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Disruption of a gene encoding glycerol 3-phosphatase from Candida albicans impairs intracellular glycerol accumulation-mediated salt-tolerance

Jinjiang Fan , Malcolm Whiteway , Shi-Hsiang Shen
DOI: http://dx.doi.org/10.1016/j.femsle.2005.02.031 107-116 First published online: 1 April 2005


Intracellular glycerol accumulation is critical for Candida albicans to maintain osmolarity, and therefore defects in glycerol homeostasis can have severe effects on the morphogenetic plasticity and pathogenicity of this fungus. The final step of glycerol synthesis involves the dephosphorylation of glycerol 3-phosphate by glycerol 3-phosphatase (GPP1). We have identified a single copy of the GPP orthologous gene (GPP1) in the C. albicans haploid genome, as well as the paralogous gene 2-deoxyglucose-6-phosphate phosphatase (DOG1); both belong to a family of low molecular weight phosphatases. A knockout of the GPP1 gene in C. albicans caused increased susceptibility to high salt concentrations, indicating a deficiency in osmoregulation. Reintroduction of the GPP1 gene complemented the impairment of salt-tolerance in the gpp1/gpp1 mutant. Northern blot analysis showed that the GPP1 gene was strongly responsive to osmotic stress, and its transcriptional expression was positively correlated with intracellular glycerol accumulation. These results demonstrate that the GPP1 gene plays an important role in the osmoregulation in C. albicans.

  • dl-Glycerol-3-phosphatase (GPP1)
  • Gene disruption
  • Glycerol
  • Candida albicans

1 Introduction

Candida albicans is a common human commensal fungal pathogen [1]. The transition from the yeast to the filamentous form has been considered a putative virulence factor for this pathogen [2,3]. Detection of Candida genes that are not homologous to human genes, and are responsible for the virulence of the pathogen, represents a promising means of identifying new drug targets. Searching among the low homology genes and/or Candida-specific genes as potential drug targets is an issue of current pharmacological interest, mainly based on the concern of recent studies on drug-resistance occurring in native and experimental populations of C. albicans[4,5].

Although osmoregulation involves a very conserved mitogen-activated protein kinase (MAPK) controlled pathway from yeast to humans, the upstream and downstream branches of the pathway appear to have some species-specific characteristics [68]. The central component of the osmoregulation pathway in yeast is the MAP kinase, Hog1p [9]. Its homologue from C. albicans has been isolated through yeast complementation [10]. The main effects of the disruption of the C. albicans Hog1 were not only on osmoregulation, but also on morphogenesis and pathogenesis [11]. These phenomena were proposed to be due to the accumulation of intracellular glycerol. However, the regulatory control mechanism of intracellular glycerol synthesis has not yet been clarified in C. albicans.

In the synthesis of glycerol in the budding yeast Saccharomyces cerevisiae, two paralogous genes, the dl-glycerol 3-phosphate phosphohydrolases GPP1 and GPP2/HOR2, encode proteins that control the dephosphorylation of glycerol 3-phosphatate into glycerol and phosphate [12]. Interestingly, two other genes, encoding the 2-deoxyglucose 6-phosphate phosphatases Dog1p and Dog2p, are highly homologous to GPP1 and GPP2, but have different defined functions. These proteins are reported to be involved in the detoxification of a non-metabolizable analogue of glucose, 2-deoxyglucose through conversion of 2-deoxy-d-glucose 6-phosphate to 2-deoxy d-glucose and orthophosphate [13]. However, only GPP2 and DOG1 in S. cerevisiae are responsive to osmoregulation [14]. In humans, glycerol is mainly derived from triacylglycerol (fat) through lipolysis, and not directly from the glycerol-3-phosphate that normally forms fat through sequential addition of three fatty acids [15,16]. In fact, the biochemical synthesis of glycerol seems to be absent in mammals.

Thus, we reasoned that the synthesis of glycerol in C. albicans could be a potential target for the drug development, given that the accumulation of intracellular glycerol affects cellular growth characteristics. Here, we report the identification of the GPP homologous genes from the human fungal pathogen C. albicans and characterize the salt sensitivity that resulted from the knockout of the GPP1 gene.

