INFECTION AND IMMUNITY, Dec. 1996, p. 4967–4975 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 64, No. 12
Cloning and Expression of the Complement Fixation Antigen-Chitinase of Coccidioides immitis C. ROGER ZIMMERMANN,* SUZANNE M. JOHNSON, GREGORY W. MARTENS, ANDREW G. WHITE, AND DEMOSTHENES PAPPAGIANIS Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, California 95616 Received 12 July 1996/Returned for modification 22 August 1996/Accepted 5 September 1996
A chitinase had been isolated from the culture filtrates of Coccidioides immitis endosporulating spherules and from hyphae and shown to be the coccidioidal complement fixation (CF) and immunodiffusion-CF antigen. In the present study, we made use of our previously determined amino-terminal (N-terminal) sequence of the CF-chitinase to design degenerate oligonucleotide primers and to amplify and sequence a PCR product that coded for the N-terminal portion of the CF-chitinase. The PCR product was used as a hybridization probe to screen a developing spherule-lZAP cDNA library, and three hybridizing clones were selected. These clones were converted into their pBluescript expression plasmid form in Escherichia coli and induced to express their recombinant proteins. Lysate from only one clone, pCTS 4-2A, yielded an enzymatically functional CF-chitinase and a line of identity with control immunodiffusion-CF-positive antigen. The pCTS 4-2A insert was sequenced and found to contain a deduced open reading frame coding for a 427-amino-acid polypeptide with an approximate molecular weight of 47 kDa. When purified by a chitin adsorption-desorption method, the recombinant protein exhibited virtually identical characteristics to those of the original C. immitis CF-chitinase. Nondenaturing gels of the pCTS 4-2A E. coli lysates and the purified C. immitis and recombinant CF-chitinase revealed proteins that had chitinase activity and similar relative electrophoretic mobilities. The appearance and relative levels of hybridizing RNA from the developing spherules-endospores (SEs) and hyphae correlated with the appearance or presence and level of CF-chitinase enzyme activity found in SEs culture filtrate and in cellular extracts of developing SE and hyphae. Thus, a functional recombinant CF-chitinase antigen was produced in E. coli and was used in serological diagnostic applications. These results also suggest a functional role for this chitinase in SE development and maturation. represented the subunits of the native CF-chitinase. Aminoterminal (N-terminal) amino acid sequencing of the CF-chitinase 48-kDa bands resulted in a single, 18-amino-acid sequence that overlapped with the reported N-terminal sequence of the IDCF antigen (12, 21). The CF-chitinase–IDCF composite sequence showed similarity to those of one fungal and three bacterial chitinases (3, 5, 8, 13, 24). In this study, we describe the cloning, characterization, and expression in Escherichia coli of the CF-chitinase mRNA. We report the use of a recombinant CF-chitinase in conventional CF and IDCF tests. In addition, the mRNA and protein expression of the native CF-chitinase during spherule-endospore (SE) development and in growing hyphae has been characterized. Our CF-chitinase DNA sequence was found to be in agreement with those of Pishko et al. (20) and Yang et al. (25).
The use of the complement fixation (CF) and the corresponding immunodiffusion-complement fixation (IDCF) tests for the diagnosis and prognosis of coccidioidomycosis is well established (reviewed in reference 19). These tests have used crude antigenic preparations, coccidioidins, that contain the reactive CF antigen as well as a multiplicity of other antigens. The characterization and production of purified antigens could enhance the effectiveness of serological tests for coccidioidomycosis. Zimmer and Pappagianis (26) described the Coccidioides immitis Silveira IDCF antigen as a protein with a median molecular weight of 110 kDa that migrated at 48 kDa when fractionated under heated, reduced sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) conditions. Subsequently, we isolated an endogenous chitinase from the culture filtrate of 48-h endosporulating spherules of C. immitis Silveira and showed it to have CF and IDCF activities (11). In its native form, this CF-chitinase from 48-h culture filtrates migrated on nondenaturing PAGE gels as a wide band with an apparent median molecular weight of 110 kDa. When fractionated under heated, reduced SDS-PAGE conditions, only a tight doublet of bands that migrated at an apparent median molecular weight of 48 kDa appeared, and protein blots of these bands retained the ability to react with control IDCFpositive serum. The size of these immunoreactive bands was similar to that of the IDCF-reactive bands observed by Zimmer and Pappagianis (26), and we concluded that these bands
MATERIALS AND METHODS Experimental outline. The methodological strategy for cloning and expressing the CF-chitinase is shown in Fig. 1. Fungal and bacterial strains. The Silveira strain (ATCC 28868) of C. immitis, in both SE and hyphal forms, was used. The E. coli strains that were used are designated below. Amplification of a CF-chitinase gene fragment by PCR. The PCR (22) was executed with the GeneAmp (Perkin-Elmer/Roche Molecular Systems, Branchburg, N.J.) protocols, reagents, and enzymes provided. Genomic DNA was extracted from 24-h developing spherules as previously described (28). C. immitis genomic DNA (0.5 mg) was incubated with the following degenerate primers (4 pmol each): those derived from the combined CF-chitinase N-terminal amino acid sequence (12, 21) sense strand, 59-ATG CC(A,C,G,T) AA(C,T) TA(C,T) TA(C,T) CC-39, and those derived from the presumed bacterial and fungal internal active-site consensus amino acid sequence (3, 5, 8, 12, 13, 24) antisense strand, 59-TA(C,T) TCC CA(A,G) TC(A,G,T) AT(A,G) TC-39. The sequences in parentheses indicate that more than one base was present. The conditions for PCR were 958C for 2 min, 558C for 2 min, and 728C for 2 min. An approximately
* Corresponding author. Phone: (916) 752-7214. Fax: (916) 7528692. Electronic mail address:
[email protected]. 4967
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FIG. 1. Outline of CF-chitinase cDNA cloning and recombinant protein expression. EIA, enzyme immunoassay.
