Infectious Diseases Section, Yale University School of Medicine, New Haven, CT 06520, USA1
Infectious Diseases Section, VA Connecticut Healthcare System, 950 Campbell Ave, Bldg 8 (111-I), West Haven, CT 06516, USA2
Author for correspondence: Brian Wong. Tel: +1 203 937 3446. Fax: +1 203 937 3476. e-mail: brian.wong{at}yale.edu
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ABSTRACT |
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Keywords: Candida albicans, protein secretion, vesicle trafficking, YPT1
The GenBank accession number for the C. albicans YPT1 sequence reported in this paper is AF330211.
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INTRODUCTION |
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Therefore, our long-term goal is to characterize key steps in the processing and secretion of virulence-associated proteins in C. albicans. In an earlier study, Mao et al. (1999) showed that the C. albicans homologue of the essential Saccharomyces cerevisiae secretion pathway gene SEC4 encodes a ras-like GTPase that is required for growth, secretion of Sap and fusion of post-Golgi secretory vesicles to the plasma membrane. In S. cerevisiae, the essential gene YPT1 encodes another ras-like GTPase that regulates a key step in pre-Golgi vesicle transport (Novick & Brennwald, 1993
). S. cerevisiae ypt1 mutants are defective in protein secretion and display intracellular accumulation of vesicles and elongated structures resembling endoplasmic reticulum (ER) (Segev et al., 1988
). In the present study, we cloned the C. albicans homologue of S. cerevisiae YPT1 and we used molecular genetic approaches to study the function of this gene and to examine its effects on Sap secretion.
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METHODS |
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Libraries and plasmids.
Plasmid p5921, which contains a hisG-URA3-hisG selectable marker, and a library of C. albicans SC5314 genomic DNA fragments in GEM-12 were obtained from W. Fonzi (Georgetown University). Plasmid YM1, which contains the C. albicans GAL1 promoter, has been previously described (Mao et al., 1999
). Plasmid YEp352 was obtained from J. Perfect (Duke University). pBluescript II SK was from Stratagene. Plasmid SFNB490 (which contains S. cerevisiae YPT1) was obtained from P. Novick (Yale University).
Isolation and analysis of C. albicans YPT1.
A 305 bp partial C. albicans gene fragment homologous to S. cerevisiae YPT1 was identified by searching the Stanford C. albicans genome sequencing database, and a 298 bp portion of this sequence was amplified from C. albicans SC5314 genomic DNA and labelled with 32P by PCR, using Taq DNA polymerase and primers P24 (5'-CGAGTGTATATGTGTCGTCAGC-3') and M11 (5'-TCTCTTTGTATGCTTGTACCCG-3'). This PCR product was used to screen a library of C. albicans genomic DNA and positive plaques were identified by autoradiography and purified. The gene of interest was localized to a 2·8 kb XbaI restriction fragment which was ligated into pBluescript SK II to generate pBSYP. Standard methods were used for restriction mapping, subcloning, Southern hybridization, DNA sequencing and transformation of S. cerevisiae mutants.
Genomic Southern blots were probed with a 210 bp 32P-labelled portion of C. albicans YPT1 that encodes a part of C. albicans Ypt1p that is analogous to the C-terminal hypervariable region of S. cerevisiae Ypt1p (Brennwald & Novick, 1993 ). The probe was amplified from C. albicans SC5314 genomic DNA by PCR, using primers Hyp1 (5'-CTATGGCAAGACAAATCAAAGCCC-3') and HCC (5'-CAACTAATCTTCCCCTGATATTTC-3'). This portion of YPT1 was used to minimize cross-hybridization to genes encoding other ras-related GTP-binding proteins. Chromosomal mapping was performed by B. B. Magee (University of Minnesota), as described by Chu et al. (1993)
.
Targeted disruption of C. albicans YPT1.