2 Materials and methods

2.1 Strains, growth conditions and plasmid preparation

The C. albicans strains used for this study are listed in Table 1. C. albicans strains were grown routinely at 30 °C in YPD medium (1% yeast extract, 2% peptone, and 2% glucose), synthetic complete medium [0.67% (wt/vol) Difco yeast nitrogen base without amino acids and 2% (wt/vol) glucose] and synthetic complete medium lacking specific nutrients. Escherichia coli strain DH5α and LB medium were used for transformation and plasmid DNA preparation. Plasmid pFLAG-Met3 was used for cloning of GPP1 in C. albicans. Vector CIp10-MAL2p was used for cloning and introducing DOG1 gene into C. albicans. Sequencing of both strands of DNA was performed by the dideoxy-chain termination method [17], using the ABI PRISM dye terminator cycle sequencing reaction kit (Perkin–Elmer), and reactions were analyzed with an automated sequencer (ABI, Model 377).

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C. albicans strains used in this study

2.2 DNA and RNA manipulations

Recombinant DNA, genomic DNA and RNA handling were carried out by standard techniques [18]. Yeast genomic DNA for PCR was prepared according to manufacturer's manual (Qiagen Inc, Germany). Southern blot hybridizations were carried out with α-32p-dCTP labeled hybrid probe as shown in Fig. 2. The 512 bp PCR amplicon with primers (GPP1-locus-R, 5′-TCCAATGATTTCCACA TTCG-3′, and GPP1-URA3-F, 5′-TTATACCATCCAAATCCCGC-3′) was radioactively labeled by random priming with Ready-To-Go DNA labeling Beads (Amersham, USA) and was used as a hybridization probe following standard procedures [18]. Total RNA from C. albicans was isolated from 50 mg (wet weight) samples by the method described in Qiagen Inc., and fractionated by electrophoresis in a 1.5% formaldehyde agarose gel. Nucleic acids were transferred to hybond-N nylon membranes as recommended by the manufacturer and Northern hybridization was performed under stringent conditions using standard protocols. The 1.5-kb ClaI–SalI fragment from the C. albicans actin gene [19] was used as a probe in control hybridization.


Disruption of the C. albicans GPP1 gene. (a) Restriction maps of the C. albicans GPP1 gene and disruption strategy for the GPP1. (b) PCR analysis of transformants with GPP1 gene disruption. M. 1 Kb ladder. Lane 1, JF1 (gpp1/GPP1); Lane 2, Rm1000; Lane 3, JF34 (gpp1/gpp1), and Lane 4, JF1 (gpp1/GPP1), Lane 5, JF34 (gpp1/gpp1); Lane 6, Sc5314. His1, PCR using His1-specific primers; Ura3, PCR using Ura3-specific primers; GPP1, using GPP1-specific primers. (c) Southern blot analysis of tranformants with PCR-based gene disruption. Lane 1, Sc5314; Lane 2, JF1 (gpp1/GPP1); Lane 3, JF34 (gpp1/gpp1). The probe used was the 512 bp PCR amplicon using primer GPP1-locus-R/GPP1-out-F (see Table 2) and is indicated in panel A as black solid bar. Genomic DNAs were digested with ClaI and HindIII. The relevant genotypes of the strains used for DNA analysis are indicated under the lanes. The exact sizes of expected hybridizing DNA fragments are indicated on the right.

2.3 Sequence analyses

The predicted amino acid sequences of GPPs and DOGs in S. cerevisiae were used to search the C. albicans genome database assembly (Version 19) at the Stanford DNA Sequencing and Technology Center (http://www-sequence.stanford.edu/group/candida). Sequencing of C. albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund. The database searches were performed using the TBLASN algorithm with default settings [20]. The predicted amino acid sequences of each selected genes were aligned using the PILEUP program of the GCG Wisconsin sequence analysis package (Wisconsin Package Version 10.0-UNIX, Genetics Computer Group (GCG), Madison, Wisc., 1999). Phylogenetic analyses were performed using programs in the Phylogeny Inference Package (PHYLIP), version 3.57c [21]. For distance-based methods, pairwise distances between each protein were calculated using PROTDIST. These distances were estimated by using the Dayhoff PAM matrix setting, and the genealogy was estimated by using the Neighbor-Joining algorithm (NEIGHBOR) in PHYLIP. Bootstrap values were calculated using the method of Felsenstein in PHYLIP [21] with 100 replications. Protein sequence motifs were identified via an iterative MEME/MAST approach (http://meme.sdsc.edu) [22].

