1 Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
2 Laboratory of Enzyme System Science, Department of Food and Nutrition, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
3 Department of Discovery Biology, Pfizer Global Research and Development, Sandwich Laboratories, Kent CT13 9NJ, UK
Correspondence
David J. Adams
d.j.adams{at}leeds.ac.uk
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AY217659 and AY217660.
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INTRODUCTION |
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Chitinases cleave the (14)-linkage between N-acetylglucosamine (GlcNAc) residues in the chitin homopolymer. Genes encoding chitinases have been cloned from a wide range of yeasts and filamentous fungi; all of these enzymes belong to glycohydrolase family 18 (Henrissat, 1999
). Within this family two distinct classes of chitinase may be identified based on the similarity of the enzymes to family 18 chitinases from plants or bacteria (Pishko et al., 1995
; Takaya et al., 1998a
). Chitinases of the plant class have been detected in Saccharomyces cerevisiae, Candida albicans and several filamentous fungi. Disruption of the CTS1 gene of S. cerevisiae led to inability of mother and daughter cells to separate during cell division (Kuranda & Robbins, 1991
) while disruption of the chiA gene of Aspergillus nidulans decreased both hyphal growth rate and the frequency of germination of conidia (Takaya et al., 1998a
). These results indicate that family 18 plant class chitinases have roles during growth and morphogenesis in fungi. Chitinases of the bacterial class have been found in filamentous fungi, but not in yeasts. Morphogenetic roles for chitinases of the bacterial class have been proposed (Takaya et al., 1998a
, b
; Reichard et al., 2000
; Kim et al., 2002
), but no such role has been demonstrated.
Previously we identified and purified a major, inducible 45 kDa chitinase from A. fumigatus (Escott et al., 1998). Here we report the cloning of the gene encoding the 45 kDa chitinase, ChiB1, which appears most closely related to other chitinases of the bacterial class. The chiB1 gene was overexpressed in the yeast Pichia pastoris and the recombinant enzyme was characterized in detail. The potential of A. fumigatus chitinases of the so-called fungal/bacterial and fungal/plant classes as targets for antifungal drugs is discussed in the light of the results of chiB1 gene disruption studies.
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METHODS |
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Isolation of genomic DNA encoding ChiB1.
Vogel's N medium (400 ml; Vogel, 1964), supplemented with 2 % sucrose, was inoculated to a final concentration of 1x106 A. fumigatus spores ml-1, incubated for 24 h at 37 °C at 200 r.p.m. and filtered through Whatman No. 3 paper under pressure to collect the mycelia as a pad on the filter paper. The mycelial pad was washed twice with 0·6 M MgSO4 and stored at -20 °C. Mycelia (1 g) were ground to a fine powder in liquid nitrogen (approx. 5 ml) and DNA was isolated from 100200 mg of this material using the Wizard Genomic DNA purification protocol (Promega). A fragment of the gene encoding ChiB1 was isolated from genomic DNA using PCR with a degenerate forward primer, 5'-GTNTAYTTYGTNAAYTGGGC-3', based on part of the N-terminal sequence (VYFVNWA) of a 45 kDa chitinase isolated previously from the culture filtrate of A. fumigatus NCPF 2140 (Escott et al., 1998
), and a degenerate reverse primer, 5'-NGGRTAYTCCCARTC-3', based on a conserved sequence (DWEYP) observed in an alignment of published Aphanocladium album (Blaiseau & Lafay, 1992
) and Trichoderma harzianum (Hayes et al., 1994
) chitinase amino acid sequences. PCR reactions (50 µl) contained buffer (Qiagen), 1·5 mM MgCl2, 200 µM each dNTP, 2 µM each primer, 100 ng genomic DNA and 1·25 units HotStarTaq polymerase. Samples were heated to 95 °C for 15 min and then cycled 25 times (95 °C, 1 min; 57 °C, 1 min; 72 °C, 1 min). A PCR product of approximately 450 bp was ligated into pGEM-T Easy (Promega) overnight at 4 °C and used to transform competent Escherichia coli JM109 cells (Promega) using standard methods (Sambrook et al., 1989
). Forward (5'-CTTCGCAAAGTTCTTCCGTC-3') and reverse (5'-CTCACACCACACCGAATACC-3') primers were designed based on the sequence of the cloned chiB1 fragment and used in a PCR reaction under conditions identical to those described above, except that digoxigenin (Dig)-labelled nucleotides were included in the reaction according to the manufacturer's instructions (Roche). The Dig-labelled probe was used to screen a
A. fumigatus ATCC 13073 genomic library (Stratagene) according to the manufacturer's instructions. Standard methods (Sambrook et al., 1989
) were used for cloning, purification and subcloning of appropriate fragments of DNA. Cloned fragments were sequenced by the DNA Sequence Service, University of Durham.
