A GAS-like gene family in the pathogenic fungus Candida glabrata

Michael Weiga,1, Ken Haynes2, Thomas R. Rogers2, Oliver Kurzai1, Matthias Frosch1 and Fritz A. Mühlschlegelb,1

Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany1
Department of Infectious Diseases and Microbiology, Imperial College of Science, Technology and Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, UK2

Author for correspondence: Fritz A. Mühlschlegel. Tel: +44 1227 82 3988. Fax: +44 1227 763912. e-mail: F.A.Muhlschlegel{at}ukc.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In fungi, the cell wall plays a major role in host–pathogen interactions. Despite this, little is known about the molecular basis of cell wall assembly in Candida glabrata, which has emerged as the second most common cause of systemic candidosis. A C. glabrata gene family, CgGAS1–3, that shares significant homologies with both the GAS1 gene of Saccharomyces cerevisiae, which is necessary for cell wall assembly, and the pH-regulated genes PHR1 and PHR2 of Candida albicans, which are involved in cell wall assembly and required for virulence, has been cloned. Among the members of this family, CgGAS1–3 display a unique expression pattern. Both CgGAS1 and CgGAS2 are constitutively expressed. In contrast, CgGAS3 transcript was not detectable under any of the assayed conditions. The C. glabrata actin gene, CgACT1, has also been cloned to be used as a meaningful loading control in Northern blots. CgGAS1 and CgGAS2 were deleted by two different methodological approaches. A rapid PCR-based strategy by which gene disruption was achieved with short regions of homology (50 bp) was applied successfully to C. glabrata. {Delta}Cggas1 or {Delta}Cggas2 cells demonstrated similar aberrant morphologies, displaying an altered bud morphology and forming floccose aggregates. These phenotypes suggest a role for CgGAS1 and CgGAS2 in cell wall biosynthesis. Further evidence for this hypothesis was obtained by successful functional complementation of a gas1 null mutation in S. cerevisiae with the C. glabrata CgGAS1 or CgGAS2 gene.

Keywords: cell wall, actin, GAS1, PHR1, PHR2

Abbreviations: CHEF, contour-clamped homogeneous electric field gel electrophoresis; GPI, glycosylphosphatidylinositol

The EMBL accession numbers for the sequences reported in this paper are AJ302061 for CgGAS1, AJ302062 for CgGAS2 and AJ302063 for CgGAS3.

a Present address: Abteilung für Bakteriologie, Universität Göttingen, 37075 Göttingen, Germany.

b Present address: Department of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The haploid opportunistic fungal pathogen Candida glabrata is now the second most common cause of systemic candidosis (Pfaller et al., 1998 ; Vazquez et al., 1998 ; Edmont et al., 1999 ). Infections caused by C. glabrata are particularly difficult to treat and have a high mortality rate in immunocompromised patients at risk. This may be related to the low level of natural susceptibility against established antifungal agents (Vanden Bossche et al., 1998 ). In common with other fungal pathogens, there is a lack of defined C. glabrata targets which can be exploited for the rational design of novel antifungal drugs (Fidel et al., 1999 ). It is anticipated that a greater understanding of the molecular mechanisms involved in virulence will yield valuable information on potential targets (Weig et al., 1998 ). However, it is only recently that the tools required for this analysis have become available in this largely unexplored organism. Auxotrophic mutant strains (Kitada et al., 1995 ), plasmid-based vectors, containing CEN/ARS sequences, and a reporter system, using lacZ, have been generated (Kitada et al., 1996 , 1997 ; El Barkani et al., 2000a ). In addition, signature-tagged mutagenesis has been applied as a genetic approach to analyse the host–pathogen interaction in C. glabrata (Cormack et al., 1999 ; Kamran et al., 2000 ). These technical advances will allow C. glabrata to be exploited as a model system for the analysis of fungal virulence.

During the last decade, the cell wall of fungi has become a major focus of scientific interest (Chaffin et al., 1998 ; Kapteyn et al., 1997 ). This highly dynamic structure, which is synthesized by enzymes specific to fungi, mediates the interaction with the external environment, including the human host, and is ultimately responsible for morphogenesis of these micro-organisms (Chaffin et al., 1998 ; De Bernardis et al., 1998 ; Mühlschlegel et al., 1998 ). It is anticipated that as our understanding of this essential structure of human pathogenic fungi increases, this knowledge may be used in the design of novel antifungal agents and the development of original diagnostic procedures (Douglas et al., 1997 ; Kurtz & Douglas, 1997 ). In Saccharomyces cerevisiae, the Gas1 protein is one of the most abundant glycosylphosphatidylinositol (GPI)-anchored cell surface proteins (Nuoffer et al., 1991 ; Popolo et al., 1993 ; Vai et al., 1990 , 1996 ). Of the five GAS genes present in the S. cerevisiae genome, only mRNA from GAS1 is detectable during vegetative growth. In addition, only mutations of the GAS1 gene lead to a phenotype which consists of an abnormal morphology, loss of 1,3-ß-glucan and increase of chitin and mannan in the fungal cell wall (Kapteyn et al., 1997 ; Popolo et al., 1997 ; Ram et al., 1998 ). In the opportunistic fungal pathogen Candida albicans, two homologous genes, termed PHR1 and PHR2, have been identified (Mühlschlegel & Fonzi, 1997 ; Saporito-Irwin et al., 1995 ). These genes display a unique counterbalanced pH-dependent expression pattern. Deletion of either PHR1 or PHR2 results in defects of growth and morphogenesis that resemble those of the gas1 null mutant of S. cerevisiae. However, in sharp contrast to S. cerevisiae, these defects are pH-conditional in C. albicans (Mühlschlegel & Fonzi, 1997 ; Saporito-Irwin et al., 1995 ). Phr1p and Phr2p are cell-wall-associated proteins that are essential for the morphological plasticity and virulence of C. albicans (De Bernardis et al., 1998 ; Mühlschlegel & Fonzi, 1997 ; Saporito-Irwin et al., 1995 ; Ghannoum et al., 1995 ). GAS1, PHR1 and PHR2 are homologous to GEL1, which encodes a 1,3-ß-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus (Mouyna et al., 2000 ). In addition, it has been shown that Gas1p, Phr1p and Phr2p display 1,3-ß-glucanosyltransferase activity similar to Gel1p (Mouyna et al., 2000 ). Recently it was demonstrated that the Phr proteins are pivotal to cell wall assembly, probably processing 1,3-ß-glucans (Fonzi, 1999 ).