To disrupt both chromosomal alleles of C. albicans YPT1, it was first necessary to construct a selectable ypt1::hisG-URA3-hisG gene disruption cassette. The first step was to use Taq DNA polymerase and primers A1 (5'-GAAGATCTAGATAAACAGTCGTGTATAAGATG-3') and A2 (5'-CGCGGATCCGTTGCTGTCTGACTTATCGG-3') to amplify 472 bp of 5' untranslated C. albicans YPT1 DNA from pBSYP and to add BglII and BamHI restriction sites (underlined). The PCR product was digested with BamHI and BglII and ligated into the BglII site in p5921, which yielded pL1. Next, primers Z001 (5'-GAAGATCTTGCTGATGCCTTGGACATTCC-3') and Z2 (5'-CGCGGATCCAATAAGAACATTACCTATTTAAAACAAC-3') were used to amplify 351 bp of DNA from the 3' end of the C. albicans YPT1 ORF in pBSYP and to add BglII and BamHI restriction sites (underlined). This PCR product was digested with BamHI and BglII and ligated into the BamHI site in pL1, which yielded pL2. Proper orientation of the inserts was confirmed at each step by PCR and restriction analysis and accuracy of the final construct was confirmed by DNA sequencing.
To disrupt the first chromosomal YPT1 allele, pL2 was digested with BamHI and BglII, and 5 µg of the linearized ypt1::hisG-URA3-hisG gene disruption cassette was introduced into C. albicans CAI4 by the lithium acetate method (Ausubel et al., 1987
). Uracil prototrophs were selected on minimal glucose and purified, and genomic DNA was extracted by vortexing with glass beads. Two methods were used to determine if homologous integration of the ypt1
::hisG-URA3-hisG gene disruption cassette at the YPT1 locus had occurred. First, allele-specific PCR with primers derived from the C. albicans YPT1 locus, P1 (5'-CGATCCAGCCAATTCATTAC-3'), and from the hisG region of p5921, G2 (5'-GCGCGGCGCGACTTCGACAGAACC-3'), was used to determine if the linearized ypt1
::hisG-URA3-hisG gene disruption cassette had integrated homologously within a YPT1 locus. Also, PCR with primers A22 (5'-CTTCTACAGTCGAACAGAGAAG-3') and HCC (5'-CAACTAATCTTCCCCTGATATTTC-3') was used to detect changes in the sizes of the chromosomal YPT1 loci. Second, C. albicans genomic DNA was digested with XmnI and transferred to nylon membranes. The membranes were hybridized in 4x SSC (1x SSC is 0·15 M NaCl, 0·015 M sodium citrate) at 65 °C with a 235 bp 32P-labelled PCR product generated from pBSYP with primers NP01 (5'-ACGGGATATTACTCATGGTGACC-3') and OUT1 (5'-CCAACAGAATGACGGAGAATAC-3'), after which the membranes were washed in 0·2x SSC/0·1% SDS at 65 °C and analysed using the DIG DNA detection kit (Boehringer Mannheim).
To disrupt the second YPT1 allele, selected C. albicans YPT1/ypt1::hisG-URA3-hisG mutants were expanded in YPD to permit loss of URA3 by cis recombination between the flanking hisG repeats, uracil auxotrophs were selected on FOA medium and the genotypes of these strains were determined by PCR and Southern hybridization as described above. The resulting C. albicans YPT1/ypt1
::hisG strains were transformed again with the linearized ypt1
::hisG-URA3-hisG gene disruption cassette, uracil prototrophs were selected and their genotypes were analysed by PCR and by Southern hybridization.
Construction of plasmids pYPT1, pN121I and pD124N.