Isolation of cDNA encoding ChiB1.
A 1 kb fragment of chiB1 was isolated from the Uni-ZAP XR cDNA library. The sequence of this fragment confirmed the position of the stop codon of chiB1. For isolation of A. fumigatus cDNA, mycelial pad was ground to a fine powder in liquid nitrogen (see above). Total RNA was extracted from 100 mg ground mycelium using the RNeasy protocol (Qiagen) and polyA+ mRNA purified from total RNA using the Oligotex protocol (Qiagen). cDNA was synthesized from 2·5 µg polyA+ mRNA using the Clontech Copykit, according to manufacturer's instructions. Sequencing of a product from a PCR reaction using cDNA, and primers and reaction conditions identical to those described below for expression of ChiB1 in P. pastoris, identified most of the remainder of the cDNA for chiB1. Finally, the 5' end of chiB1 cDNA was determined using 5'-RACE, according to manufacturer's instructions (Roche).
Preparation and purification of recombinant ChiB1.
Forward primer CHIBXF (5'-CATTCGCTTTACGTAAGCTCCGGTTATCGCTCGGTC-3') and reverse primer CHIBXR (5'-CGAGCTCAATCTAGATTATCAGGTTTGCATGCCATT-3') were used to amplify a product, with the 5' region corresponding to sequence encoding the N terminus of native ChiB1 purified from an A. fumigatus culture filtrate (Escott et al., 1998), from cDNA. This product lacks the first 39 aa of the enzyme, including an apparent N-terminal signal peptide which was replaced with the S. cerevisiae
-factor for expression in P. pastoris. PCR reactions (50 µl) contained buffer (Qiagen), 1·5 mM MgCl2, 200 µM each dNTP, 2 µM each primer, 100 ng cDNA and 1·25 units HotStarTaq polymerase. Samples were heated to 95 °C for 15 min, then cycled 25 times (95 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min) and finally heated to 72 °C for 2 min. The restriction sites, underlined in the primer sequences, SnaBI in CHIBXF and XbaI in CHIBXR, were included to facilitate cloning into a modified pPICZ
A P. pastoris expression plasmid kindly provided by Dr Lars Hesse (University of Heidelberg, Germany). A single product of approximately 1200 bp was subcloned into the pPICZ
A vector and ChiB1 was expressed in the supernatant of P. pastoris X-33, according to manufacturer's instructions. Supernatant (10 ml) was desalted using a Sephadex G-25 M column (Prepacked P-10; Amersham Biosciences) and elution buffer (0·1 M potassium phosphate buffer, pH 6). Ammonium sulphate was added to a final concentration of 1 M and the sample was subjected to hydrophobic interaction chromatography using a Butyl Sepharose Fast Flow 4 column (20 ml) equilibrated with equilibration buffer (0·1 M potassium phosphate buffer, 1 M ammonium sulphate, pH 6). The sample was applied at a flow rate of 0·1 ml min-1 and the column was eluted with equilibration buffer (100 ml) and elution buffer (100 ml) at a flow rate of 1 ml min-1. Fractions (1 ml) were analysed by SDS-PAGE and those fractions containing the 45 kDa protein were pooled (3 ml), applied to a Sephacryl S-100 (Amersham Biosciences) gel filtration column (100 ml) and eluted with elution buffer. The purity of the eluted proteins was assessed by SDS-PAGE and MS. Polyclonal antibodies against purified recombinant ChiB1 were raised in rabbits by Charles River UK Ltd.
