1 Robert Koch-Institut, Nordufer 20, D-13353, Berlin, Germany
2 Molecular Phytopathology and Genetics, University of Hamburg, Biocenter Klein Flottbek, Ohnhorststr. 18, D-22609 Hamburg, Germany
3 Microbiology Research Division, School of Dental Science, University of Dublin, Trinity College, Dublin 2, Republic of Ireland
4 Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland
5 Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
Correspondence
Bernhard Hube
hubeb{at}rki.de
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ABSTRACT |
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INTRODUCTION |
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Mammalian PI-Plcs are grouped into four subfamilies (,
,
and
) whose structural organization shares an X and a Y domain, but differs in elements necessary for their specific functions and localizations (Rhee, 2001
). In lower eukaryotes, such as yeasts, only
isoforms have been found, leading to the hypothesis that this isoform represents an archetypal phospholipase C (Ochocka & Pawelczyk, 2003
; Rhee, 2001
). Similarities between mammalian and bacterial PI-Plcs are reduced to a few residues within the N-terminal part, representing the X domain (Griffith & Ryan, 1999
).
A gene encoding a PI-Plc (PLC1) has been identified in Saccharomyces cerevisiae (Flick & Thorner, 1993; Payne & Fitzgerald-Hayes, 1993
; Yoko-o et al., 1993
) and depending on the genetic background, PLC1 in S. cerevisiae can be essential (Yoko-o et al., 1993
). In other studies, disruption of PLC1 revealed a large number of phenotypic responses. For example, plc1 mutants showed osmosensitivity at high concentrations of carbohydrates or NaCl, chromosome mis-segregation, temperature sensitivity and reduced growth rates in media containing non-glucose carbon sources (Flick & Thorner, 1993
; Payne & Fitzgerald-Hayes, 1993
). Some of these phenotypes (chromosome missegregation, temperature sensitivity) could be partially restored with exogenous Ca2+ ions, which supported the view that PLC1 is involved in Ca2+-signal transduction of S. cerevisiae. PLC1 was shown to be essential for glucose-stimulated PI turnover and subsequent activation of plasma-membrane H+-ATPases (Coccetti et al., 1998
), and mutants lacking PLC1 failed to regulate intracellular Ca2+ concentrations in response to extracellular glucose, suggesting a role for Plc1p in transducing glucose signals (Tisi et al., 2002
). Furthermore, Ansari et al. (1999)
showed that Plc1p interacts with the receptor-like protein Gpr1p as a component of the nitrogen-signalling pathway for formation of pseudohyphae in S. cerevisiae. Based on these results it was suggested that Plc1p acts in two filamentation pathways, the cAMP- and the Ras2-controlled (mitogen-activated protein kinase, MAPK) pathway.
While traditionally, Plc activity has been thought to be associated with the plasma membrane, a putative nuclear localization sequence was described in an early report of PLC1 (Payne & Fitzgerald-Hayes, 1993), and York et al. (1999)
and Odom et al. (2000)
demonstrated a nuclear function of Plc1p within an inositol polyphosphate pathway for mRNA export and regulation of the transcriptional complex ArgR-Mcm1 (reviewed by York et al., 2001
). Moreover, Plc1p physically interacts with the nuclear protein Sgd1p, with Ndc10 and Cep3 (two proteins of the kinetochore complex), and with Tor2, a protein involved in actin structure formation (Lin et al., 1998
, 2000
, 2002
). These results show that Plc1p acts as an important multifunctional protein at several locations within the yeast cell.
Candida albicans is a human-pathogenic fungus of increasing clinical importance closely related to S. cerevisiae. Among the most prominent virulence factors of C. albicans are extracellular hydrolytic enzymes and the ability to grow either in a yeast or a hyphal form (dimorphism) (Calderone & Fonzi, 2001). Extracellular hydrolases include proteinases (Naglik et al., 2004
), lipases (Hube et al., 2000
) and phospholipases (Ghannoum, 2000
), the latter with phospholipase A and B activities (Ghannoum, 2000
). The dimorphic transition is regulated by a number of signal transduction pathways such as the MAPK and cAMP/protein kinase A pathways (reviewed by Sudbery et al., 2004
; Whiteway & Oberholzer, 2004
). In addition, lipid signalling pathways with second messengers such as DAG may play a role during morphogenesis of C. albicans (Hube et al., 2001
).