2.4 Gene knockout procedure

Single and double allelic knockouts of the GPP1 gene (orf19.5437/orf19.12892) were achieved by the procedure described previously [23]. In brief, URA3-dpl200 and/or His1 cassettes were PCR-amplified with the introduction of flanking 60 bp of target genes at 5′-end and 3′-end of each open reading frame (ORF). The PCR products were directly used to sequentially transform a suitable host strain through electroporation [24]. Primers were designed based on the predicted genomic sequence of each target gene (Fig. 2), and are listed in Table 2. Correct insertion and deletion of the Ura3 and/or His1 marker were checked by PCR using specific primers (Fig. 2(a)). The colonies producing a correct PCR pattern were further checked by Southern blot analysis using a hybrid probe originating from the Ura3/His1 marker and target gene to rule out possible off-target insertion (Fig. 2 (c)). Loop-outs of the Ura3-marker from the chromosome were selected by growing the cells overnight in SD medium without adding histidine and uridine, and then plating the cells on minimal medium supplemented with uridine (50 μg/ml) and 5-fluoroorotic acid (5-FOA) (200 μg/ml). A conditional knockout of the DOG1 gene (orf19.10895/Orf19.3392) was made by introducing a Mal2-DOG1 linearized plasmid derived from vector CIp10-MAL2p [25]. The plasmid, named as CIp10-Mal2-DOG1, contains a fragment of the 5′ end of the DOG1 ORF at the cut-sites of MuI/XbaI that was then linearized by NcoI before transformation.

View this table:

Oligonucleotides used in this study

2.5 PCR amplifications

Pfu polymerase (New England Biolabs, USA) was used for PCR to generate DNA fragments used in gene knockout, cloning steps and/or as probes. PCR buffers and conditions were those suggested by the manufacturer. PCR was carried out in a thermal cycler 480 (Perkin–Elmer) with a first cycle at 94 °C for 2 min, followed by 30 cycles of annealing at 55 °C for 1 min. Elongation was performed at 72 °C for 2 min. The different primers used for PCR are described in Table 2. C. albicans DNA templates for PCR were prepared from overnight cultures by mechanical breakage with glass beads, and then used DNeasy Plant Mini Kit following the procedure in the manufacturer's manual (Qiagen Inc, Germany).

2.6 Re-introduction of the wild-type GPP1 gene

For re-integration of a single copy of the GPP1 gene into the gpp1/gpp1 deletion mutant JF34, the C. albicans wild-type GPP1 gene on a 765-bp fragment was amplified from genomic DNA of strain SC5314 by standard PCR with primers Flag-GPP1R (5′-GCCTGCAGATGACAAAGACTCAACAACC-3′) and Flag-GPP1F (5′-GCGCATGCAGCAGATTCTTGTAAAAATTGC-3′). The resulting PCR product was digested with PstI and SphI, and ligated into plasmid pFlag-Met3 at the PstI and SphI sites [26], resulting in plasmid pFlag-Met3-GPP1. Plasmid pFlag-Met3-GPP1 was digested with StuI and transformed into the gpp1/gpp1 strain JF34lp1 to generate the gpp1/gpp1+GPP1 complemented strain JF34lp+. Integration was confirmed by PCR with primers Flag-GPP1R/Flag-GPP1F.

2.7 C. albicans transformation

C. albicans strains were transformed by electroporation [24]. PCR products were directly used for the GPP1 gene deletion, and the plasmids were linearized with StuI and then transformed for reintroduction of the GPP1 gene into C. albicans. Uridine and/or histidine prototrophic transformants were selected on SD agar plates without uridine and/or histidine.

2.8 Quantitative dilution assays and plate assay

Saturated cultures of C. albicans strains were diluted into SD liquid medium. A serial dilutions of cell suspensions from an initial OD600= 0.5 were spotted onto SD solid medium with or without uridine and/or histidine and incubated at 30 or 37 °C for 3 days.

2.9 Measurement of intracellular glycerol

Determination of intracellular glycerol was done spectrophotometrically with a commercial glycerol determination kit (Boehringer-Mannheim Biochemicals) as described in the instructions from the manufacturer. In brief, cells were grown overnight in selection medium (SD) with supplements of uridine or histidine. A 10 ml culture of YPD was re-inoculated with 1:50 of the overnight culture, and was grown to OD600= 0.5 (about 4 h). Then the cells were treated with 0.5 M NaCl for 45 min by adding 10 ml of 1 M NaCl in YPD, or treated with YPD only as control. To collect the cells, the 20 ml cultures were spun down and the pellets were washed and resuspended in 2 ml of 0.5 M Tris–HCl, pH 7.5, by vortexing. One ml was heated at 95 °C for 10 min and then centrifuged at low-speed (about 5000 rpm for 30 s). The supernatant was used for glycerol determination according to the manufacturer's specifications. Glycerol concentrations were normalized to the weight of each pellet. Data represent the average of three independent experiments.