Immunoblot analysis.
Protein blotting onto nitrocellulose was as described by Sambrook et al. (1989) and antibodyantigen conjugates were visualized using a monoclonal anti-rabbit immunoglobulin alkaline phosphatase-conjugate (SigmaAldrich) and NBT/BCIP enzyme substrate (Roche).
Chitinase assays
Fluorimetric assays.
Fluorimetric assays with 4-methylumbelliferyl-,D-N,N'-diacetylchitobioside [4MU-(GlcNAc)2] or 4-methylumbelliferyl-
,D-N,N',N''-triacetylchitotrioside [4MU-(GlcNAc)3] as substrates were used to measure chitinolytic activity in A. fumigatus culture filtrates, essentially as described by McCreath & Gooday (1992)
. All assays were performed in triplicate.
Anomeric form of products.
The anomeric form of the reaction products was determined by HPLC using a TSK Amide 80 column (Koga et al., 1998; Fukamizo et al., 2001
) and the cleavage pattern was assessed from the anomer formation (Koga et al., 1998
). Briefly, the enzyme reactions contained 4·8 mM (GlcNAc)6, 0·4 µM recombinant ChiB1 and 50 mM sodium acetate, pH 5, and were incubated at 30 °C. For HPLC, the elution solvent was 70 % acetonitrile and the flow rate was 0·7 ml min-1. Oligosaccharides were detected by UV absorption at 220 nm.
Reaction time-course.
Products of the hydrolysis of each of (GlcNAc)4, (GlcNAc)5 and (GlcNAc)6 by recombinant ChiB1 were analysed and quantified by gel filtration HPLC using a TSK-GEL G2000PW column (Tosoh) essentially as described by Fukamizo et al. (2001). Briefly, enzyme reactions contained 4·8 mM (GlcNAc)n substrate, 0·4 µM enzyme and 50 mM sodium acetate, pH 5, and were incubated at 40 °C. For HPLC, the elution solvent was distilled water and the flow rate was 0·3 ml min-1. Oligosaccharides were detected by UV absorption at 220 nm. The amounts of saccharides produced were determined by computer-aided integration of individual peaks using a standard curve obtained from authentic saccharide solutions.
Zymogram procedure.
Chitinase activity was detected in polyacrylamide gels containing chitin as described by Escott et al. (1998).
Construction of gene replacement vector (pchiB1) for disruption of the chiB1 gene.
Plasmid pchib1 was designed as a gene replacement vector for the removal of a 484 bp fragment of the chiB1 gene containing the putative active site (Robertus & Monzingo, 1999
). The vector was constructed using pAN7.1 (Punt et al., 1987
), a fragment of chiB1 incorporating the 5' end of the gene (from 1251 bp upstream of the start codon to 110 bp into the translated sequence) and a fragment of chiB1 incorporating the 3' end of the gene (from 594 bp downstream of the start codon to 70 bp downstream of the stop codon) (Fig. 1
ac). Plasmid p
chiB1 was used to transform E. coli JM109 and identified in transformants following restriction digestion with StuI and SapI, and agarose gel electrophoresis. Construction of the disruption cassette was confirmed by DNA sequencing. The disruption plasmid was excised using FspI and HpaI, and used to transform A. fumigatus ATCC 13073 essentially as described by Paris (1994)
. Transformants were subcultured onto MMS agar (Paris, 1994
) containing hygromycin (200 µg ml-1) twice and spore suspensions were prepared as described above.