500-bp PCR product was generated, ligated into plasmid pCRII, and transformed into E. coli INVaF9, using the Invitrogen Original TA Cloning kit (Invitrogen, San Diego, Calif.) according to the manufacturer’s instructions. One clone, designated pCTS TA-7, that was judged to contain the entire PCR product was selected. E. coli cultures containing pCTS TA-7 were grown in Luria-Bertani medium (Difco, Detroit, Mich.) with 50 mg of ampicillin (Sigma, St. Louis, Mo.) ml21 overnight at 378C, and plasmid DNA was isolated with the materials and protocols of a Plasmid Midi kit (Qiagen, Chatsworth, Calif.). Both DNA strands of the pCTS TA-7 insert were sequenced with the Sequenase Rapidwell DNA Sequencing kit (Amersham, Arlington Heights, Ill.) according to the manufacturer’s protocols. RNA extraction, gel, and blot. RNA was extracted from first-generation, synchronously developing SE cultures and actively growing hyphae according to the method of Zimmermann and Pappagianis (27). To our knowledge, no mRNA has been reported to date whose level of expression is sufficiently constant to use as a loading control for C. immitis, a problem previously noted for Candida albicans (9). Therefore, mRNA levels were measured relative to the rRNAs (which make up 94 to 98% of the total RNA), by loading approximately equal amounts of total RNA in each lane of the denaturing gel. Preliminary gels were run, stained with ethidium bromide, and examined on a UV transilluminator to ensure that the RNA exhibited sharp, defined bands and that the rRNA bands appeared to have approximately equal ethidium bromide staining intensity. The size of the CF-chitinase transcript was derived from a standard curve prepared by using the lengths of the large and small subunit rRNA bands of C. immitis whose approximate lengths (1.8 and 3.3 kb) had been previously determined by comparison to known RNA length standards (Promega, Madison, Wis.). Ten micrograms of total RNA from each of the spherule development time points and actively growing hyphae were fractionated on denaturing 0.6 M formaldehyde– 0.7% agarose gels, stained with 0.5 mg of ethidium bromide ml21, destained, photographed, and blotted onto nitrocellulose. The blots were hybridized to 32 P-labeled RNA probes that were synthesized with T7 RNA polymerase, using the DNA of clone pCTS 4-2A as the template and the materials and protocols provided by a Riboprobe Transcription System kit (Promega). Hybridization of the probe to the blots was at 558C for 16 h in 50% formamide–0.8 M NaCl–1 mM EDTA–50 mM sodium phosphate buffer (pH 6.8)–50 mg of poly(rA) RNA per ml–100 mg of sheared, single-stranded herring testes DNA per ml and 0.2% of bovine serum albumin–Ficoll type 400–polyvinylpyrrolidone type 40 (103 Den-
INFECT. IMMUN. hardt’s solution) (all from Sigma). Following hybridization, the blots were washed in 23 SSC (13 SSC 5 0.15 M NaCl, 0.015 M sodium citrate z 3H2O, pH 7.0) at room temperature (RT), in 50 mM NaCl–20 mM sodium phosphate buffer (pH 6.8)–1 mM EDTA–0.1% SDS at 688C until the wash buffer was less than two times background counts per minute, and in 23 SSC at RT. Blots were allowed to dry at RT and autoradiographed with XAR-5 X-ray film (Kodak) at 2708C with intensifying screens (Kodak). cDNA library construction and screening. RNA was extracted from 24-h developing spherules, and poly(A)1 RNA was isolated by using materials from a PolyATract mRNA Isolation System kit (Promega) according to the manufacturer’s protocol. The poly(A)1 RNA was used as the template for a cDNA library constructed in lZAP, using the Uni-ZAP XR Cloning kit (Stratagene, La Jolla, Calif.) according to the manufacturer’s instructions. The library was screened by using a modification of the method of Benton and Davis (2), and the 32 P-labeled PCR product was used as a selection probe. The probe was synthesized with GeneAmp (Perkin-Elmer/Roche Molecular Systems) protocols, reagents, and enzymes by incubating about 50 ng of the gel-purified PCR product DNA with 4 pmol each of the following primers: those derived from the nucleotide sequence of the 59 end of the CF-chitinase 511-bp PCR product sense strand, 59-ATG CCC AAT TAT TAT CCA GTC-39, and those derived from the nucleotide sequence of the 39 end of the CF-chitinase 511-bp PCR product antisense strand (starting 5 bases from the end of the amplimer), 59-CCA ATC GAT GTC AAT CCC ATC-39. The conditions for PCR were the same as those noted above. Hybridization of the probe to the filters was at 688C for 16 h in 1.0 M NaCl–50 mM Tris-HCl (pH 7.6)–103 Denhardt’s solution–10 mM EDTA– 0.1% SDS–and 100 mg of sheared, single-stranded herring testes DNA per ml. Following hybridization, the blots were washed in 23 SSC at RT, in a series of washes as follows: 23 SSC, 13 SSC, and 0.53 SSC plus 0.1% Na4P2O7 z 10H2O– 0.1% SDS