Interestingly, while PLC1 is the only known gene encoding a PI-Plc in S. cerevisiae (Jun et al., 2004), at least two PLC genes have been identified in C. albicans. The first PLC gene identified in C. albicans (named CAPLC1) is an orthologue of PLC1 of S. cerevisiae (Bennett et al., 1998
). CAPLC1, which has been shown to be transcribed in both the yeast and hyphal forms of C. albicans, encodes a large protein of 1099 amino acids with classical X and Y domains (27 % homology to ScPlc1p), with phopsholipase C activity when expressed in Escherichia coli. The protein encoded by the second gene (named PIPLC) is more similar to extracellular bacterial phospholipases C than to the phospholipases C from higher eukaryotes (Andaluz et al., 2001
). PIPLC was described as a significantly smaller gene compared to CAPLC1 (1029 bp), has a presumptive homologue in the neighbourhood of the gene LIG4 and produced DAG when expressed in E. coli. The fact that this gene has no counterpart in S. cerevisiae may suggest that it evolved during adaptation of C. albicans to the human host. However, the function of PLC genes in C. albicans is unknown.
The aim of this project was to study the function of these two different types of PI-PLC genes in C. albicans. Therefore, we have investigated the role of CAPLC1 (named CaPLC1 in this study), PIPLC (renamed CaPLC2 in this study) and a third PLC gene (named CaPLC3) in growth, cellular functions, morphology and virulence.
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METHODS |
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For studying the PI-Plc inhibitor 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosporylcholine (ET-18), hyphal formation was induced by pH- and temperature-shift (Buffo et al., 1984) and inhibitor was added at concentrations of 10, 20 and 200 µM. Three independent samples were taken after 1, 2, 3, 4 and 5 h to determine the percentages of hyphal formation. For phenotypic screening of mutant strains a serial drop dilution test was used. Samples (5 µl) of suspensions with different concentrations of cells were dropped onto the corresponding solid media. For screening the conditional mutant
Caplc1/pMET3-CaPLC1, wild-type and mutant cells were pre-cultured in SD supplemented with 2·5 mM methionine and 2·5 mM cysteine at 37 °C for 1 day prior to counting. To analyse growth of the heterozygous mutant Caplc1/CaPLC1 in non-glucose media, cells were incubated in SD without glucose, but with 3 % (v/v) glycerol or 2 % (w/v) potassium acetate. For expression studies the following growth conditions were used. For expression of CaPLC1 and genes possibly associated with CaPLC1 functions, CAF2-1 was grown overnight at 30 °C in SD medium, diluted into fresh SD medium to give a concentration of 106 cells ml1 and incubated at 37 °C to an OD600 of 0·6. The culture was then divided into 40 ml aliquots, centrifuged and the pellet resuspended in 40 ml of the following media: SD at 37 °C, SD at 42 °C (heat shock), SD containing 60 µM cadmium at 37 °C and SGlyc [SD with 2 % (v/v) glycerol instead of 2 % (w/v) glucose] at 37 °C. After 1 h incubation, cells were harvested for RNA extraction (see below). For expression of PLC2 and PLC3, an overnight culture of SC5314 was grown in SD at 30 °C, diluted into SD (1 : 100), YPD (1 : 100), or Sabouraud glucose (SG) (1 : 1000) and incubated at 37 °C until the appropriate OD600 was reached (see Fig. 5
). Aliquots (10 ml) were harvested for RNA extraction. For SLAD [6·7 g l1 YNB (Difco) without amino acids and ammonium sulfate plus 0·05 mM (NH4)2SO4] and YPD plus 5 % (v/v) fetal calf serum (YSer), 2x107 cells ml1 were inoculated from a preculture grown in SD at 37 °C and 25 ml aliquots were harvested for RNA extraction. For induction of the PCK promoter we used SGlycAc [SD without glucose but with 2 % (w/v) potassium acetate and 3 % (v/v) glycerol] and succinate medium (B-medium) (Leuker et al., 1997
; Stoldt et al., 1997
).