Until recently, very little information was available about C. glabrata genes involved in cell wall biosynthesis (B. A. Junginger, M. Weig & F. Mühlschlegel, GenBank accession no. AF229171; Nagahashi et al., 1998 ). In this manuscript, we report the identification and characterization of a gene family termed CgGAS1–3 (C. glabrata GAS homologues 1–3). CgGAS1 and CgGAS2 of C. glabrata demonstrate a unique expression pattern. In clear contrast to S. cerevisiae, both genes, CgGAS1 and CgGAS2, are expressed. CgGAS3, similar to GAS2–5 in S. cerevisiae, is not expressed during vegetative growth. The expression patterns of CgGAS1 and CgGAS2 are also different to that of the two C. albicans homologues. Expression of CgGAS1 and CgGAS2 is constitutive and not influenced by the environmental pH. CgGAS1 and CgGAS2 encode putative cell surface proteins that are homologous to the GPI-anchored proteins Gas1p of S. cerevisiae, Phr1p and Phr2p of C. albicans and Gel1p of A. fumigatus. Deletion of the genes results in characteristic growth and morphological defects. The functional homology of CgGAS1 and CgGAS2 was shown by their ability to reverse the aberrant phenotype of an S. cerevisiae {Delta}gas1 mutant.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and growth conditions.
The C. glabrata and S. cerevisiae strains used in this study are listed in Table 1. The strains were routinely cultured on YPD plates at 30 °C. For the growth of the auxotrophic strains, the media were supplemented with 25 µg uridine ml-1, 20 µg histidine ml-1 or 40 µg tryptophan ml-1 when needed. pH-dependent effects were studied in either medium 199 (Gibco-BRL) or YNB medium (2% glucose, 1x Difco yeast nitrogen base) buffered with 150 mM HEPES as described previously (Mühlschlegel & Fonzi, 1997 ). The cell morphology and growth rates were determined in medium 199, YPD and YNB medium at 26 and 37 °C.


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Table 1. C. glabrata and S. cerevisiae strains used in this study

 
Construction of the C. glabrata genomic library.
Genomic DNA was isolated from the C. glabrata ATCC 90876 strain as previously described (Saporito-Irwin et al., 1995 ). The DNA was partially digested with Sau3A and fragments ranging from 3 to 12 kb were excised and purified. The inserts were ligated into BamHI-digested, dephosphorylated ZapExpress vector and packaging was done using the Gigapack III Gold kit (Stratagene).

Isolation of the CgGAS1, CgGAS2 and CgGAS3 genes.
Fragments of C. glabrata genes, homologous to GAS1 of S. cerevisiae and PHR1/PHR2 of C. albicans, were isolated by PCR amplification using the degenerate inosine-containing primers HO-1 and HO-2 (Table 2) (Mühlschlegel & Fonzi, 1997 ) and genomic DNA of C. glabrata ATCC 90876 as the template. The resulting 0·24 kb PCR products were purified and cloned into pBSK+ (Stratagene). Sequence analysis was performed on both strands of the inserts from 30 plasmids isolated from different clones. DNA sequences of three homologous genes, termed C. glabrata GAS homologues (CgGAS1, CgGAS2 and CgGAS3), were identified and subsequently used for a hybridization screening of the C. glabrata genomic library. The hybridizing DNA fragments were isolated by in vivo excision of the pBK-CMV phagemid vectors, to generate pBK-MW1–3. pBK-MW1 harboured a 6 kb DNA fragment that included the CgGAS1 gene lacking the 5'-portion of the predicted open reading frame, pBK-MW2 harboured a 3 kb fragment that included the CgGAS2 gene lacking the 5'-portion of the predicted open reading frame and pBK-MW3 included a 5 kb fragment with the entire CgGAS3 gene and flanking regions.


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Table 2. Primers used in this study

 
Inverse PCR.
Inverse PCR was performed in order to obtain the missing 5' coding regions as well as flanking sequence information of CgGAS1 and CgGAS2. In Southern blot hybridization experiments, digestion of genomic DNA from C. glabrata ATCC 90876 with AccI revealed a 2·4 kb CgGAS1 fragment, whereas digestion with StuI produced a 1·7 kb CgGAS2 fragment. The digested DNA fragments were circularized and subjected to PCR amplification. The necessary primers were deduced from the known DNA sequences 3' to the AccI/StuI restriction sites. The PCR products were purified and cloned into pCR2.1 to generate plasmids pIPMW-1 (CgGAS1) and pIPMW-2 (CgGAS2).