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RESULTS |
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Recombinant ChiB1 was purified from the culture filtrate of P. pastoris following heterologous expression of a truncated form of the chiB1 gene. SDS-PAGE analysis of the purified preparation identified a single protein of molecular mass in excess of 45 kDa (data not shown), while QTOF electrospray MS analysis indicated that the molecular mass of the recombinant protein was approximately 45 529 Da. This value is similar to the molecular mass of native ChiB1 (Escott et al., 1998). Recombinant ChiB1 exhibited chitinase activity on a glycol chitin gel and polyclonal antibodies raised against the pure protein identified a single band on a Western blot (data not shown); no immunoreactive protein was detected using pre-immune serum from the rabbit used for antibody production. When the deduced amino acid sequence of recombinant ChiB1 was subjected to primary structure analysis using the proteomics server of the Swiss Institute of Bioinformatics (SIB; http://ca.expasy.org/), predicted molecular mass and pI values for ChiB1 were 43 653 Da and 4·93, respectively. The difference between theoretical and measured molecular mass values is most likely attributable to post-translational modification of the ChiB1 polypeptide in A. fumigatus and in the heterologous host P. pastoris. A protein motif search, using the Scan Prosite program at the SIB website, identified several potential sites for post-translational modification, each with a high probability of occurrence; included among these is an Asn N-glycosylation site containing residues 266269. It is of interest to note, in this regard, that purified native ChiB1 reacted positively with the carbohydrate stain Periodic acidSchiff's reagent (Escott et al., 1998
).
Kinetic properties of recombinant ChiB1
Anomeric form of the reaction products.
The anomers of ChiB1 products from (GlcNAc)6 substrate at pH 6·0 were analysed using HPLC (TSK Amide 80 column). The major products were (GlcNAc)2 and (GlcNAc)4 and the two anomers of each product eluted separately as a function of time (Fig. 2). After a 5 min incubation virtually all of the newly formed disaccharide was formed as the
-anomer. Over a much longer incubation period (up to 90 min) the
-anomer gradually mutarotated to the
-anomer. Similarly, after a 5 min incubation, the ratio of
:
anomer for the (GlcNAc)4 reaction product was approximately 1 : 1, but after 90 min this ratio was close to the standard mutarotation equilibrium value for GlcNAc oligomers of approximately 5 : 2 (Fukamizo et al., 2001
). These results indicate that ChiB1 has a retaining mechanism of action typical of family 18 chitinases with the
-conformation of the glycosidic linkage preserved in the reaction products. The predominance of the (GlcNAc)2 product indicates that ChiB1 preferentially cleaves (GlcNAc)6 between the second and third sugars from the non-reducing end of this substrate.
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Loss of ChiB1 chitinase activity by the chiB1 transformant and comparative analysis of growth and morphogenesis of the parental and
chiB1 strains
Mutant and wild-type strains were cultured for 8 h in Vogel's N medium containing crab shell chitin and culture filtrates isolated as described in Methods. Chitinolytic activity in culture filtrates was assayed using a zymogram procedure and the presence/absence of ChiB1 was determined using antibodies raised against recombinant ChiB1 and Western blotting. A chitinase of approximately 45 kDa was detected in the wild-type, but not in the mutant strain containing the disrupted chiB1 gene (Fig. 4, lanes 1 and 2). The results of the Western blot analysis confirmed disruption of chiB1: the ChiB1 protein was detected in the wild-type, but not in the mutant strain (Fig. 4
, lanes 3 and 4).