Mutant ypt1 alleles encoding an Asn to Ile substitution at position 121 (N121I) and an Asp to Asn substitution at position 124 (D124N) of C. albicans Ypt1p were constructed using the QuickChange PCR-based site-directed mutagenesis method (Stratagene). The N121I mutation was generated with Pfu DNA polymerase, pBSYP as the template, and oligonucleotides N121I5 (5'-CCGGTGTTGGGAAAAATTGTTTATTATTGCGTTTTG-3') and N121I3 (5'-CAAAACGCAATAATAAACAATTTTTCCCAACACCGG-3') as primers. The D124N mutation was generated in analogous fashion using oligonucleotides D124N5 (5'-GTTGGTAATAAGGCTAATTTGTCTGATAAAAAAATC-3') and D124N3 (5'-GATTTTTTTATCAGACAAATTAGCCTTATTACCAAC-3'). Primers 5P1 (5'-CCTTAATTAAATGAATAACGAATACGAC-3') and 3K1 (5'-GGGGTACCAAATAAGAACATTACCTATT-3') were used to amplify the wild-type YPT1, ypt1(N121I) and ypt1(D124N) ORFs, and the 1·0 kb PCR products were cloned into the PacI and SpeI sites of the GAL1-regulated C. albicans expression plasmid pYM1, yielding plasmids pYPT1, pN121I and pD124N, respectively (Fig. 1). The accuracy of these DNA constructs was confirmed by DNA sequencing.
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Whether differences in growth rates were plasmid-mediated was examined by plasmid curing. Representative ura+ transformants were expanded for 40 generations in liquid YPD medium and ura- clones were selected and purified on FOA plates. Growth of these ura- clones was then compared to growth of the ura+ transformants on minimal glucose and minimal galactose.
Sap expression was induced by growing the C. albicans transformants to stationary phase in minimal glucose, after which the cells were washed and resuspended at OD600 10 in glucose-BSA-YE or galactose-BSA-YE. The cell suspensions were shaken at 30 °C and cell-free supernatants obtained after 224 h were tested for residual BSA by SDS-PAGE with Coomassie blue staining and for immunoreactive Sap by Western blotting, using polyclonal rabbit antibodies to Sap (from N. Agabian, University of California at San Francisco).
The ultrastructural morphology of C. albicans cells overexpressing ypt1(N121I) was determined by transferring glucose-grown SL21, SL22 and SC5314 cells to minimal glucose or minimal galactose media for 6 h. Cells from 10 ml of culture were harvested by centrifugation, fixed in 3% glutaraldehyde/0·1 M sodium cacodylate (pH 6·8) overnight at 4 °C, washed with 0·1 M sodium cacodylate and post-fixed in 1% OsO4 for 12 h. The fixed cells were embedded in Epox 812x and polymerized at 60 °C for 48 h, sectioned, stained with lead citrate/uranyl acetate and examined with a Philips 300 electron microscope.
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RESULTS |
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Targeted disruption of C. albicans YPT1
Since the results described above indicated that C. albicans YPT1 is present in a single copy per haploid genome, we next attempted to construct C. albicans ypt1 null mutants by homologous gene targeting. When the linearized insert from pL2 (which contains the ypt1::hisG-URA3-hisG gene disruption cassette) was introduced into C. albicans strain CAI4, homologous gene replacement was demonstrated by Southern analysis and allele-specific PCR in 19 of 20 transformants analysed (Fig. 4
).
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Strain T7F4 was transformed again with the linearized ypt1::hisG-URA3-hisG gene disruption cassette. Twenty of 20 transformants derived from strain T7F4 were found by Southern hybridization and allele-specific PCR to retain one wild-type YPT1 allele. Twelve of the transformants demonstrated simple allelic replacement of the ypt1
::hisG allele with gene disruption cassette DNA and eight revealed more complex integration events. When no null mutants were found among the T7F4 transformants, we also transformed strain T8F11 with the same ypt1
::hisG-URA3-hisG gene disruption cassette. Ten of 10 transformants analysed were found by allele-specific PCR and Southern hybridization to retain one wild-type YPT1 allele (Fig. 4
).