Cloning of the C. glabrata actin gene.
Primers OK1 and OK2 were used to amplify a 1 kbp fragment of the C. albicans ACT1 coding sequence (Losberger & Ernst, 1989 ), suitable for hybridization screening of the C. glabrata genomic library. Hybridizing phage clones were excised as phagemid vectors. Clone p4-28a contained the entire C. glabrata ACT1 gene (CgACT1) with 5' and 3' flanking sequences. Double-strand sequencing revealed a 1128 bp ORF interrupted by a single intron of 891 bp (exon 1 10 bp, exon 2 1118 bp; GenBank accession no. AF069746). Splicing of the intron was confirmed via RT-PCR. The CgACT1 nucleotide sequence is 90% homologous to the ACT1 gene of S. cerevisiae. The second exon of CgACT1 was amplified with primers CgACTf and CgACTr and used as a probe in control hybridizations.

Construction of auxotrophic C. glabrata strains.
Previously we observed that homologous recombination at the marker locus occurs in C. glabrata strains harbouring only partial deletions of this locus (data not shown). Consequently a strain completely lacking HIS3 was constructed. PCR primers CGHIS3/1 and CGHIS3/2 were used to amplify the C. glabrata HIS3 locus from -468 to +890. The HIS3 ORF ends at +633. This fragment was blunt-ended and cloned into EcoRV-digested pBSK+. The resulting plasmid (pTW23) was amplified, cut with BclI, blunt-ended and digested with SpeI leaving bp -467 to -254 on the XbaI arm and +777 to +890 on the XhoI arm. Into it a 3·5 kb SpeI–SmaI fragment from plasmid pU1b, containing the entire CgURA3 gene, was ligated (Zhou et al., 1994 ). The resulting CgHIS3/URA3 disruption cassette was amplified by PCR using the same primers. The fragment was used to transform C. glabrata 2001U (Kitada et al., 1995 ), utilizing a modified lithium acetate protocol (Gietz et al., 1992 ), and uracil prototrophs were selected. Ten of these strains were subcultured on histidine drop out medium and all exhibited auxotrophy. No other auxotrophies were detected. Confirmation of insertion at the CgHIS3 chromosomal locus and gene replacement was obtained using PCR and Southern analysis (data not shown). A single strain was selected and designated {Delta}H1, which has a complete removal of CgHIS3 from -254 to +777. This includes the entire ORF. {Delta}HT6 ({Delta}ura3 {Delta}trp1 {Delta}his3) was constructed in the same way using C. glabrata 2001TU instead of C. glabrata 2001U as the recipient strain (Kitada et al., 1995 ).

Construction of C. glabrata {Delta}Cggas1 mutants.
To generate a CgGAS1 knockout strain a disruption cassette was constructed in which the selection marker HIS3 was flanked by CgGAS1 sequences for targeted integration. A 918 bp fragment of the C. glabrata HIS3 gene was amplified by PCR using the primers HIS-XbaI-f and HIS-HindIII-r, which included integrated XbaI–HindIII restriction sites, and pTW23 as a template. The PCR product was subcloned into plasmid pCR2.1 (Invitrogen) and excised with XbaI and HindIII. It was then integrated into the respective sites of pBSK+ to generate plasmid pMW-1. A 524 bp CgGAS1 fragment (-235 to +289) was amplified using primers BL5-XbaI-f and BL6-XbaI-r and using embedded XbaI sites was cloned into pMW-1 to generate pMW-2. The orientation of the fragment within pMW-2 was confirmed by PCR analysis.

A 566 bp CgGAS1 fragment (+1418 to +1984, the CgGAS1 stop codon is at +1680) was amplified with the primers BL7-HindIII-f and BL8-HindIII-r and using embedded HindIII sites was cloned into pMW-2 to generate pMW-3. The desired orientation of the HindIII fragment was confirmed by PCR analysis.

Using pMW-3 as a template, a 2·0 kb disruption cassette containing HIS3 and 0·5 kb CgGAS1 flanking sequences was amplified by PCR with the primers Ko-MW1-f and Ko-MW1-r (Fig. 1a). C. glabrata {Delta}HT6 was transformed with the disruption cassette by the lithium acetate method (Gietz et al., 1992 ). Transformants were selected on YNB plates supplemented with tryptophan. Integration of the transforming DNA was analysed by PCR with primers RMW-31 and HIS-Ver-f (Fig. 1a). Primer RMW-31 binds upstream of the integration locus and HIS-Ver-f is homologous to the selection marker. The disruption of CgGAS1 was confirmed by Southern and Northern analysis using the CgGAS1 probe to verify deletion. To unequivocally verify the correct integration event, a PCR product amplified with primers IMW-7 and RMW-31 and genomic DNA of the representative {Delta}Cggas1 mutant, CGMW2, was sequenced (Fig. 1a). These primers anneal at sites 105 bp upstream and 128 bp downstream of the expected integration locus. A CgGAS1 null mutant was also constructed using two 70 bp oligonucleotides (CGG1-Ko70-f and CGG1-Ko70-r). Each oligonucleotide was composed of 50 bp flanking the start (-51 to -1) or stop (+1681 to +1731) codon of the CgGAS1 gene and 20 bp homologous to the C. glabrata HIS3 gene. These oligos were used to amplify the HIS3 gene using pTW23 as a template. The PCR product was purified and used to transform strain {Delta}HT6. Transformants were selected and confirmed as described for CGMW2. A representative {Delta}Cggas1 mutant, termed CGMW1, was chosen for further analysis.



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Fig. 1. Design and construction of CgGAS1 and CgGAS2 deletion mutants. (a) Restriction map of the CgGAS1 deletion construct as cloned in plasmid pMW-3 (dotted boxes) and the amplified deletion cassette as well as the region deleted and replaced in the genome of the C. glabrata strain {Delta}HT6. The open reading frame of CgGAS1 is indicated in black. Sequences flanking CgGAS1 are indicated as striped boxes. Primers used for screening are indicated as arrows. (b) Restriction map of the CgGAS2 deletion construct as cloned in plasmid pMW-5 (dotted boxes) and the amplified deletion cassette as well as the region deleted and replaced in the genome of {Delta}HT6. The open reading frame of CgGAS2 is indicated in black. Sequences flanking CgGAS2 are indicated as striped boxes. Primers used for screening are indicated as arrows.