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DISCUSSION |
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The results of our gene disruption studies argue against a major morphogenetic role for ChiB1. Growth and morphogenesis for all three of the mutant strains harbouring a disrupted chiB1 gene appeared identical to growth and morphogenesis of the wild-type organism under a range of growth conditions. However, the levels of lytic activity detected in the growth medium of the wild-type strain using 4MU-(GlcNAc)2 and 4MU-(GlcNAc)3 substrates were markedly higher than the levels of activity detected in a chiB1 disruptant during a 16-day batch culture (Fig. 5c). These results suggest strongly that ChiB1 is secreted into the growth medium in response to nutrient depletion, indicating that the principal role for this enzyme is the degradation and recycling of fungal chitin during autolysis. ChiB1 is a family 18 chitinase of the fungal/bacterial class and the results obtained during the present study are similar to those obtained by Reichard et al. (2000)
. These workers were unable to detect any effect on endosporulation or virulence of the pathogenic fungus C. immitis following disruption of CTS1, a gene encoding a fungal/bacterial chitinase in this organism. Taken together these results suggest that family 18 chitinases of the fungal/bacterial class may not themselves represent targets for antifungal agents. However, mechanisms that regulate the activities of these or other chitinases may be exploitable in the design of novel drugs as excessive expression of chitinolytic activity during normal growth may trigger cell lysis. Currently we are investigating the transcriptional and post-translational regulation of A. fumigatus chitinase activities.
Family 18 chitinases of the fungal/plant class appear to have morphogenetic roles in S. cerevisiae and A. nidulans (Kuranda & Robbins, 1991; Takaya et al., 1998a
). During the present study we cloned a gene, chiA1, which encodes a chitinase of the fungal/plant class in A. fumigatus (GenBank accession no. AY217659). The deduced amino acid sequence was found to have 50·3 % identity with the deduced sequence of ChiA, a fungal/plant chitinase from A. nidulans (Takaya et al., 1998a
) and 36·8 % identity with the deduced sequence for the C. immitis fungal/plant chitinase CTS2 (Pishko et al., 1995
). Further analysis of the deduced amino acid sequence for A. fumigatus ChiA1, using the DGPI facility at the SIB website, identified putative GPI-anchor and cleavage sites at the C terminus of the protein; no such sites were identified for A. fumigatus ChiB1. GPI-anchoring of fungal chitinases may provide at least a partial explanation for the close association of chitinases with membrane and cell-wall fractions isolated from A. fumigatus and other species (Dickinson et al., 1991
; Rast et al., 1991
; Hearn et al., 1998
). When the gene encoding ChiA, a closely related chitinase from A. nidulans, was disrupted, the mutant strain exhibited a decreased rate of germination of conidia and a lower hyphal growth rate when compared to the wild-type organism (Takaya et al., 1998a
). Analysis of the deduced amino acid sequence of A. nidulans ChiA indicates that this protein is also likely to have GPI-anchor and cleavage sites at the C terminus. Recently, Mouyna et al. (2000)
demonstrated that a glucanosyl-transferase, with an important role in A. fumigatus cell-wall biosynthesis, is GPI-anchored to the cell membrane. Moreover, Bruneau et al. (2001)
identified a further nine GPI-anchored proteins in A. fumigatus; five of these proteins are homologues of putatively GPI-anchored proteins from Candida albicans or S. cerevisiae with apparent roles during cell-wall construction or maintenance. Fungal/plant chitinases like ChiA1, GPI-anchored to the fungal cell membrane or wall, may also perform essential roles during growth and morphogenesis in filamentous fungi.
A. fumigatus ChiA1 contains a serine/threonine-rich domain located primarily at the C terminus of the protein (no such domain is present in ChiB1). Frieman et al. (2002) found that a serine/threonine-rich region of Epa1p, a GPI-anchored cell-wall adhesin of Candida glabrata, was required for the projection of the N-terminal ligand-binding domain of this protein into the external environment. It is possible that the serine/threonine-rich domain of ChiA1 performs a similar role in projecting the N-terminal active site of the GPI-anchored enzyme into, or through, the cell wall of A. fumigatus.
Future gene disruption experiments with chiA1 and genes encoding other chitinases of the fungal/plant class should allow us to identify precise roles for these enzymes in A. fumigatus. However, as the A. fumigatus genome sequencing project nears completion it should be noted that the elucidation of roles for individual chitinases is likely to be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database.
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ACKNOWLEDGEMENTS |
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Received 9 May 2003;
revised 10 July 2003;
accepted 11 July 2003.