at 688C and in 23 SSC at RT. The blots were dried at RT and autoradiographed as described above. Positively hybridizing l clones were plaque purified by rescreening several times, and the clones were converted to their pBluescript SK(2) plasmid form in E. coli XL1-Blue by in vivo excision according to the manufacturer’s protocol (Stratagene). Cultures of E. coli XL1Blue containing the plasmid clones and the plasmid vector alone, grown in Luria-Bertani medium with 50 mg of ampicillin ml21, were induced with 1.0 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG). Clones producing lysates with an IDCF-positive and enzymatically active CF-chitinase were selected as outlined below. ID plates. The ID tests were carried out in 50-mm-diameter plastic petri dishes as described by Huppert and Bailey (10) with modification (19), including substitution of the gellan gum Gelrite (Schweizerhall, South Plainfield, N.J.) for agarose. Antibody-containing human control sera were used with coccidioidin as the reference antigen. Twenty-seven micrograms total protein of the control antigens and 1 to 2 mg total protein of the E. coli-derived preparations or 15 mg total protein of C. immitis culture filtrates was added per well. CF. The clone and vector-only lysates were tested as CF antigens by the method of C. E. Smith (reviewed in reference 19). Isolation of chitinase. The native C. immitis CF-chitinase was isolated from SE-phase culture filtrates and cells. Culture filtrates from various time points were collected by centrifugation and filtration through a 0.2-mm-pore-size cellulose acetate filter (Nalgene, Rochester, N.Y.). Phenylmethylsulfonyl fluoride (Boehringer Mannheim, Indianapolis, Ind.) and thimerosal were added to 1.0 mM and 0.01%, respectively. The culture filtrates were concentrated 200-fold in a stirred pressure cell on ice with a Diaflo YM-10 molecular membrane (exclusion limit, 10 kDa; Amicon, Danvers, Mass.). Developing spherules, from time points bracketing the appearance of CF-chitinase in the culture filtrates, were collected by centrifugation as described above, fixed in 0.5% formaldehyde, and suspended in 50 mM potassium phosphate buffer, pH 6.3, containing 0.01% thimerosal. Phenylmethylsulfonyl fluoride to 1 mM and an equal volume of sterile 100- to 300-mm-diameter glass beads were added, and the spherules were broken by vortexing at high speed with intervals of cooling on ice. Cell debris and glass beads were removed by centrifugation, and the supernatant was filtered through a 0.2-mm-pore-size filter. The supernatant was concentrated as described above. The protein concentration was estimated according to the method of Lowry et al. (16). The recombinant CF-chitinase was produced in, and extracted from, whole E. coli cells by an osmotic shock lysis procedure (15). The supernatant was filtered through a 0.2-mm-pore-size filter and concentrated 100-fold as described above. The protein concentration was estimated according to the method of Lowry et al. (16). Heat lability and susceptibility to digestion by pronase were determined as previously described (11, 26). pCTS 4-2A DNA isolation and sequencing. The cDNA insert of one clone, pCTS 4-2A, that produced both IDCF and chitinase activity was sequenced. Plasmid DNA was isolated and purified from cultures of E. coli grown in LuriaBertani broth as outlined above, using the materials and protocols of a Qiagen Plasmid Midi kit as outlined above. Both strands of the cDNA insert were sequenced with the same kits and methods used to sequence the PCR product described above, with the addition of sequence-derived, primer-walking sequencing primers replacing the primers supplied with the kit as needed. Chitinase affinity purification. Affinity purification of chitinase was carried out by an adsorption-desorption method previously described (11). The supernatant
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FIG. 2. CF-chitinase PCR amplimer clone pCTS TA-7 DNA sequence and deduced amino acid sequence. Amino acid positions are labeled down the left side, and nucleotide positions are labeled down the right side. Each plus sign in the broken line indicates a group of 10 nucleotides. Nucleotides that code for amino acids in the presumed ORF are capitalized. The nucleotide sequence of the putative intron is in lowercase letters. Thirty of 35 amino acids of the combined amino-terminal amino acid sequence determined for the CF-chitinase are underlined. The exact sequence of nucleotides from C. immitis that were complementary to the degenerate PCR primers are underlined. The italicized, nonunderlined amino acids are those deduced amino acids that differ from the reported combined CF-chitinase sequence (12). The boldface amino acid Q at position 149 differs from the equivalent amino acid in clone pCTS 4-2A.