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Disruption of CaPLC1
Four different disruption cassettes were produced to disrupt CaPLC1, as follows.
Construction of pDB104.
A 716 bp PCR fragment containing the region between 1043 bp and 1759 bp of CaPLC1 (accession no. Y13975), was cloned into pMB7 (Fonzi & Irwin, 1993) upstream of the hisG-URA3-hisG cassette. A second PCR fragment representing 2668 bp and 3445 bp of CaPLC1 was ligated into the downstream region of the hisG-URA3-hisG cassette of pMB7 and named pDB104. pDB104 was linearized with PvuII prior to transformation and used to disrupt the first allele of CaPLC1.
Construction of pDK-4.
pDK-4 contains an allele-specific CaPLC1-disruption cassette. PCR products for the gene-specific regions of the cassette were amplified using the primers PLC1-3 and PLC1-4 (Table 1) with DNA from the heterozygous
Caplc1/CaPLC1 mutant as template. This PCR fragment was ligated into pCR2.1-TOPO (Invitrogen) to produce pDK-1. The fragment was subcloned into pGEM-T Easy (Promega) to give pDK-2. The selection marker URA3 was amplified from pMB7 with primers URA-1 and URA-2 and ligated into pCR2.1-TOPO (pDK-3). Finally, the URA3 fragment of pDK-3 was cloned into the CaPLC1 fragment within pDK-2 to give pDK-4. pDK-4 was linearized prior to transformation of the Ura heterozygous
Caplc1/CaPLC1 mutant.
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Construction of cassette 4 (oligomer-based).
We also aimed to disrupt CaPLC1 with a PCR product that had homologous oligonucleotide sequences to the CaPLC1 gene sequence on each end of the URA3 marker as described by Wilson et al. (1999). A primer pair was designed in which each primer overlapped both the CaPLC1 and URA3 genes. Primer PLC1-5 shared 58 bp and 22 bp homology to the CaPLC1 and URA3 genes, respectively and PLC1-6 had 57 bp and 23 bp homology to the CaPLC1 and URA3 genes, respectively (Table 1
). This disruption cassette was amplified with the plasmid pGEM-URA3 as template.
Construction of a conditional CaPLC1 mutant.
In order to produce a conditional CaPLC1 mutant we used primers PLC1-7 and PLC1-8 flanked by a BamHI and a PstI site to amplify a 1350 bp PCR product containing a fragment of the 5' end of CaPLC1 beginning with the ATG start codon. This fragment was cloned into the BamHI/PstI sites of pCaDis (Care et al., 1999) to give pIM14. pIM14 was linearized with ClaI and transformed into the Ura heterozygous
Caplc1/CaPLC1 mutant. Integration into the non-disrupted allele was confirmed by Southern analysis and PCR using primers pMET1 and PLC1-9. Integration into the non-disrupted allele was shown by the amplification of a 2·8 kb PCR product containing a HindIII site, while integration into the disrupted allele resulted in an amplicon of 3 kb without a HindIII site.
Cloning and sequencing of CaPLC2 and CaPLC3.
The genes were amplified using the Expand Long Template PCR System (Roche), genomic DNA of SC5314 and the primers PLC2-9, PLC2-10, PLC3-2 and PLC3-3. PCR products were ligated into pGEM-T or pGEM-T Easy and transformed into E. coli TOP 10 (Invitrogen). PCR fragments were sequenced using T7 or gene-specific primers (PLC2-0/PLC2-10 and PLC3-2/PLC3-3) (Table 1). Sequences were compared with sequences of PI-PLC (AJ277538) (Andaluz et al., 2001
) and the Candida database (http://www.Pasteur.fr/recherche/unites/Galar-Fungail) of the Institut Pasteur, France, using the BLASTN search mode.
Disruption of CaPLC2 and CaPLC3.