Effects of ypt1(N121I) overexpression on growth and viability
Since the results above suggested, but did not prove that C. albicans YPT1 might be an essential gene, we used an alternative approach to study the functions of C. albicans YPT1. One strategy that is often used to examine the functions of essential genes in S. cerevisiae and other organisms is to determine the phenotypic consequences of overexpression of dominant-negative alleles. Because C. albicans Ypt1p shares some key structural features with S. cerevisiae Ypt1p and other Rab-family GTPases, we used this approach to analyse the functions of C. albicans YPT1. When Asn-121 of S. cerevisiae Ypt1p is replaced by Ile or when Asp-124 is replaced with Asn, the resulting proteins function as trans-dominant inhibitors of YPT1 function (Schmitt et al., 1986 ; Jones et al., 1995
). Since C. albicans YPT1 encodes Asn and Asp in the analogous positions (Fig. 2
), we constructed mutant C. albicans ypt1 alleles encoding the N121I and the D124N substitutions and we used a GAL1-regulated expression plasmid to express these alleles in C. albicans.
Multiple attempts to introduce pD124N into C. albicans yielded only four ura+ transformants and all of these transformants grew very poorly both on minimal glucose and minimal galactose. In contrast, C. albicans was readily transformed with plasmid pN121I [which encodes the GAL1-regulated mutant ypt1(N121I) allele]. These pN121I transformants grew normally on minimal glucose, but 10 of 11 transformants tested grew poorly on minimal galactose (Fig. 5). Moreover, plasmid curing experiments verified that growth inhibition in galactose was plasmid-mediated. When four C. albicans pN121I transformants were cured by culturing for 40 generations in YPD and by plating on FOA medium, four of four FOA-resistant clones derived from each transformant (16 total) exhibited wild-type growth on minimal glucose and minimal galactose media.
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DISCUSSION |
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In S. cerevisiae, YPT1 encodes a small ras-like GTPase (Ypt1p) that is required for fusion of pre-Golgi secretory vesicles to the Golgi apparatus (Segev et al., 1988 ). This protein is found associated with the Golgi and ER-Golgi carrier vesicles. Because YPT1 is essential in S. cerevisiae, ypt1 null mutants are non-viable. However, temperature-sensitive and dominant-negative mutations in S. cerevisiae YPT1 inhibit growth and protein secretion and cause pre-Golgi secretory vesicles to accumulate intracellularly (Schmitt et al., 1986
; Jones et al., 1995
; Segev & Botstein, 1987
; Becker et al., 1991
; Jedd et al., 1995
). In the present study, we cloned the C. albicans YPT1 homologue by screening a
library of C. albicans genomic DNA with a radiolabelled probe based on a C. albicans gene fragment highly homologous to S. cerevisiae YPT1 that was identified by the C. albicans genome sequencing project and we showed that C. albicans YPT1 complemented a temperature-sensitive S. cerevisiae ypt1 mutant.
Because C. albicans has a diploid genome and no well-defined sexual cycle, classical genetic approaches are of limited use for studying this organism. One powerful experimental approach is to use homologous gene targeting to delete specific C. albicans genes, after which the phenotypic consequences of the resulting mutations can be ascertained (Kelly et al., 1987 , 1988
; Gorman et al., 1991
). A method for homologous gene disruption in a C. albicans ura3
strain (CAI4) whose virulence can be restored to wild-type levels by reintroducing URA3 is now widely used (Fonzi & Irwin, 1991
) and this and related methods have been used to study the effects of several putative C. albicans virulence factors (Ghannoum et al., 1995
; Hube et al., 1997
; Leidich et al., 1998
). However, an important limitation of this general experimental approach is that disruption of essential genes by definition results in non-viability, thereby preventing subsequent investigation of the function of these genes. In the present study, one chromosomal allele of C. albicans YPT1 was readily disrupted by homologous gene targeting. However, efforts to disrupt the second chromosomal YPT1 allele in two different YPT1/ypt1
::hisG strains yielded no viable ypt1 null mutants among a total of 30 transformants analysed. This result suggested that C. albicans YPT1 may be essential, but a firm conclusion on this point based only on inability to generate a null mutant would have required analysis of many more transformants and/or use of a second gene targeting construct with no sequence homology to the first.