 
Complementation of C. glabrata {Delta}Cggas1 mutants.
Complemented C. glabrata strains were generated by reintroduction of the CgGAS1 gene into the {Delta}Cggas1 mutants CGMW1 and CGMW2. Complementation was achieved by targeted integration of CgGAS1, using linearized plasmid pMW-7. To construct pMW-7, the C. glabrata TRP1 gene was released from plasmid pCgACT14 (Kitada et al., 1996 ) by XhoI digestion and introduced into the respective sites of pBSK+, to generate pMW-6. Next a 3108 bp fragment (-1103 to +2006) encompassing the CgGAS1 open reading frame was amplified from genomic DNA of C. glabrata 2001 with primers BL12-BamHI-f and BL13-BamHI-r and cloned into the unique BamHI site of pMW-6 to generate pMW-7, which was linearized at its unique SphI site, within the 5' untranslated region of CgGAS1, and used to transform CGMW1 and CGMW2 to tryptophan prototrophy. Successful integration of the transforming DNA was verified by Southern and Northern analysis plus PCR with primers CGG1-Ver-f and CGG1-Ver-r. Two representative transformants, CGMW5 and CGMW6, were chosen for further analysis.

Construction of C. glabrata {Delta}Cggas2 mutants.
A CgGAS2 knockout strain was constructed using a similar approach to that described for CGMW2 (Fig. 1). A 499 bp CgGAS2 fragment (-221 to +278) was amplified from genomic DNA of C. glabrata 2001 with primers BL1-XbaI-f and BL2-XbaI-r, each including an XbaI restriction site. Next a 423 bp fragment (+1487 to +1810, stop codon at +1698) was amplified with primers BL3-HindIII-f and BL4-HindIII-r, containing HindIII restriction sites. To generate plasmid pMW-5, the two CgGAS2 fragments were cloned in pMW-1 (HIS3 in pBSK+) via their XbaI–HindIII restriction sites, thus flanking the HIS3 selection marker. The orientation of the fragments was confirmed by PCR analysis and the deletion cassette was amplified with primers Ko-MW4-f and Ko-MW4-r (data not shown). Integration of the transforming DNA was analysed by PCR with primers IMW-9 and HIS-Ver-r (Fig. 1b). Primer IMW-9 binds upstream of the integration locus and HIS-Ver-r is homologous to the selection marker. The disruption of CgGAS2 and verification of the integration event, using the primers IMW-9 and RMW-30, was done as described for the {Delta}Cggas1 mutants (Fig. 1b). These primers bind 69 bp upstream and 56 bp downstream of the expected integration locus. A single disrupted strain, CGMW3, was selected for further study.

Another Cggas2 knockout strain, termed CGMW4, was constructed and confirmed in a similar procedure to that described for CGMW3. However, TRP1 was used as selection marker in order to be able to utilize the remaining histidine auxotrophy for an additional null mutation of CgGAS1.

Complementation of {Delta}Cggas2 mutants.
CGMW3 was complemented with CgGAS2 by targeted integration of the linearized plasmid pMW-8. Plasmid pMW-8 was constructed by ligating a 3·3 kb CgGAS2 fragment into pMW-6, which already harboured TRP1. The 3·3 kb CgGAS2 fragment was amplified by PCR with the primers BL9-EcoRI-f and BL10-EcoRI-r and cloned into the unique EcoRI site of pMW-6. pMW-8 was linearized at its multiple cloning site using XbaI and transformed into CGMW3. Tryptophan prototrophs were selected and further characterized. Successful targeted integration of the transforming DNA was verified by Southern and Northern analysis plus PCR with primers CGG2-Ver-f/CGG2-Ver-r. A representative transformant was designated CGMW7.

Complementation of S. cerevisiae {Delta}gas1 mutants.
CgGAS1 and CgGAS2 were used to transform the S. cerevisiae {Delta}gas1 mutant WB-2d (Popolo et al., 1993 ). Plasmid pMW-9 was constructed by ligation of the 3108 bp CgGAS1 fragment that was previously described for generation of pMW7, into the BamHI site of YEp24 (NEB). Plasmid pMW-10 was constructed by ligation of a 3506 bp CgGAS2 fragment (amplified with primers BL14-AatII-f and BL11-AatII-r) to the unique AatII site of YEp24. pMW-9 and pMW-10 were transformed into S. cerevisiae WB-2d. Uracil prototrophs were selected and transformants were verified by both PCR and Northern analysis. Representative clones containing CgGAS1 or CgGAS2 were selected and designated SCMW1 and SCMW2, respectively.

Phenotypic analysis.
Mutant strains were grown on YNB supplemented with the appropriate amino acids, YPD and medium 199 at 26 and 37 °C. Phenotypic analysis was performed at pH 4, 5, 6, 7 and 8 using a Leitz Aristoplan microscope.

CHEF.
For the separation of intact chromosomes, contour-clamped homogeneous electric field gel electrophoresis (CHEF) was used. The preparation of the C. glabrata chromosomes was done as previously described for C. albicans (Fonzi & Irwin, 1993 ). The conditions for electrophoresis were a linear ramp from 300 to 1400 s, a duration of 144 h and 75 V. The separated chromosomes were blotted and consecutively used for Southern blot analysis, with CgGAS1 and CgGAS2 as hybridization probes.