Using primers PLC2-5 and PLC2-6 almost the complete ORF of CaPLC2 was amplified using genomic DNA of SC5314, and the corresponding PCR product was ligated into pGEM-T Easy, generating pDK-6. The fragment was sequenced and the sequence compared with published data. A HindIII restriction site downstream of the insert was deleted by linearizing the plasmid with HindIII, treatment with mung bean nuclease (MBI Fermentas) followed by religation, to generate pDK-7. The deletion was confirmed by sequencing. Primers PLC2-7 and PLC2-8 were then used to amplify a PCR fragment that contained pDK-7 except for 135 bp within the ORF by inverse PCR. The fragment was ligated with the hisG-URA3-hisG cassette to give pDK-8. pDK-8 was linearized prior to transformation into C. albicans. Due to the high similarity of PLC2 and PLC3, pDK-8 was used for the deletion of both genes.
Overexpression of CaPLC2 and CaPLC3.
In order to rescue the wild-type phenotype in mutants lacking CaPLC2 and CaPLC3, and to overexpress CaPLC2 or CaPLC3, both genes were cloned behind the PCK1 promoter of plasmid pBI-1 (Leuker et al., 1997; Stoldt et al., 1997
). CaPLC2 and CaPLC3 were amplified using the primers PLC3-2 and PLC2-10, and PLC3-2 and PLC3-3, respectively, with SC5314 genomic DNA as template and cloned into the BglII site of pBI-1, generating pDK-16 and pDK-18. Integration of CaPLC2 and CaPLC3 downstream of the PCK1 promoter was confirmed by analysing a number of restriction digests of pDK-16 and pDK-18.
Transformation of C. albicans.
Two modified lithium acetate protocols (Sanglard et al., 1996) and one electroporation protocol (De Backer et al., 1999
) were used. Transformants were screened using PCR and Southern blot analysis as described previously (Felk et al., 2002
).
RNA extraction and RT-PCR.
Liquid cultures were grown as described for each experiment, cells harvested and RNA isolated as described previously (Felk et al., 2002). For RT-PCR, 1·5 µg RNA was DNase treated and cDNA synthesized (Felk et al., 2002
). Controls included cDNA synthesis without addition of reverse transcriptase and amplification of an intron-containing fragment of the EFB1 gene (Schaller et al., 1999
) (with primers EFB1-1 and EFB1-2, Table 1
). To ensure that samples from the exponential phase of PCR amplification were examined, we used 20, 25, 30, 35 and 40 cycles, respectively, for genes of interest. All RT-PCR experiments were done at least in duplicate with samples from two independent biological experiments. The housekeeping gene ACT1, which was expressed to a similar extent under all conditions investigated, was used as an additional internal standard control.
Microarray hybridization and analysis.
For transcript profiling, we used C. albicans microarrays (Eurogentec) containing 6039 ORFs. Arrays were designed as described under http://www.pasteur.fr/recherche/unites/Galar_Fungail/arrays.html. Information about coding sequences and proteins was obtained from the CandidaDB database (http://genolist.pasteur.fr/CandidaDB/). RNA was reverse transcribed into cDNA and labelled with Cy3 or Cy5 (dye swap) (Sigle et al., 2005). Labelled cDNA was hybridized to the C. albicans arrays as described by Sigle et al. (2005)
. Hybridized slides were scanned with an Axon 4000B scanner at a 10 µm resolution. Data were extracted by GenePix 4.1 software (Axon). An intensity-dependent data normalization (LOWESS) was performed in GeneSpring 6.0. The different sets of data were compared to each other by a one-way analysis of variance (ANOVA) test with a P-value cut-off of 0·099 for genes that had an intensity in both channels higher than 100. Each gene that passed this test and showed at least 1·5-fold change in two arrays was defined as differentially expressed.
Systemic mouse infection.
C. albicans strains (plc2/
plc3, CAF2-1) were used in a mouse model of systemic infection as described previously (Fradin et al., 2005
).