Alternative approaches used to study the functions of essential genes include generation of conditional lethal mutants, overexpression of dominant-negative alleles and replacement of a native promoter with a tightly repressible heterologous promoter. Although these approaches are commonly used in S. cerevisiae, they have only recently been used in C. albicans. One group used homologous gene targeting to introduce a temperature-sensitive mutation in the C. albicans homologue of the essential protein myristoylation gene NMT and subtle temperature-dependent differences in gene function were seen (Weinberg et al., 1995 ). Another group developed a method for using the tightly regulable MET3 promoter to study essential genes in C. albicans (Care et al., 1999
). Also, two groups used overexpression of dominant-negative alleles to define the functions of C. albicans genes. Feng et al. (1999)
use this approach to show that a non-essential ras gene is required for serum-induced hyphal differentiation. Mao et al. (1999)
overexpressed a dominant-negative allele of the secretion pathway gene SEC4 to show that this gene is required in C. albicans for growth, protein secretion and fusion of post-Golgi secretory vesicles to the plasma membrane.
Since we found that C. albicans YPT1 was identical to S. cerevisiae YPT1 at two positions where known amino acid substitutions (N121I and D124N) encode trans-dominant inhibitors of S. cerevisiae Ypt1p, we used PCR-based site-directed mutagenesis to construct mutant C. albicans ypt1 alleles encoding the analogous amino acid substitutions. Despite multiple repeated attempts, only four pD124N-bearing C. albicans transformants were obtained and they grew too poorly for meaningful analysis. One possible explanation is that the D124N mutant allele may cause stronger dominant-negative effects than the N121I allele. Since the C. albicans GAL1 promoter is incompletely repressed in glucose (Brown et al., 1996 ), low-level expression of a strong dominant-negative ypt1 allele might have inhibited growth sufficiently to prevent isolation of transformants. Although this could have been tested by replacing the GAL1 promoter with the more tightly repressible MAL2 or MET3 promoters, this was not necessary because numerous transformants were obtained when C. albicans CAI4 was transformed with plasmids encoding the ypt1(N121I) mutant allele.
The C. albicans pN121I transformants showed poor growth when grown in galactose medium and plasmid curing experiments verified that this effect was mediated by the ypt1(N121I) allele. When pN121I transformants were incubated at high cell density, degradation of Sap was markedly reduced compared to pN121I transformants grown in glucose or pYPT1 transformants in glucose or galactose. These results imply a profound defect in protein secretion, since multiple Saps are normally secreted by C. albicans. Lastly, electron microscopy demonstrated marked accumulation of large membrane-bound structures containing many vesicles and some elongated forms. Since some of these structures resembled the elongated ER that accumulates in S. cerevisiae ypt1 mutants (Schmitt, 1988 ) and since they were very different from the 90110 nm spherical post-Golgi vesicles that accumulate in S. cerevisiae and C. albicans sec4 mutants (Salminen & Novick, 1987
; Mao et al., 1999
), we concluded that the dominant-negative ypt1 allele blocked protein secretion at an early, pre-Golgi stage. Thus, use of a dominant-negative strategy allowed us to ascertain in considerable detail the functions of C. albicans YPT1.
In summary, we have cloned and sequenced the C. albicans homologue of the essential S. cerevisiae secretory gene YPT1 and we have shown that this gene and its protein product are essential for growth and that they function early in the protein secretory pathway. Furthermore, secretion of the virulence-associated enzyme Sap was markedly reduced when YPT1 function was inhibited by overexpression of a dominant-negative allele. These results confirm the usefulness of dominant-negative inhibitors for studying the functions of essential genes in C. albicans. Lastly, since many virulence-associated C. albicans proteins are either secreted into the extracellular environment or localize at the cell surface, the general experimental approach used in this study could be used to analyse the post-translational processing and intracellular trafficking of multiple proteins that are involved in virulence and/or pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 5 January 2001;
revised 21 February 2001;
accepted 5 March 2001.