Southern and Northern blot analysis.
Isolation of C. glabrata genomic DNA and RNA, followed by Southern and Northern blot analysis, was done as described previously (Mühlschlegel & Fonzi, 1997 ). Inserts of pBK-MW1, pBK-MW2 and pBK-MW3, a 694 bp fragment of the C. glabrata HIS3 gene and exon 2 of the C. glabrata actin gene were used as hybridization probe for Southern and Northern blot analysis under high stringency conditions (68 °C). Labelling of the probes was done using the Prime-It kit (Stratagene). An RNA ladder (0·25–9·5 kb; Gibco-BRL) was utilized to determine the transcript size in Northern blots.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of the C. glabrata CgGAS1, CgGAS2 and CgGAS3 genes
Using PCR with degenerate inosine containing primers, the sequences of which were deduced from conserved regions of the Gas and Phr proteins from S. cerevisiae and C. albicans, three different amplification products of approximately 0·24 kb were generated. Sequence analysis of the cloned PCR products revealed uninterrupted coding sequences for all three fragments. Comparison of the deduced amino acid sequences showed homologies to Gas1p of S. cerevisiae, and the Phr1p or Phr2p proteins of C. albicans (data not shown). We termed the corresponding genes C. glabrata GAS homologues 1–3 (CgGAS1–3). Southern blot experiments were carried out with the cloned CgGAS1–3 PCR products as hybridization probes under high stringency conditions at 68 °C. Each probe hybridized to a major band in total DNA digests with EcoRI from C. glabrata ATCC 90876 or C. glabrata ATCC 2001. The size of this band was 12 kbp in the case of CgGAS1, and 5·8 kbp and 7 kbp in the case of CgGAS2 and CgGAS3, respectively (data not shown). Using the same probes, DNA digests with XbaI revealed bands with sizes of 4·6, 5·6 and 9 kbp in the case of CgGAS1, CgGAS2 and CgGAS3, respectively (data not shown). This indicates the presence of three distinct genes. Examination of the electrophoretic karyotypes of the wild-type C. glabrata strains ATCC 90876, C. glabrata ATCC 2001 and its auxotrophic derivatives {Delta}HT6, {Delta}H1, HTU and TU revealed no major chromosomal abnormalities between the strains and derivatives (data not shown). Southern blot analysis of the CHEF gels showed that CgGAS1 and CgGAS2 can be assigned to different chromosomes (Fig. 2).



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Fig. 2. Chromosomes of the C. glabrata strain {Delta}HT6 separated by CHEF (lane 1). The gel was blotted and probed with the 0·24 kbp CgGAS1 and CgGAS2 PCR fragments (lane 2).

 
Expression of CgGAS1 and CgGAS2 is constitutive
The cloned CgGAS1–3 0·24 kbp PCR products were used as hybridization probes for Northern blot analysis under high stringency conditions at 68 °C. The specificity of the probes was assessed by cross-hybridization of the probes with pBK-MW1, pBK-MW2 and pBK-MW3. No cross-hybridization signal was observed in these control experiments (data not shown). RNA was prepared from C. glabrata ATCC 90876 and ATCC 2001, after the cells were grown at pH 4, 5, 6, 7 and 8 for 1 h. In both strains, a single band, with an approximate size of 1·6 kb, was detected with the CgGAS1 and CgGAS2 probes. High levels of expression were observed at all pH values for CgGAS1 (Fig. 3) and for CgGAS2 (data not shown). In contrast, no CgGAS3 mRNA was detectable (data not shown). The expression pattern of CgGAS1 and CgGAS2 was not affected by the different growth media (M199, YPD, YNB) or the incubation temperatures (26 °C, 37 °C) (data not shown).



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Fig. 3. Effect of culture pH on CgGAS1 expression. Cells of C. glabrata strain 2001 were cultured at pH 4·0 and 8·0. Total RNA was isolated and examined by Northern blot analysis. The blots were hybridized with either CgGAS1 or CgACT1 probes.

 
CgGAS1, CgGAS2 and CgGAS3 share structural features common to the PHR/GAS family of genes
The cloned CgGAS1–3 gene fragments were used for a hybridization screening of a C. glabrata genomic library. Three DNA fragments were identified harbouring CgGAS1 (6 kb), CgGAS2 (3 kb) and CgGAS3 (5 kb). Missing 5' portions of CgGAS1 and CgGAS2 were complemented using inverse PCR. Sequence analysis of the cloned genes revealed uninterrupted open reading frames of 1680 nucleotides for CgGAS1, 1698 nucleotides for CgGAS2 and 1929 bp for CgGAS3. The nucleotide sequence data have been submitted to the EMBL nucleotide sequence database under the accession numbers AJ302061 for CgGAS1, AJ302062 for CgGAS2 and AJ302063 for CgGAS3. The predicted structural features and the overall identity of the putative CgGas proteins and of the homologous Gas1p, Phr1p and Phr2p proteins are listed in Table 3. The deduced amino acid sequences of CgGas1–3p were colinear with Gas1p, Phr1p and Phr2p (Fig. 4). Furthermore, CgGas1–3p share regions structurally similar to the signal sequences of secretory proteins from other prokaryotic and eukaryotic cells. The hydrophobic amino- and carboxy-termini that serve as transmembrane helices in Gas1p, Phr1p and Phr2p are also present in CgGas1–3p (Hirokawa et al., 1998 ). The presence of a COOH-terminal hydrophobic sequence is common to genes that undergo GPI anchor addition after translation (Gerber et al., 1992 ). A hydrophobic sequence was predicted at the amino-terminus of the CgGas1p open reading frame extending between amino acids 4 and 26 (SLVSFITAATLLLSSVMADDLPA). The predicted cleavage site for the signal sequence (Nielsen et al., 1997 ) is between position 21 and 22 (VMA-DD). The last 23 amino acids of the CgGas1 protein sequence (position 534–556) encompass another hydrophobic region (QVGLYQLLFSAFITLGAVAGAGF) for which the formation of a transmembrane helix is predicted. Corresponding regions were found in the CgGas2 protein sequence [hydrophobic regions: 1–23, MFFKNTLAALTAASALFSTVKAD; 544–565, FQVIATSVISISMLAGLGFVLA; cleavage site between position 22 and 23 (VKA-DD)] and in the CgGas3 protein sequence (hydrophobic regions: 4–24, LLYFALSVVATSAQLADEVLL; 519–541, NTLSGTKLLVITVLNTLVVLLIA; cleavage site between position 16 and 17: TSA-QL).