was filtered with a 0.2-mm-pore-size syringe filter and concentrated as described above. The protein concentration was estimated according to the method of Lowry et al. (16). CF-chitinase enzyme activity. CF-chitinase enzymatic activity was detected by measuring the fluorescence produced by the release of 4-methylumbelliferone (4-MU) from the conjugated glycoside 4-MU-b-D-N,N9-diacetylchitotriose (4MU-GlcNAc3) (Sigma) (1, 11). When used to detect the presence of chitinase activity in E. coli cell lysates and on ID plates, the substrate was added to microtiter plate wells containing the chitinase preparations or to washed, phosphate buffer-equilibrated ID plates containing precipitated antigen-antibody complexes. A buffer-substrate-only control was similarly prepared. Both types of plates were incubated at 378C for 10 min and illuminated with a 300-nm UV transilluminator (Fotodyne, Hartland, Wis.), and the appearance and relative amounts of blue fluorescence produced by the lysates, controls, and precipitates were estimated visually. To characterize the chitinase enzyme specific activity, triplicate reaction mixtures containing the substrate, phosphate buffer, and CF-chitinase enzyme preparations were mixed together in a final volume of 400 ml (the enzyme being added to the prewarmed buffer-substrate mixtures last), and the mixture was incubated at 378C. Triplicate buffer-substrate-only controls were similarly prepared and incubated. The reactions were stopped by the addition of 600 ml of 0.5 M glycine, pH 10.4, and three 250-ml portions of each reaction mixture and the buffer-substrate-only control were placed into wells of a 96-well, flat-bottom, untreated microtiter plate (Costar, Cambridge, Mass.). Fluorescence was measured with a Millipore Cytofluor 2300 microtiter plate fluorometer, and the net fluorescence was determined in triplicate by subtracting the mean of three buffer-substrate-only control wells from the mean of each set of three reaction wells. Nondenaturing PAGE. Proteins in their native state were fractionated by PAGE as previously described (11). Approximately 20 mg of each E. coli cell lysate from clone pCTS 4-2A (uninduced and induced) and from pBluescript (uninduced and induced), 30 mg of chitin affinity-purified CF-chitinase from induced clone pCTS 4-2A, and 15 mg of chitin affinity-purified CF-chitinase from C. immitis were fractionated in a 12% gel. To demonstrate chitinase activity, the gel was equilibrated in 50 mM potassium phosphate buffer, pH 6.3, for 5 min at RT. The buffer was removed, enough 4-MU-GlcNAc3 substrate solution was added to cover the gel, and the gel was incubated for 10 min at 378C. The gel was illuminated on a 300-nm UV transilluminator and photographed through a Wratten no. 3 filter (Kodak). The gel was then stained with Coomassie blue (11), destained, and photographed through a Wratten no. 15 filter (Kodak). Microscopy. SE-phase cells at various stages of development were stained with Calcofluor White, using a modification of the Maeda and Ishida procedure (17). Cells were killed with 0.2% formaldehyde, washed twice with phosphate-buffered saline (PBS), and collected by centrifugation. The cell pellet was suspended in PBS plus 300 mg of Calcofluor White per ml, allowed to incubate for 5 min at RT, washed twice with PBS, and suspended in PBS. Wet mounts of the cells were examined and photographed with a Zeiss Universal microscope set up to detect epifluorescence, with excitation at 365 nm and the bandpass set at 420 nm. All micrographs were taken with the same 3400 magnification and film exposure settings. Nucleotide sequence accession numbers. The pCTS TA-7 and pCTS 4-2A insert DNA sequences were deposited in GenBank under the accession numbers U60806 and U60807, respectively.
RESULTS Amplification of a CF-chitinase gene fragment by PCR. A single, approximately 500-bp DNA PCR product appeared on ethidium bromide-stained agarose gels (data not shown), and this band was excised, cloned, and sequenced (Fig. 2). The plasmid pCTS TA-7 had a 511-bp insert with two, discontinuous, deduced open reading frames (ORFs), 25 and 124 amino acids long, respectively, that used the same reading frame. These ORFs were separated by a presumptive mRNA precursor intron of 61 bp that contained 59, internal, and 39 splice signal sequences typical of introns of filamentous fungi and other eukaryotes (4, 7). Hypothetically, the excision of this intron would result in a contiguous ORF of 149 amino acids, placing the first 30 amino acids adjacent to one another in the same order as the equivalent amino acid residues of the reported composite CF-chitinase (12, 21). When aligned to achieve the best fit, there was a 90% sequence homology (27 of 30 amino acids) between similarly positioned amino acids of the deduced sequence of the PCR product and the reported sequence of the composite CF-chitinase (12, 21). The amino acid differences were at amino acid positions 9, 18, and 25. Despite these differences, the PCR product was judged to have enough homology to be used as a hybridization probe to select CF-chitinase cDNA clones, and exactly matching, complementary sequence PCR primers for the 59 sense and 39 antisense ends of the PCR product were constructed. Library screening and clone selection. Approximately 2 3 105 clones from a 24-h developing spherule lZAP cDNA library were screened. Six clones that produced positive signals were chosen for rescreening and plaque purification. After three rounds of rescreening at progressively lower plaque densities, three plaque-purified, positively hybridizing l clones remained. These clones were converted into the pBluescript SK(2) plasmid form and used to transform E. coli XL1-Blue. To select a clone that produced a functional CF-chitinase antigen, the three plasmid clones along with the E. coli XL1Blue transformed with pBluescript plasmid alone were grown in liquid culture and induced with IPTG to express any b-galactosidase or b-galactosidase-linked fusion protein DNA coding sequence present. As shown in Fig. 3, only the extract from clone pCTS 4-2A produced a line of identity with the unheated, control IDCF-positive antigen. As previously noted for the native coccidioidal CF-chitinase (11), the induced lysate
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FIG. 3. ID reactivity of total whole-cell extracts from IPTG-induced E. coli containing recombinant plasmids and plasmid vector alone. Well 1, control IDCF-positive serum; well 2, unheated, control IDCF-positive antigen (total protein, 27 mg per well); well 3, clone pCTS 4-2A (total protein, 1 to 2 mg per well); well 4, clone pCTS 1-1A (total protein, 1 to 2 mg per well); well 5, unheated, control IDCF-positive antigen (total protein, 27 mg per well); well 6, pBluescript; well 7, clone pCTS 9-2A (total protein, 1 to 2 mg per well).