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RESULTS |
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In order to investigate the function of CaPlc1p we prepared a disruption cassette to delete CaPLC1 in a wild-type background (strain CAI4). Plasmid pDB104 was used to delete an internal 900 bp fragment, containing a HindIII site, of CaPLC1. After a first round of transformation with pDB104, a number of clones isolated had this fragment deleted in one allele of CaPLC1 as shown by PCR and Southern analysis. However, even after screening more than 190 clones following a second round of transformation with pDB104, no clone was identified in which both alleles of CaPLC1 were deleted. Since it has been shown that allele-specific gene targeting in C. albicans may result from heterozygosity between alleles (Yesland & Fonzi, 2000), we constructed an allele-specific cassette with gene-specific sequences that were deleted in the heterozygous mutant. Therefore, integration by homologous recombination should only be possible into the second non-disrupted allele. Furthermore, we used a third cassette with flanking sequences representing non-translated regions of CaPLC1 and a fourth cassette based on allele-specific flanking oligonucleotides (Wilson et al., 1999
). None of the transformants obtained with these disruption cassettes had all alleles disrupted. In total, an additional 500 clones obtained by different transformation protocols using these cassettes in more than 28 transformation experiments were screened. Although the vast majority of clones showed integration into the first allele (non-allele-specific cassettes) or random integration into the genome (allele-specific cassette), we identified seven clones where integration into the second allele occurred. However, in these clones, additional DNA fragments were identified which contained the non-disrupted CaPLC1 allele, suggesting duplication of these regions prior to integration of the cassettes into the corresponding region. These data strongly suggest that CaPLC1 is an essential gene.
Construction of a conditional CaPLC1 mutant
Since all attempts to generate a CaPLC1 null mutant failed and disruption of the second allele was only possible in mutants where the chromosomal CaPLC1 region had been duplicated, we decided to produce a conditional mutant. Since the MET3 promoter of C. albicans (pMET3), first analysed by Care et al. (1999), was shown to be a tightly regulated promoter and appropriate for studying essential genes, we used the plasmid pCaDis (Care et al., 1999
) to prepare a CaPLC1 mutant with a disrupted allele and a non-disrupted allele under the control of the MET3 promoter. Addition of 2·5 mM methionine and cysteine was shown to repress the MET3 promoter (Care et al., 1999
). However,
Caplc1/pMET3-CaPLC1 mutants were still viable on media containing methionine and cysteine (2·5 mM each). In order to investigate the transcript levels of CaPLC1 in these mutants under conditions that repress the MET3 promoter, we used RT-PCR and CaPLC1-specific primers. Although expression levels of CaPLC1 were significantly reduced in media containing methionine and cysteine, transcripts were still detectable (not shown). Since the level of CaPLC1 transcripts in wild-type strains was very low compared to housekeeping genes (Fig. 1
), we reasoned that the low level of transcription of CaPLC1 in the controllable mutant was sufficient for survival. However, since the level of transcription of CaPLC1 in the
Caplc1/pMET3-CaPLC1 mutant was reduced under repression of the MET3 promoter, compared to CaPLC1 levels in wild-type cells, we used this conditional mutant for a broad phenotype screening.
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In addition to reduced growth at elevated temperature and in the presence of nocodazole, the heterozygous mutant showed reduced growth at 30 °C and 37 °C in media supplemented with itraconazole (1 µg ml1) or hydrogen peroxide (880 µM), and in media with glycerol and acetate as sole carbohydrate sources (not shown). Furthermore, hyphal formation was impaired at 37 °C on blood agar, serum agar or solid Spider medium, as filaments appeared delayed in comparison to the CAF2-1 wild-type strain (Fig. 3). Both the wild-type and the heterozygous mutant produced secreted aspartic proteinases (Saps) that permitted growth on YCB-BSA medium. However, hyphal formation and the white zone around colonies due to acidification and precipitation of BSA were retarded for
Caplc1/CaPLC1 colonies (Fig. 3
).
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Transcriptional analysis of CaPLC1 and genes putatively linked to CaPLC1 function
Phenotypic screening of mutants lacking PLC1 in S. cerevisiae (Odom et al., 2000) and C. albicans (this study) revealed sensitivity to different environmental stresses. Therefore, we questioned whether CaPLC1 is regulated in response to such stresses. In S. cerevisiae the products of the PLC1, GLE1, IPK1 and IPK2 (ARG82) genes are known to be involved in nuclear mRNA export (York et al., 2001
) and may be co-regulated. A mutant lacking GLE1 in S. cerevisiae showed temperature sensitivity similar to mutants lacking PLC1. Therefore, we examined the transcript level of CaPLC1 and the orthologous genes of GLE1, IPK1, IPK2 in C. albicans wild-type cells (CaGLE1, IPF15261, CA2378; CaIPK1, IPF10566, CA0280; CaIPK2, IPF16498, CA2312) under different stress conditions such as heat shock, glycerol as sole source of carbon and exposure to cadmium (Fig. 1
). None of the investigated genes had significantly modified transcript levels in these media, although mRNA levels for CaPLC1, GLE1 and IPK2 seemed moderately increased at 42 °C and for CaPLC1 moderately reduced in medium containing cadmium (Fig. 1
).