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Table 3. Percentage identity of CgGas1p, CgGas2p, CgGas3p and homologous proteins

 


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Fig. 4. Comparison of the predicted amino acid sequences of the CgGAS1, CgGAS2, CgGAS3, PHR1 (accession no. M90812), PHR2 (accession no. AF011386), ScGAS1(accession no. X53424) and GEL1 (accession no. AF072700) products using CLUSTAL W (Thompson et al., 1994 ). Identical residues are boxed.

 
A serine-rich region located near the carboxy-terminus is present in CgGas1p and in CgGas2p but is absent in CgGas3p. The serine (and threonine) residues could provide possible sites for O-glycosylation (Jentoft, 1990 ). All 12 of 13 cysteine residues that are conserved in Phr1p and Phr2p are also conserved in CgGas1–3p. Furthermore, all three CgGasp sequences contain the requisite motifs of GPI-anchored proteins, i.e. signal sequence, hydrophobic amino- and carboxy-terminus plus potential cleavage residues at position {omega} (Fig. 4).

Recently, two conserved potentially catalytic sites, Ala-166 Gly Asn Glu Val-170 and Phe-267 Phe Ser Glu Tyr Gly-272, were reported in Phr1p and Phr2p (Fonzi, 1999 ). The identical amino acid motifs were also detected in CgGas1p and CgGas2p (Fig. 4). A single amino acid exchange was found in CgGas3p at position 270, where a charged Tyr was replaced by a hydrophobic Phe.

Construction of CgGAS1 and CgGAS2 null mutants
To evaluate the functional role of CgGAS1 and CgGAS2, two null mutants lacking the respective genes were constructed. CgGAS1 was mutated by homologous recombination applying two different techniques. Firstly, a 2·0 kb knockout cassette was constructed, consisting of the C. glabrata HIS3 gene flanked by approximately 0·5 kb CgGAS1 sequence on each side. This construct was expected to replace 1075 bp of the CgGAS1 ORF. Genomic DNA from 15 transformants, resulting from two independent transformations, was subjected to PCR analysis. Three clones were identified that resulted in PCR fragments which were consistent with the predicted integration event. Sequencing of a PCR product generated with primers binding 105 bp upstream and 128 bp downstream of the expected integration locus of a representative {Delta}Cggas1 mutant, CGMW2, confirmed the predicted integration event (data not shown). Genomic DNA of this strain was digested with HindIII or SspI. Southern blot analysis, using HIS3 as a probe, revealed the predicted bands of 1·8 kb and 1·45 kb, respectively (data not shown). Sequencing of the PCR product and Southern blot analysis confirmed that no ectopic integration had taken place.

A rapid PCR-based knockout procedure was applied as an alternative approach to delete CgGAS1. The deletion cassette was generated using the primers CGG1-Ko70-f and CGG1-Ko70-r. These amplify the C. glabrata HIS3 gene and are tagged with 50 bp CgGAS1 flanking sequences. The purified amplification product was used to transform the C. glabrata strain {Delta}HT6. Fifty histidine prototrophs were analysed by PCR and one mutant was detected that had undergone the desired recombination event. This mutant, termed CGMW1, was confirmed by Southern blot analysis following SspI digestion and PCR analysis (data not shown). When tested in Northern blot analysis, using CgGAS1 as a probe, no transcripts could be detected in the {Delta}Cggas1 mutants CGMW1 and CGMW2, providing further proof of the successful deletion (data not shown).

A {Delta}Cggas2 null mutant was generated by transforming strain {Delta}HT6 to histidine prototrophy using a deletion cassette with 0·5 kb and 0·42 kb CgGAS2 sequences, flanking HIS3. Fifteen histidine prototrophs were screened by PCR and four clones were isolated that gave bands indicative of the desired recombination event (data not shown). Sequencing of a PCR product generated with primers binding 69 bp upstream and 56 bp downstream of the expected integration locus of a representative {Delta}Cggas2 mutant, CGMW3, confirmed the predicted integration event (data not shown). Genomic DNA of this strain was digested with SspI or AccI. Southern blot analysis, using HIS3 as a probe, revealed the predicted bands of 2 kb and 1·3 kb, respectively (data not shown). Sequencing of the PCR product and Southern blot analysis confirmed that no ectopic integration had taken place. Northern blot analysis confirmed the loss of CgGAS2 expression in CGMW3 (data not shown).