from clone pCTS 4-2A was the only extract that produced the characteristic blue fluorescence following the addition of the 4-MU-GlcNAc3 substrate (data not shown). Serological activity. Additional ID plate assays of clone
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pCTS 4-2A E. coli lysate produced a precipitated line of identity with the IDCF reference antigen but did not produce a line corresponding to the immunodiffusion-tube precipitin (IDTP) reference antigen (Fig. 4A). As previously noted for the coccidioidal chitinase (11), this same IDCF antigen-antibody precipitate also showed the characteristic blue fluorescence following the addition of the 4-MU-GlcNAc3 substrate, and no fluorescence was associated with the IDTP line (data not shown). The chitin affinity-purified recombinant CF-chitinase gave similar ID results (Fig. 4B). As with the CF-chitinase produced by C. immitis (11, 26), enzymatic activity was destroyed when the recombinant CF-chitinase was heated for 30 min at 568C, when it was heated for 4 min at 1008C, or when it was treated with pronase. Similarly, the recombinant CF-chitinase lost IDCF activity after heating for 30 min at 568C. Both the total pCTS 4-2A lysate and chitin affinity-purified recombinant CF-chitinase demonstrated satisfactory activity as the antigen in the CF tests with human sera known to be positive for coccidioidal antibody by CF and ID. pCTS 4-2A DNA sequence analysis. Clone pCTS 4-2A plasmid DNA was isolated and sequenced (Fig. 5). The pCTS 4-2A clone contained a cDNA insert of 1,637 bp including a 29-bp 39 poly(A) tail. There was a single, contiguous deduced ORF of 427 amino acids that was calculated to code for an approximately 47-kDa polypeptide, which was similar to the measured value of the reported CF-chitinase obtained by denaturing PAGE. At the 59 end of the DNA sequence there was a 26-bp untranslated leader region whose bases were in the same translation frame alignment as those of the ORF. Taken together, the DNA sequence contributed by the vector’s b-galactosidase N terminus and the cloned insert could code for a deduced approximately 54-kDa fusion protein. The ATG at nucleotides 27 to 29, which coded for the first and presumably initiating methionine, appeared to be in the correct sequence context for a filamentous fungus consensus translation start site (7, 14). The ORF contained a contiguous deduced 35-amino-acid se-
FIG. 4. (A) ID reactivity of total whole-cell lysates from IPTG-induced E. coli containing pCTS 4-2A (total protein, 1 to 2 mg per well). (B) ID reactivity of chitin affinity-purified CF-chitinase from clone pCTS 4-2A (total protein, 1 to 2 mg per well). Wells 1, lysate or purified CF-chitinase; wells 2, unheated, control IDCF-positive antigen (total protein, 27 mg per well); wells 3, control IDCF-positive serum; wells 4 and 5, unheated, control IDCF-positive antigen (total protein, 27 mg per well); wells 6, control IDTP-positive serum; wells 7, heated, control IDTP-positive antigen (total protein, 27 mg per well).
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FIG. 5. CF-chitinase cDNA clone pCTS 4-2A DNA sequence and deduced amino acid sequence. Amino acid positions are labeled down the left side, and nucleotide positions are labeled down the right side. Nucleotides that code for amino acids in the presumed ORF are capitalized. The nucleotide sequences of the 59 untranslated leader region and 39 UTR are in lowercase letters. The contiguous 34 amino acids of the combined amino-terminal sequence reported for the CF-chitinase are underlined. The missing serine of the reported amino acid sequence is indicated by a nonunderlined, boldfaced, larger-case S. The sequence of nucleotides from C. immitis that were complementary to the PCR primers used to synthesize the labeled PCR hybridization probe are underlined. The three amino acids defining a possible N glycosylation site are overlined. The italicized, nonunderlined amino acids are those deduced amino acids that differ from the reported CF-chitinase sequence. The boldfaced amino acids and overlined nucleotides at nucleotide positions 102 to 103 and 126 to 127 are those that differ from the ones reported for CTS1 by Pishko et al. (26) and CF/chitinase by Yang et al. (32), respectively.
quence that was homologous to the reported combined amino acid sequence of the CF-chitinase (12, 21). An amino acid, at position 21, that previously had not been resolved during amino acid sequencing of the CF-chitinase, was deduced to be a serine residue. Along with the three amino acid differences shared with the CF-chitinase PCR product, one additional difference between the reported and deduced amino acid sequences of the PCR product and full cDNA insert was found at amino acid number 19. A presumptive consensus signal sequence peptide (7, 23), which included a core of hydrophobic amino acids, was found in the first 17 amino acids of the ORF. Cleavage of the peptide bond between amino acids 17 and 18 would account for the generation of the N terminus of the reported purified CF-chitinase amino acid sequence (12) and yield a secreted protein of approximately 45 kDa. Asparagine (N) residues in the amino acid sequence N-X-S or T are usually N glycosylated (18), and one such site at amino acid positions 386 to 388 was found. After the first deduced translation
