupted as predicted, loss of gene function was confirmed, andthe mutated gene remained stable after animal passage, reiso-lation, and serial transfer in vitro in the absence of hygromycin.Results of Southern hybridization confirmed the predicted ho-mologous integration of the plasmid construct and provideddefinitive evidence that this diphasic fungus is haploid. Immu-noblot analyses of the mycelial culture filtrate of the putativeDcts1 transformant, using either antibody raised against therecombinant CTS1 protein or serum from a CF antibody-positive patient with a known coccidioidal infection, failed todetect the 48-kDa band. Patient sera also failed to recognizethe CF-Ag in ID assays of the Dcts1 culture filtrate. Examina-tion of chitinase activity present in the mycelial culture filtrateof the Dcts1 strain by substrate gel electrophoresis revealed theabsence of a major enzyme band which was prominent in theparental strain and identified by immunoblot analysis as theCTS1 protein. Finally, Southern hybridization studies of di-gests of genomic DNA isolated from the Dcts1 strain afteranimal passage and reisolation in vitro revealed the same pat-tern of bands as the original, selected Dcts1 transformant. Themutated gene, therefore, appears to be mitotically stable. Onthe basis of these data obtained from genotypic and phenotypicstudies of Dcts1, we argue that use of protoplasts for introduc-tion of exogenous DNA into C. immitis and the gene disruptionmethod for homologous integration of the plasmid constructinto chromosomal DNA are viable approaches to the transfor-mation of this pathogen. This is the first successful targetedgene disruption in C. immitis.

The virulence of the parental and mutant strains were com-pared in our murine model of coccidioidomycosis to determinewhether disruption of the CTS1 gene resulted in loss or signif-icant reduction of the pathogenicity of the fungus. Our resultssuggested that the Dcts1 strain showed no significant change invirulence compared to the parental strain. Histological sec-tions of infected mouse tissues suggested that morphologicalfeatures of spherule formation, endospore differentiation, andendospore release from ruptured spherules were comparableafter challenge with either the parental or Dcts1 strain. Loss ofCTS1 gene function had no apparent effect on endosporula-tion. A possible explanation for the absence of a morphoge-netic phenotype which defines the Dcts1 strain is that one ormore of the other C. immitis chitinases compensate for loss offunction of the CTS1 gene. In fact, substrate gel electrophore-sis suggested that expression of a high-MW chitinase, identi-fied as CTS2 (29) in Fig. 7, was distinctly elevated in themycelial culture filtrate mutant compared to the parentalstrain. The deduced molecular size of the CTS2 chitinase of C.immitis is 91.4 kDa, and it shows 47% amino acid sequencesimilarity to the S. cerevisiae chitinase reported to play a role incell separation (18). In both CTS2 and the yeast chitinase, thededuced catalytic domain lies upstream of an amino acid vari-able region which is adjacent to a serine-threonine-rich do-main. The latter has been suggested to serve as a potential sitefor O-mannosylation in both cases. The Ser-Thr-rich domain isfollowed by a cysteine-rich, high-affinity chitin-binding region(18, 22, 29). The similarity between the structures of the S.cerevisiae and C. immitis CTS2 chitinases suggests that CTS2 playsa similar role in cell separation during the reproductive cycle.



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However, preliminary data from RT-PCR analyses of tran-script levels of CTS2 during spherule-endospore formationsuggested that the gene is constitutively expressed (unpub-lished data). In contrast, CTS1 expression is sharply increasedat the onset of endosporulation. Thus, it is possible that CTS1and CTS2 function synergistically in C. immitis to modify thestructure of the segmentation wall complex of spherules inpreparation for endospore differentiation and release from theparasitic cell. In order to test this hypothesis, our strategy willbe to disrupt the CTS2 gene in the Dcts1 host. A secondselective marker (i.e., sensitivity of C. immitis to phleomycin)will be used together with the E. coli plasmid (pAN8.1) whichcarries the phleomycin resistance gene (31). The results ofthese studies will be the basis of a future report.

ACKNOWLEDGMENTS

We are grateful to Alejandro Nila and Kalpathi R. Seshan for theirtechnical support in performance of the chitinase assays and histolog-ical studies, respectively. We also thank P. F. Lehmann for his sugges-tions during the preparation of the manuscript.

This work was supported by Public Health Service grant AI19149from the National Institute of Allergy and Infectious Diseases.

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Disruption of the Gene Which Encodes a Serodiagnostic Antigen and

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