Genome-wide transcriptional profiling of the conditional Caplc1/pMET3-CaPLC1 mutant
PI turnover due to the hydrolytic activity of phospholipase C produces not only DAG, but also IP3, another intracellular second messenger that stimulates release of Ca2+, activates calmodulin-dependent enzymes and is a substrate for inositol polyphosphates. Therefore, reduced levels of CaPLC1 transcripts may have an influence on multiple cellular processes, which in turn could cause changes in the global transcriptional profile of the cell. To analyse the transcriptional profile of the conditional mutant we chose the condition that caused the strongest phenotype in the conditional mutant: elevated growth temperature. CAF2-1 and the conditional mutant were precultured in SD at 37 °C, diluted into SD medium supplemented with 2·5 mM methionine and cysteine at a density of 1x107 cells ml1 and incubated for 3 h at 43 °C. RNA was isolated from both the wild-type and the conditional mutant and used to prepare labelled cDNA.
Forty-five genes were found to be differentially regulated in the conditional mutant as compared with the wild-type strain under repressed conditions. Only four genes (GDH3, MET18, IPF12399 and CDC46) were found to be downregulated and 41 genes upregulated (Table 3).
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None of the orthologues of GLE1, IPK1 or IPK2 were significantly up- or downregulated, confirming the results obtained with RT-PCR in our heat-shock experiment.
At least three genes in the genome of C. albicans encode phospholipases C
Andaluz et al. (2001) described a gene encoding a phospholipase C (CaPI-PLC, renamed CaPLC2 in this study) (accession no. AJ277538) and a presumptive truncated homologous gene that differed in size by 145 bp, on chromosome 2 (named CaPLC3 in this study) in the genome of C. albicans, in addition to the CaPLC1 gene first cloned by Bennett et al. (1998)
. CaPLC2 was described as an ORF of 1029 bp encoding a protein of 343 amino acids (Andaluz et al., 2001
) with no obvious signal peptide, transmembrane region or GPI-anchor-like sequences, suggesting that CaPLC2 encodes an intracellular phospholipase C. The deduced protein sequence of CaPLC2 is more similar to bacterial PI-specific phospholipases C, is very different from CaPlc1p (Andaluz et al., 2001
) and has no homologous counterpart in S. cerevisiae. The higher similarity to bacterial phospholipases may be explained by the fact that CaPlc2p only consists of the X domain of mammalian phospholipases C. However, an alignment of the X domain of CaPlc1p and the entire CaPlc2p sequence showed only 8·5 % similarity (Fig. 4
).
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In order to investigate whether the prokaryotic-type phospholipase genes CaPLC2 and CaPLC3 are distributed among other Candida species, we used CaPLC2 as a probe for a Southern blot analysis. Genomic DNA of different Candida species was digested with three restriction enzymes (EcoRI, BamHI and SalI). A CaPLC2 probe covering the whole ORF revealed no signal for Candida krusei and Candida glabrata, one band for Candida parapsilosis and Candida tropicalis and two bands for Candida albicans and Candida dublinensis, suggesting that only Candida species closely related to C. albicans contain genes similar to CaPLC2 or CaPLC3 (not shown).
The transcriptional profiles of CaPLC2 and CaPLC3 are similar
Several gene families have been identified in the genome of C. albicans (d'Enfert et al., 2005; Fradin & Hube, 2003
). These families include gene members with high similarity and similar functions, but individual expression profiles. For example, expression of the SAP and PHR genes was shown to be tissue specific (Hube, 2004
). Since CaPLC2 and CaPLC3, although highly similar in their ORFs, differed significantly in their promoter regions, we reasoned that these two genes might be differentially expressed in response to different environmental conditions.