termination codon triplet, there was a 300-bp untranslated region (UTR), not including the poly(A) tail. The consensus polyadenylation signal AATAAA, found in most higher eukaryotic mRNAs, was not found in the 39 UTR. However, as reported for other filamentous fungal genes (7), the 39 UTR was rich in T and A, resulting in a number of short sequence motifs that could supply the polyadenylation signal. A consensus transcription termination signal described for several other fungal genes (7) was not found near the poly(A) site in the 39 UTR. DNA and deduced ORF amino acid sequence comparison between the CF-chitinase cDNA clone and the reported CTS1 gene clone (20) and the CF/chitinase clone of Yang et al. (25) revealed that there was one nucleotide difference that resulted in an amino acid change between our CF-chitinase and each of the other two reported sequences. Our CF-chitinase contained an aliphatic serine at position 26 in place of an aromatic tyrosine found in CTS1 (20). The CF-chitinase also contained an
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TABLE 1. Temporal appearance of CF-chitinase IDCF and chitinase enzymatic activity in SE-phase culture filtrates of C. immitis Age (h)
IDCFa
Relative chitinase enzymatic activityb
24 28 32 36 40 48 52 56
No No No Yes Yes Yes Yes Yes
2 2 1 11 1111 11 11 11
Tested at a concentration of 15 mg of protein per well. Determined fluorometrically, on the basis of detection of 4-MU released per milligram of protein present. The lower limit of detection was ,4.4 3 1025 mmol of 4-MU. The detected chitinase activity ranged from 0.99 3 1023 to 3.1 3 1022. a b
alanine at position 34 instead of the glycine found in the CF/chitinase clone of Yang et al. (25). In addition, there was one other nucleotide difference between the CTS1 and our cDNA sequences of the ORF that did not result in an amino acid change. There were also some differences found in the nucleotide sequence of the 39 UTR and 59 untranslated leader region between the CF-chitinase and the two other reported sequences. Temporal appearance of chitinase. CF-chitinase was not detected in the culture filtrates by the IDCF test until 36 h after the cultures had been inoculated (Table 1). However, CFchitinase enzymatic activity was detected earlier in the culture filtrates at 32 h of development (Table 1). The total CF-chitinase activity increased between 32 and 40 h. CF-chitinase activity then decreased after 40 h. Similar enzymatic analysis of aqueous extracts of mechanically disrupted developing spherules showed little or no CF-chitinase activity before 32 h of development (Table 2). Nondenaturing PAGE. When the gel was incubated in the 4-MU-GlcNAc3 solution, fluorescent bands of similar mobility appeared in the lanes that contained the C. immitis and recombinant chitin affinity-purified CF-chitinase (Fig. 6). However, the recombinant band migrated slightly ahead of and was less diffuse than the C. immitis band. Fluorescent bands with the same mobility as the chitin affinity-purified recombinant CF-chitinase also appeared in lanes containing the clone pCTS 4-2A uninduced and induced E. coli lysates. No fluorescent bands appeared in the vector pBluescript lanes. Coomassie blue staining demonstrated a single band for the chitin affinitypurified CF-chitinase from C. immitis. The lane containing the chitin affinity-purified recombinant CF-chitinase had one prominent stained band with an additional faint, more rapidly migrating component, while multiple protein bands were evident in the other preparations from E. coli. RNA blot. As shown in Fig. 7, a strongly hybridizing band corresponding to an RNA of approximately 1.7 kb was ob-
FIG. 6. Nondenaturing PAGE gel. (A) Coomassie blue stained; (B) 4-MUGlcNAc3 treated. Lanes 1, induced pBluescript lysate; lanes 2, uninduced pBluescript lysate; lanes 3, induced clone pCTS 4-2A lysate; lanes 4, uninduced clone, pCTS 4-2A lysate; lanes 5, chitin affinity-purified fraction clone pCTS 4-2A lysate; lanes 6, chitin affinity-purified fraction C. immitis culture filtrate.
served in the 36-h SE-phase RNA lane. A relatively weak band of approximately 1.6 kb was detected in the hyphal RNA lane. A faint band of approximately 1.3 kb was noted in each of the lanes containing RNA from 6- and 17-h SE-phase cells. Microscopy. In order to characterize the presence and location of chitin present in C. immitis cell walls during SE development using our in vitro culture system, cells sampled at time intervals during the entire SE cycle were stained with the fluorescent chitin-specific staining agent Calcofluor White. As shown in Fig. 8, at 6 h after inoculation, the enlarging endospores had little chitin in the cell wall. By 24 h, the enlarged endospores had some fluorescence in the walls. At 28 h, the immature spherule wall was uniformly fluorescent and the newly formed endospore segmentation walls were highly fluorescent. The spherule wall was no longer fluorescent at 32 h, while the endospore crosswalls remained intensely fluorescent. After 32 h, the spherules began to rupture, and by 48 h, only clumps of endospores remained. These new endospores exhibited a fluorescence almost as low as that observed at 6 h.
TABLE 2. Chitinase activity in cell extracts of SE-phase C. immitis Agea (h)
Sp act (1023) (IUb/mg)
SD (1023) (IUb/mg)
24 28 32
None detected 0.055 0.314
60.052 60.026
a b
Extracts were prepared from an equal packed cell volume. 1 IU 5 1 mmol of 4-MU released min21 from triplicate determinations.
FIG. 7. Total RNA gel and blot. Ten micrograms of gel-fractionated total RNA from each morphological form or developmental time point were blotted, hybridized to a 32P-labeled RNA probe spanning the entire insert of pCTS 4-2A, and autoradiographed. Calibrated C. immitis rRNA subunit molecular-length standards are depicted on the left. (A) Ethidium bromide-stained gel before blotting. (B) Autoradiograph of blot after hybridization. Lanes 1, hyphae; lanes 2, 7-day-old endospores; lanes 3, 6-h SE phase; lanes 4, 17-h SE phase; lanes 5, 24-h SE phase; lanes 6, 36-h SE phase.
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FIG. 8. Fluorescent photomicrographs of representative Calcofluor White-stained SE-phase cells at intervals after inoculation. All photomicrographs were taken at a magnification of 3400, using the same film exposure time.