3 Results and discussion

3.1 Identification of S. cerevisiae GPP orthologues in C. albicans

In S. cerevisiae, two GPPs are closely related to each other with 95% amino acid identity. These form a new family of low molecular weight phosphatases together with two DOGs, which encode highly homologous enzymes capable of dephosphorylating 2-deoxyglucose-6-phosphate [14]. To identify the GPP homologous sequences from C. albicans, we used BLAST programs to search the C. albicans genome database for S. cerevisiae GPP1 homologues and adopted an established phylogenetic method to predict the functions of each homologous sequence from C. albicans. Two genes were found that possessed, respectively, 46% and 38% identities in amino acid sequence to that of the GPPs and DOGs from S. cerevisiae. We used MEME as a motif discovery tool and obtained five motifs, which occur in most of the selected sequences (Fig. 1(a)). Interestingly, motif 1 with a consensus sequence DXDG(T/V/L), contains a hydrolase signature DXDXT/V [28]. This motif at the N-terminus of GPP1 and/or DOG1 and most of the other sequences selected may indicate a functional importance within the class of low molecular weight phosphatases (Fig. 1(a)). Four sequences in the alignment, including GS1 that encodes a human protein of unknown function [27], have a threonine replaced by a leucine at the position +5 in the motif DXDXT/V (Fig. 1(a)); this may affect their conserved functions. The first aspartate in the motif was previously reported to be phosphorylated and strictly conserved in a large family of hydrolases comprising phosphatases and haloacid dehalogenases [28,29].


Comparison of the C. albicans homologues of Saccharomyces cerevisiae GPP1/GPP2 and DOG1/DOG2 to other sequences of GPP- and DOG-related proteins. (a) Deduced amino acid alignments for GPP- and DOG-sequences from C. albicans and 13 members of the haloacid dehalogenase hydrolase/phosphatase superfamily from other species. The GenBank Accession Nos. of the sequences used here are as follows: Aspergillus nidulans (GPP_an), AF043232; Sinorhizobium meliloti (GPP_sm), CAB01954; Escherichia coli (GPP_ec), AAG57422; Drosophila melanogaster (GPP_dm), AE003586; D. melanogaster (GS1L_dm), Q94529; Human (GS1), XP_010289; Schizosaccharomyces pombe (Hydro_sp), T40833; Streptomyces coelicolor (GPP_sco), CAB76079; Mycobacterium tuberculosis (Hydro_mt), (AAK47845); S. cerevisiae (ScDOG1), NP_011910; S. cerevisiae (ScDOG2), NP_011909; S. cerevisiae (ScGPP1/rh2), NP_012211; S. cerevisiae (ScGPP2/hor2), NP_010984. Alignments were made using the PILEUP program of the GCG Wisconsin sequence analysis package. Residues conserved in complete or 100% conserved, 80% or greater conserved, and 60% or greater conserved, are highlighted in black, dark grey, and light grey, respectively. The top two levels are also distinguished by either upper or lower case characters on the consensus line. Similar amino acids are defined by Higgins et al. [34]. Dashes indicate gaps introduced to facilitate alignment. Solid bars indicate the conserved region in motif, DXDXT/V of the hydrolases/phosphatases, and other motifs obtained from the MEME/MAST searches. (b) Unrooted protein phylogeny for GPP- and DOG-like sequences from C. albicans and other organisms. This tree was produced using the neighbor-Joining algorithm with the PHYLIP 3.5 package [21] on the core 246 aa of sequence conserved between all selected hydrolases/phosphatases. The numbers at the nodes represent the bootstrap percentages (100 bootstrap resamplings), showing only those larger than 50%. The bold value on the node and thicker branch represent the two main phylogenetic clades. The scale bar indicates the estimated number of amino acid substitutions per site.

Phylogenetic analysis demonstrated that the two genes from C. albicans were GPP1 and DOG1 orthologues, suggesting that the two genes in C. albicans have diverged from each other before species separation from S. cerevisiae. GPP1 (orf19.5437/orf19.12892) from C. albicans contains an open reading frame (ORF) encoding a putative protein of 254 amino acids with a calculated molecular mass of 28.1 kDa, while DOG1 (orf19.10895/Orf19.3392) from C. albicans contains an ORF with 240 amino acids and a molecular mass of a 26.1-kDa. The predicted amino acid sequences encoded by GPP1 and DOG1 exhibit high similarities with the sequences of other members of the haloacid dehalogenase hydrolase/phosphatase superfamily from other organisms including humans, but the GPP1 and DOG1 genes belong to one of the two main distinct phylogenetic clades with 99% bootstrap support, suggesting that they are distantly related to the homologous gene GS1 from humans (Fig. 1 (b)).