In an attempt to generate a {Delta}Cggas1/{Delta}Cggas2 double knockout strain, we first constructed a histidine auxotrophic {Delta}Cggas2 null mutant in {Delta}HT6. To achieve this, a deletion cassette consisting of 0·5 kb of CgGAS2 sequence on each side of the C. glabrata TRP1 gene was constructed. The construction of the cassette and the verification of the homologous integration was done as described for CGMW1 or CGMW3 (data not shown). The resulting {Delta}Cggas2 strain, CGMW4, was then transformed with the HIS3-based disruption cassette used for deleting CgGAS1. In addition, the {Delta}Cggas1 strains CGMW1 and CGMW2 were transformed with a TRP1-based CgGAS2 disruption cassette (data not shown). Plates were examined, on a daily basis, for 7 d in order to allow detection of the potentially slow growing double mutants. Although 50 clones from each of the three transformations were screened by PCR and selected strains were analysed by Southern blotting, no double {Delta}Cggas1/{Delta}Cggas2 mutant was isolated.

The loss of CgGAS1 or CgGAS2 results in defects of growth and cell morphology
In comparison to the parental strain (Fig. 5a), CGMW1 and CGMW2 formed aggregates (Fig. 5b and data not shown). The formation of aggregates has been previously reported for the C. albicans {Delta}phr1 and {Delta}phr2 double mutant CAP-2 (Mühlschlegel & Fonzi, 1997 ). In a similar fashion to S. cerevisiae, but distinct from C. albicans, the growth and morphologic abnormalities in the {Delta}Cggas1 mutants were not pH-dependent (data not shown). In comparison to the parental strain {Delta}HT6, the {Delta}Cggas1 mutants, CGMW1 and CGMW2, showed a twofold increased doubling time (data not shown). No phenotypic differences were observed between CGMW1 and CGMW2.



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Fig. 5. Effect of CgGAS1 null mutation on the morphology of C. glabrata. The CgGAS1 strain is shown in (a) ({Delta}HT6) and the {Delta}Cggas1 mutant strain in (b) (CGMW2). The phenotype was completely reversed after reintroduction of CgGAS1 into the mutant (CGMW5) (c). Bar, 10 µm.

 
The same growth rate and cell morphology phenotypes described for CGMW1 and CGMW2 were observed in the {Delta}Cggas2 mutant CGMW3 (data not shown). Again the mutant phenotype was independent of the environmental pH.

To confirm that the mutant phenotypes were directly attributable to the loss of CgGAS1 in strains CGMW1 and CGMW2, or the loss of CgGAS2 in strain CGMW3, the respective wild-type genes were reintroduced by targeted integration. Plasmid pMW-7, harbouring CgGAS1, was used to transform CGMW1 and CGMW2 to tryptophan prototrophy. Fifteen transformants were subjected to Southern analysis and the expected integration event was seen in all transformants including strains CGMW5 and CGMW6 (data not shown). Similarly the {Delta}Cggas2 mutant CGMW3 was transformed with plasmid pMW-8, harbouring CgGAS2. Southern blot analysis of 15 transformants showed that they all contained the expected integration event including strain CGMW7 (data not shown).

Northern analysis using CgGAS1 as probe and total RNA from strains CGMW5 and CGMW6 gave a single band of the expected size. Similar results were obtained when total RNA from strain CGMW7, into which CgGAS2 was introduced, was probed with this gene (data not shown). Introduction of CgGAS1 (Fig. 5c) and CgGAS2 (data not shown) into the respective {Delta}Cggas1 and {Delta}Cggas2 mutants reversed the growth and morphological defects.

CgGAS1 and CgGAS2 are functionally homologous to GAS1
Sequence similarities between C. glabrata CgGAS1 and CgGAS2 and S. cerevisiae GAS1, in addition to the constitutive expression level of CgGAS1 and CgGAS2, suggested that the encoded proteins may be functional homologues of the Gas1p protein. Therefore we hypothesized that CgGas1p and CgGas2p should be able to complement the growth and morphological defects of the S. cerevisiae {Delta}gas1 null mutant SCWB-2d. As shown in Fig. 6(a), the mutant cells become extremely large, highly vacuolated, and granulated, and often carry more than one bud. To test our hypothesis, CgGAS1 and CgGAS2 were ligated to the S. cerevisiae vector YEp24 and introduced into SCWB-2d. Fifteen leucine prototrophs from each transformation were subjected to PCR analysis to confirm the presence of CgGAS1 or CgGAS2 (data not shown). The representative S. cerevisiae mutants containing CgGAS1 and CgGAS2, termed SCMW1 and SCMW2, were subsequently subjected to Northern analysis. Using CgGAS1 or CgGAS2 as probes, a strong band of the expected size was detected in the respective transformants (data not shown). Paralleling the expression of CgGAS1 and CgGAS2, the growth and morphologic defects, resulting from the deletion of the GAS1 gene, were reversed (Fig. 6b).



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Fig. 6. S. cerevisiae strain SCWB-2d harbouring the gas1 null mutation (a) and strain SCMW1 that was complemented with the CgGAS1 gene (b). Bar, 10 µm.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have identified a family of genes named CgGAS13. The deduced amino acid sequences and predicted structural homology with the Gas1 protein of S. cerevisiae and the Phr1 and Phr2 proteins of C. albicans suggested similar functions of the CgGas proteins in C. glabrata. Gas1p of S. cerevisae, Gas1p homologues in Schizosaccharomyces pompe, Epd1p and Epd2p from Candida maltosa, Gel1p from A. fumigatus and Phr1p and Phr2p from C. albicans are members of this family of homologous proteins. It has been shown that most of these are involved in cell wall biosynthesis (Fonzi, 1999 ; Kapteyn et al., 1997 ; Mouyna et al., 2000 ; Mühlschlegel & Fonzi, 1997 ; Nakazawa et al., 1998 , 2000 ; Popolo et al., 1997 ; Saporito-Irwin et al., 1995 ; Vai et al., 1996 ). Phr1p and Phr2p have been shown to process ß-glucans of the cell wall and are required for proper cross-linking of 1,3-ß and 1,6-ß glucans (Fonzi, 1999 ).