DISCUSSION Only three positively hybridizing clones remained after the screening was completed, and the clones were judged to be plaque pure. Of these three, only one clone that produced an enzymatically functional, IDCF-reactive recombinant CF-chitinase was found. The small number of clones detected in the initial screening of the library may have been due to a combination of the relatively small total number (about 2 3 105) of clones screened and the low abundance, as judged from the RNA blot analysis, of CF-chitinase mRNA present in the poly(A)1 RNA used to construct the l library. More sensitive methods of detection, such as quantitative, reverse transcriptase PCR, may be able to detect the small amount of CFchitinase mRNA that must be present in the 24-h spherule RNA. The cloned PCR product DNA sequence revealed the presence of a presumptive intron near the 59 end of the clone insert whose location was in agreement with the sequence of Pishko et al. (20). There were three differences in amino acid residues between the deduced amino acid sequence of the PCR product and the native CF-chitinase N-terminal amino acid sequence. These differences could be accounted for by the method used to derive the reported amino acid sequence of the CF-chitinase. When sequencing is carried out on protein blot membranes, the identity of some amino acid residues may be equivocal. For example, we were never able to identify an unambiguous
amino acid residue in position 4 of the reported CF-chitinase amino acid sequence (12). The DNA and deduced amino acid sequences of our cDNA clone were found to be almost identical to the sequences of the CTS1 gene characterized by Pishko et al. (20) and CF antigen cDNA clone isolated by Yang et al. (25). It was reasonable to expect that the CTS1 gene would be similar, because the method and hybridization probes used in the clone isolation were similar. That a cDNA clone with an almost identical sequence was isolated using the method and immunological reagents of Yang et al. (25) provides additional validation. There was one amino acid difference (with an accompanying single nucleotide change in the DNA sequence) between our CF-chitinase and each of the other two reported sequences. In addition, there was one other nucleotide difference between the CTS1 and CF-chitinase DNA sequences of the ORF that did not result in an amino acid change. The amino acid difference in the ORF between our CF-chitinase and CTS1 is the substitution of an aliphatic serine in the former for an aromatic tyrosine in the latter. The consequences of this change are unknown, since the enzymatic and immunological characteristics of the CTS1 protein have not been reported. Since the CTS1 gene was isolated from a C. immitis isolate designated C735, the differences between the CF-chitinase and CTS1 sequences may be due to differences between the two strains. The amino acid difference between our CF-chitinase and the
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CF/chitinase clone of Yang et al. (25), an alanine in the former for a glycine in the latter, does not appear to change the immunological or enzymatic properties of the protein (25). There were also some differences found in the nucleotide sequence of the 39 UTR and 59 untranslated leader region between our CF-chitinase and the two other sequences (20, 25). These differences may not be important, because they do not influence the sequence or translation frame of the ORF and do not occur in or at known sequence motifs important for mRNA transcription and translation (4, 7). The 39 UTR trailer sequences of the filamentous fungi are usually short but show considerable variation (from 5 to 408 nucleotides) (7). The 300-bp length found for the 39 UTR trailer of our CF-chitinase clone falls within this range. Although only a handful of C. immitis cDNA clones have been totally sequenced, there is presently a tendency toward long 39 UTRs. A recently reported cDNA clone coding for a potentially immunogenic protein had a 39 UTR of 468 nucleotides (6), and our laboratory has sequenced a cDNA clone coding for a chitin synthase that has a 506-nucleotide 39 UTR (unpublished results). It is not known whether these long 39 UTRs have a specific function. On ID plates with sera from patients with coccidioidomycosis, our recombinant CF-chitinase generated in E. coli produced a line of identity with the coccidioidal IDCF antigen control. It also had chitinase activity in the antibody-antigen precipitates on ID plates, as had been reported for the CFchitinase isolated from C. immitis SE-phase culture filtrates (11). The persistence of enzymatic activity in the antigen-antibody precipitate may indicate that the epitopes for antibody reaction are distinct from the enzymatically active site(s). The recombinant protein also served as a CF antigen reactive with sera from humans with coccidioidomycosis. The recombinant chitinase enzymatic activity was associated with a protein found to have electrophoretic mobility on nondenaturing gels slightly faster than that of the native C. immitis CF-chitinase which had migrated on nondenaturing PAGE gels at an apparent median molecular weight of 110 kDa (11, 26). The calculated size of the recombinant fusion protein was approximately 54 kDa. This included contributions from the vector and the insert of approximately 4.5 and 49.5 kDa, respectively. In view of the similarity of the mobilities of the enzymatically active recombinant protein and the native C. immitis CF-chitinase, it appears possible that the active recombinant product is assembled from subunits represented in the 54-kDa fusion protein. When RNA blot analysis and enzymatic activity were compared, the results showed that the appearance and relative levels of hybridizing RNA from the developing SEs and hyphae correlated well with the appearance or presence and level of chitinase enzyme activity found in SE culture medium and in extracts of developing SEs and hyphae. These results suggest that the appearance of the CF-chitinase is developmentally regulated. This regulation may enable the timed production of mRNA and, thus, the appearance of the active enzyme at the appropriate time of spherule development, resulting in the release of the new endospores from the mature spherule. The blot of hyphal RNA showed a weakly hybridizing band that appeared to migrate slightly faster than the band in the 36-h SE-phase lane. This difference may be the result of a technical artifact or may represent a real difference in the size of the CF-chitinase mRNA in hyphae. Alternatively, the weakly hybridizing band may be the result of hybridization to the smaller transcript of another chitinase gene. The discovery of an additional, unrelated chitinase gene (CTS2) in C. immitis by Pishko et al. (20) raises the possibility of multiple chitinase
INFECT. IMMUN.