3.2 Chromosomal deletion of GPP1

To illuminate the functions of GPP1, we deleted both chromosomal copies of the gene in C. albicans (Fig. 2). The central part of the GPP1 coding regions was replaced sequentially by PCR products of His1 and URA3-dpl200 with using two flanking sequences of the target gene for gene deletion through homologous recombination (Fig. 2(a)). Out of 60 transformants from the first round of transformation, 50% had the PCR amplicon insertion at the GPP1 locus based on a PCR screening strategy (Fig. 2 (b)). The colonies with a right sized PCR product were picked for further Southern blot analysis. The pattern of Southern hybridization with the 512 bp target gene/Ura3 hybrid probe was consistent with integration of the Ura3 and His1 cassettes at the GPP1 loci uniquely (Fig. 2 (c)). To re-introduce the deleted genes, the mutants were grown on FOA-containing SD agar plates to obtain Ura strains through eliminating the URA3 marker, and then a plasmid construct containing a functional gene was transformed into the cells. The revertants were confirmed by direct PCR of the GPP1 encoding gene (data not shown).

3.3 GPP1 can account for the high osmo-resistance of C. albicans

The ability to obtain the homozygous gpp1/gpp1 mutant in C. albicans suggests that GPP1 is not essential for cell viability. However, a gpp1/gpp2 double mutant in S. cerevisiae is hypersensitive to stress growth conditions [12]. To determine whether the gpp1/gpp1 mutant of C. albicans is sensitive to NaCl, the homozygous mutant was tested against a serial salt dilution (Fig. 3). The result showed that the growth of the gpp1/gpp1 mutant, when compared to the heterozygote and wild-type parental strains, was strongly inhibited by increased salinity.


Sensitivity of GPP1 knockout strains to NaCl compared to wild types. C. albicans cells were grown to exponential phase in selection medium and then diluted to OD600= 0.5. A serial dilution of cells from 10−3 to 10−7 were made as show above each panel, and then spotted onto SD plates (10 μl each). The plates were incubated at 30 °C for 3 days (a), and incubated at 37 °C for 3 days (b) before microphotographs were taken. (c) Reintroduction of the GPP1 gene restored its salt resistance. The revertant strain was constructed from the null mutant for GPP1 with a functional GPP1 gene driven by the MET3 promoter. The GPP1 ORF was cloned into a plasmid pFLAG-Met3[26], and then the plasmid was used to transform the null mutant strains for GPP1 gene to make a revertant (JF34lp+). Wild type, parental, revertant, and mutant strains were grown in SD medium, diluted, and spotted on SD plates. The plates were incubated at 30 °C for 3 days.

Using similar disruption strategies, we disrupted a single allele of DOG1 gene, but were not able to obtain the dog1/dog1 homozygous mutant, which indicates either that DOG1 gene is essential for C. albicans survival, or that deletion may damage other essential genes adjacent to its locus. In support of the later assumption there are only 241 bp between the terminus of the DOG1 gene and the presumed promoter region of its neighboring gene encoding a dead box helicase (orf19.3393/orf19.10896). Nevertheless, we constructed a conditional mutant using the MAL2 promoter (data not shown) and compared this strain's growth under the same saline conditions. It appears that DOG1 has a different role from that of GPP1 (Fig. 3), which is consistent with their distinct physiological functions in S. cerevisiae[13].

To examine whether the reduced resistance to salt observed above was directly caused by the deletion of the GPP1 gene, we reintroduced GPP1 under control of the MET3 promoter on an integrating plasmid into the gpp1/gpp1 homozygous mutant. In the reintegrant the reduced resistance to salt was reversed back to that of the wild type and parental strains (Fig. 3 (c)). The gene products of GPP1 and GPP2 in S. cerevisiae have been shown to be similar in enzymatic activities, but only GPP2 is regulated by osmotic stress [12,14]. In C. albicans, it seems that the unique GPP1 gene is essential for salt resistance.