Northern blot analysis revealed that both CgGAS1 and CgGAS2 were constitutively expressed under all environmental conditions tested. In contrast, no CgGAS3 mRNA was detected. This expression pattern is unique among the reported members of the PHR/GAS gene family. Indeed, in S. cerevisiae of the five GAS genes detected in the sequenced genome only GAS1 is expressed during vegetative growth. Similar to CgGAS3 the other four members are not expressed under standard laboratory conditions and their deletion does not result in any aggravation of the mutant phenotype (Ram et al., 1998 ). Similar to C. glabrata two members of this family, PHR1 and PHR2, are expressed in C. albicans. However, in contrast to CgGAS1 and CgGAS2, PHR1 and PHR2 expression responds to alterations of the ambient pH (Mühlschlegel & Fonzi, 1997 ; Saporito-Irwin et al., 1995 ). Expression of PHR1 and PHR2 is observed in the context of a morphogenic transition from yeast to hyphal growth, triggered by variations of the ambient pH. This transition, termed pH-regulated dimorphism, as well as expression of PHR1 and PHR2 is controlled by the transcriptional regulator Prr2p/Rim101p (Ramon et al., 1999 ; Davis et al., 2000 ; El Barkani et al., 2000b ). C. glabrata does not respond to changes of the environmental pH with a change in cell shape and this may account for the differences in expression patterns observed for CgGAS1 and CgGAS2. In contrast, the recently described fungal species Candida dubliniensis, which has been shown to be a common cause of oropharyngeal candidosis in HIV-infected individuals, responds to changes of the environmental pH with a change in cell shape as well as the pH-dependent differential expression of the PHR homologues CdPHR1 and CdPHR2 (Schorling et al., 2000 ; Kurzai et al., 1999 , 2000 ; Heinz et al., 2000 ).

In order to assess whether the unique regulation of CgGAS1 and CgGAS2 was paralleled by distinct functional features, single deletions of each gene were constructed. To accomplish this goal we used two methodological approaches. In one, the selection marker was flanked by several hundred base pairs of the targeted genomic locus, thus providing ample sequence for homologous recombination. In the second approach, we used short flanking regions of 50 bp, homologous to the targeted genomic locus. Both methods allowed the construction of mutants with the correct integration events. Primer-based disruption of genes of interest has been described for S. cerevisiae and was recently reported in C. albicans (Belli et al., 1998 ; Huang et al., 1997 ; Wilson et al., 1999 ). Our data suggest that this approach may be added to the methodological repertoire available for the molecular analysis of C. glabrata.

Deletion of CgGAS1 or CgGAS2 led to growth and morphological aberrations that were indistinguishable from each other. The cells formed aggregates and their doubling time was increased. Reintroduction of either CgGAS1 or CgGAS2 into the corresponding C. glabrata mutants completely reversed the defective phenotypes.

PHR1 and PHR2 are functionally homologous and both complement a {Delta}gas1 deletion in S. cerevisiae (Vai et al., 1996 ; O. Kurzai & F. A. Mühlschlegel, unpublished data). Likewise, CgGAS1 and CgGAS2 of C. glabrata were able to complement the phenotypic defects of the S. cerevisiae {Delta}gas1 mutant, demonstrating their functional homology. Based on this observation, we speculate that the growth and morphological defects in the C. glabrata CgGAS1 or CgGAS2 single mutants may represent an intermediate phenotype that results from the persistent expression of the remaining homologue. This explanation is reasonable since an intermediate phenotype was observed for the heterozygous S. cerevisiae {Delta}gas1 mutant ND1 (Popolo et al., 1993 ). In addition, intermediate phenotypes are observed in C. albicans {Delta}phr2 mutants, while shifting the ambient pH from permissive neutral to restrictive acid values, gradually reducing the expression of the functional homologue PHR1 (F. A. Mühlschlegel, unpublished data).

We therefore attempted to construct a C. glabrata {Delta}Cggas1/{Delta}Cggas2 double mutant. Although several different approaches were tested and a large number of transformants were analysed, we were not able to isolate transformants containing the double deletion. This may indicate that a C. glabrata {Delta}Cggas1/{Delta}Cggas2 double mutant is not viable or capable of sustained growth. The growth defect that results from the deletion of the PHR/GAS gene family is not identical in the different yeast species. In C. albicans, a {Delta}phr2 mutant will divide once and then stop growing, when transferred to the non-permissive pH 4·0. In contrast, {Delta}phr1 cells, albeit highly reduced, do grow (Mühlschlegel & Fonzi, 1997 ; Saporito-Irwin et al., 1995 ). In S. cerevisiae {Delta}gas1 mutants, growth is reduced several-fold, when compared to wild-type cells (Popolo et al., 1993 ). Since C. glabrata is haploid and lacks a sexual cycle, definitive proof of the viability of a {Delta}Cggas1/{Delta}Cggas2 double mutant will require submitting CgGAS1 to the control of a regulatable promoter in a {Delta}Cggas2 background or vice versa. This will be attempted using the only recently described tetracycline control element (Nakayama et al., 1998 ).


   ACKNOWLEDGEMENTS
 
We acknowledge the expert technical assistance of Barbara Landeck and Stefanie Müksch. We thank Kunio Kitada for strains and plasmids and Elaine Bignell for critically reading the manuscript. This work was supported by grants MU1212/2-1 from the Deutsche Forschungsgemeinschaft (to F.A.M.) and MRC 3856 from the Medical Research Council (to K.H. and T.R.R.). O.K. is supported by a student fellowship from the Studienstiftung des Deutschen Volkes.


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Received 13 October 2000; revised 28 March 2001; accepted 9 April 2001.