genes that are differentially regulated in the hyphal and SE forms of the fungus. In the course of coccidioidal disease, the titer of the antibody to the CF-chitinase rises with increasing extent of tissue involvement. Whether the chitinase represents an incidental inducer of antibody to the CF-chitinase alone or whether the CF-chitinase complexed with antibody contributes to pathogenesis can be studied with the availability of the recombinant CF-chitinase. For example, it has been demonstrated that, in a fibrin matrix, a CF antigen-antibody precipitate forms (19), and this could represent a chemotactic complex to which inflammatory leukocytes are attracted. Our results demonstrate that a functional CF antigen can be produced in E. coli and used in serological diagnostic applications. These results also suggest a functional role for this chitinase in SE development and maturation. Future studies involving genetic disruption experiments will provide more substantial evidence for the role of this chitinase in morphogenesis. REFERENCES 1. Barth, M. G. M., and P. D. Bridge. 1989. 4-Methylumbelliferyl substituted compounds as fluorogenic substrates for fungal extracellular enzymes. Lett. Appl. Microbiol. 9:177–179. 2. Benton, W. D., and R. W. Davis. 1977. Screening lambda gt recombinant clones by hybridization to single plaques in situ. Science 196:180–182. 3. Blaiseau, P.-L., and J.-F. LaFay. 1992. Primary structure of a chitinaseencoding gene (chi1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases. Gene 120:243–248. 4. Breathnach, R., C. Benoist, K. O’Hare, F. Gannon, and P. Chambon. 1978. Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc. Natl. Acad. Sci. USA 75:4853– 4857. 5. Cabib, E. 1988. Chitinase from Serratia marcescens. Methods Enzymol. 161: 460–462. 6. Dugger, K. O., K. M. Villareal, A. Nguyen, C. R. Zimmermann, J. H. Law, and J. N. Galgiani. 1996. Cloning and sequence analysis of the cDNA for a protein from Coccidioides immitis with immunogenic potential. Biochem. Biophys. Res. Commun. 218:485–489. 7. Gurr, S. J., S. E. Unkles, and J. R. Kinghorn. 1987. The structure and organization of nuclear genes of filamentous fungi, p. 93–139. In J. R. Kinghorn (ed.), Gene structure in eukaryotic microbes. IRL Press, Oxford, United Kingdom. 8. Harpster, M. H., and P. Dunsmuir. 1989. Nucleotide sequence of the chitinase B gene of Serratia marcescens QMB 1466. Nucleic Acids Res. 17:5395. 9. Hube, B., M. Monod, D. A. Schofield, A. J. P. Brown, and N. A. R. Gow. 1994. Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol. Microbiol. 14:87–99. 10. Huppert, M., and J. W. Bailey. 1965. The use of immunodiffusion tests in coccidioidomycosis. I. The accuracy and reproducibility of the immunodiffusion test which correlates with complement fixation. Am. J. Clin. Pathol. 44:364–368. 11. Johnson, S. M., and D. Pappagianis. 1992. The coccidioidal complement fixation and immunodiffusion-complement fixation antigen is a chitinase. Infect. Immun. 60:2588–2592. 12. Johnson, S. M., C. R. Zimmermann, and D. Pappagianis. 1993. Aminoterminal sequence analysis of the Coccidioides immitis chitinase/immunodiffusion-complement fixation protein. Infect. Immun. 61:3090–3092. 13. Jones, J. D. G., K. L. Grady, T. V. Suslow, and J. Bedbrook. 1986. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. EMBO J. 5:467–473. 14. Kozak, M. 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292. 15. LaVallie, E. R., E. A. DiBlasio, S. Kovacic, K. L. Grant, P. F. Schendel, and J. M. McCoy. 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formulation in the E. coli cytoplasm. Biotechnology 11:187–193. 16. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275. 17. Maeda, H., and N. Ishida. 1967. Specificity of binding of hexopytanosyl polysaccharides with fluorescent brightener. Biochemistry 62:276–278. 18. Marshall, R. D. 1972. Glycoproteins. Annu. Rev. Biochem. 41:673–702. 19. Pappagianis, D., and B. L. Zimmer. 1990. Serology of coccidioidomycosis. Clin. Microbiol. Rev. 3:247–268. 20. Pishko, E. J., T. N. Kirkland, and G. T. Cole. 1995. Isolation and characterization of two chitinase-encoding genes (cts1, cts2) from the fungus Coccid-
VOL. 64, 1996 ioides immitis. Gene 167:173–177. 21. Resnick, S., B. Zimmer, D. Pappagianis, A. Eakin, and J. McKerrow. 1990. Purification and amino-terminal sequence analysis of the complement-fixing and precipitin antigens from Coccidioides immitis. J. Clin. Microbiol. 28:385– 388. 22. Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim. 1988. Enzymatic amplification of b-globin genomic sequences and restriction site analysis for the diagnosis of sickle-cell anemia. Science 230:1350–1354. 23. von Heijne, G. 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14:4683–4690. 24. Watanabe, T., K. Suzuki, W. Oyanagi, K. Ohnishi, and H. Tanaka. 1990. Gene cloning of chitinase A-1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to type III homology units
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of fibronectin. J. Biol. Chem. 265:15659–15665. 25. Yang, C., Y. Zhu, D. M. Magee, and R. A. Cox. 1996. Molecular cloning and characterization of the Coccidioides immitis complement fixation/chitinase antigen. Infect. Immun. 64:1992–1997. 26. Zimmer, B. L., and D. Pappagianis. 1988. Characterization of a soluble protein of Coccidioides immitis with activity as an immunodiffusion-complement fixation antigen. J. Clin. Microbiol. 26:2250–2256. 27. Zimmermann, C. R., and D. Pappagianis. 1994. Extraction and purification of poly(A)1 RNA from Coccidioides immitis spherule-endospore and hyphal phases, p. 257–263. In B. Maresca and G. S. Kobayashi (ed.), Molecular biology of pathogenic fungi. Telos Press, New York. 28. Zimmermann, C. R., C. J. Snedker, and D. Pappagianis. 1994. Characterization of Coccidioides immitis isolates by restriction fragment length polymorphisms. J. Clin. Microbiol. 32:3040–3042.