3.4 GPP1 null mutant cells exhibit reduced accumulation of intracellular glycerol

Glycerol is of great importance as an osmolyte in the cellular response to stresses. To synthesize large amounts of glycerol in response to stress, the pathway-related enzymes have to be activated. To examine whether the transcription of the GPP1 gene in the wild-type strain is related to stress conditions, Northern hybridization with a GPP1 probe was analyzed and compared with that using an Act1 probe as a control. GPP1 transcription was weakly detected in the untreated cells, whereas cells treated with higher salt concentrations exhibited a higher lever of GPP1 mRNA (Fig. 4(a)). The results demonstrated that GPP1 was highly up-regulated by the stress response, consistent with a function in the osmoregulation.


The expression of the GPP1 is responsive to different concentrations of NaCl. (a) Northern blot analysis of GPP1 expression in response to different concentrations of NaCl. Total RNA was isolated from exponential phase Sc5314 cell grown in YPD containing 0, 0.75, 1.0, and 1.5 M of NaCl. The blot was probed using GPP1 full ORF. A PCR-amplified DNA fragment of the C. albicans actin gene (ACT1) was used as control. (b) Comparison of intracellular glycerol accumulation among wild type (1), parental (2), single chromosomal copy mutant (3), and double chromosomal copies mutant (4), and revertant (5) strains, subjected to NaCl in YPD medium. The intracellular glycerol content was measured from 3 independent experiments after the addition of 0.5 M NaCl (solid bars) to exponentially growing cultures. As a non-omostressed control, one-half of the culture received just YPD medium (open bars).

In the 5′-nontranslated regions of the GPP1 gene from C. albicans there were two canonical nuclear factor 1-like proteins (NF1) binding sites (TGGCA), and two hexameric repeats (HR), TTGCTA, which are found in WHI1 gene from C. albicans[30,31] (data not shown). It seems that GPP1 gene may also plays a role in white/opaque transition. Even though the potential role of GPP1 in the white/opaque transition has not been well established, these motifs, together with two putative TATAA boxes and two CAATA boxes in the same region, suggest that the expression of the GPP1 gene has the potential to be highly regulated. In fact, genome-wide transcriptional profiling has showed that the GPP1 gene is up-regulated in white cells [35], and a comprehensive microarray analysis has reported the GPP1 gene to be significantly repressed upon induction of hyphal growth [32].

Because the ultimately controlled product of GPP1 is the accumulation of glycerol, we measured the intracellular concentrations of glycerol in the gpp1/gpp1 mutant and in its parental strains in the presence of 0.5 M NaCl (Fig. 4 (b)). The results showed that glycerol accumulation in the gpp1/gpp1 mutant upon exposure to salt was significantly lower than that of its parental strain, the heterozygous mutant or the revertant (Fig. 4 (b)), indicating that GPP1 significantly contributes to glycerol synthesis in C. albicans. The revertant possesses a single copy of the GPP1 gene inserted into the RP10 locus; the glycerol values in the revertant were similar to that of heterozygous mutant, consistent with our expectations. The C. albicans GPP1 has a function similar to that of GPP2 in S. cerevisiae, the gene is responsible for osmoregulation in yeast, whereas GPP1 in S. cerevisiae appears to play a different role in the physiology of the organism [12]. Nevertheless, the level of glycerol accumulation is a mechanism essentially responsible for the salt-tolerance in C. albicans. Since intracellular accumulation of glycerol also plays a role in germination of some fungi and in penetration of the cuticles of rice blast's host plants [33], a further study on the function of glycerol in hyphal formation and in other stress conditions should help provide us with new insights into the virulence factors of C. albicans.

4 Conclusions

We have identified two GPP homologues (GPP1 and DOG1) from C. albicans by phylogenetic sequence analysis. The gene disruption of the GPP1 orthologue from C. albicans showed that it plays an important role in the intracellular glycerol-mediated osmoregulation. Consequently, the GPP orthologue of C. albicans may contribute to pathogenesis in humans. Since the expression of the GPP1 gene is significantly up-regulated by high salinity, but down-regulated during the yeast/hyphae- and white/opaque-transitions in C. albicans[32,35], further study on the mutant GPP1 gene should clarify the biological function of glycerol in this human pathogenic yeast.


This work was supported by Visiting Fellowships in Canadian Government Laboratories from the Natural Sciences and Engineering Research Council of Canada to J.F. through the NRC GHI program. We acknowledge help and advice received from Denis Banville and Zhenbao Yu (BRI, NRC, Canada). We wish to thank Denis L'Abeé for technical support.


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