The wild-type strain SC5314 was grown in different media (SD, YPD, YSer, SLAD or SG) and RNA samples from different time points were analysed by RT-PCR. To discriminate between CaPLC2 and CaPLC3 transcripts we used two gene-specific forward primers (24 bp in length, identical to regions upstream of the ATG start codon of CaPLC2 and CaPLC3) in combination with a single primer that recognized a sequence in the ORFs of both genes.
Under all conditions tested, both genes were expressed (Fig. 5). However, expression levels of CaPLC2 were higher under all conditions as compared with CaPLC3. Expression levels rose in the order SG>SLAD>YPD>YSer for CaPLC2, indicating a lower level of expression in hyphal cells (YSer) as compared with yeast cells (SG, SLAD, YPD) (it should be noted that under the conditions used we observed only yeast cells in SLAD medium, although SLAD medium is known to be a hyphal induction medium) (Fig. 5
). Similarly, expression of CaPLC2 was higher in Lee's medium at pH 4·5 (which supports yeast growth) as compared with Lee's medium at pH 6·5 (which supports hyphal growth) (not shown). In contrast, the expression level of CaPLC3 was highest in SLAD as compared with all other growth conditions. Therefore, transcriptional regulation of CaPLC2 and CaPLC3 is at least partially different.
CaPLC2 and CaPLC3 are not essential for growth and virulence
In order to analyse the function of CaPLC2 and CaPLC3 we constructed mutants that lacked these genes. Mutants were generated using homologous recombination with only one disruption cassette in a multi-step procedure. Neither single mutants lacking CaPLC2 or CaPLC3 respectively nor double mutants lacking both genes had any growth defects in minimal or complex growth media, suggesting that CaPLC2 and CaPLC3 are not essential for growth.
Mutants lacking CaPLC2 or CaPLC3 or both genes showed no change in phenotype at reduced or elevated temperatures (25, 30, 37 or 43 °C), in media with different carbohydrate sources (glucose, galactose or sucrose), under anaerobic conditions, in media with high NaCl or carbohydrate concentrations causing osmotic stress, or at different pH values (pH 5·78·0) as compared with the parental wild-type (CAF2-1). In addition, no growth differences were observed in SD medium supplemented with benomyl, calcofluor white, Congo red, nocodazole, hygromycin B, cycloheximide, EDTA, 5-flucytosine, tetracycline, amorolfine, SDS, butanol, 1-propranolol or calcium (up to 300 mM) at elevated concentrations to achieve distinct effects of stresses to the cells. Furthermore, extracellular lipolytic activity was not reduced, as growth on blood or egg-yolk agar showed the same characteristic halo around colonies as compared with the wild-type, and serum-induced hyphal production was indistinguishable from the wild-type.
However, growth of the Caplc2/
Caplc3 double mutant was inhibited at 30 °C in SD medium supplemented with 20 mM caffeine, or at 37 °C supplemented with 1 µg itraconazole ml1 (Fig. 6a
) or in the presence of 880 µM hydrogen peroxide (not shown). Hyphal formation of the
Caplc2/
Caplc3 double mutant was delayed at 30 °C on CAA medium, and at 37 °C on M199 agar (Fig. 6a
), on Spider medium and on YCB-BSA agar (not shown). Furthermore, the production of the characteristic white zone around colonies of the double mutant was delayed on YCB-BSA agar. None of the phenotypes observed for the double mutant were detected in the single mutants, suggesting that both genes have the potential to compensate for the loss of the homologous CaPLC gene and that the observed phenotypes are not due to a position effect of the URA3 marker.
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Delayed formation of hyphae is restored by extrachromosomally expressed CaPLC2
In order to rescue the wild-type phenotype in mutants lacking CaPLC2 and CaPLC3, and to overexpress CaPLC2 or CaPLC3, both genes were cloned behind the PCK1 promoter of plasmid pBI-1 (Leuker et al., 1997). No obvious phenotypes were observed in media where the PCK1 promoter is induced (CAA medium, succinate medium or SGlycAc medium). However, when CaPLC3 was overexpressed at 30 °C on CAA medium in the
Caplc2/
Caplc3 double mutant, hyphal formation was similar to wild-type (CAF2-1) (Fig. 6b
).
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DISCUSSION |
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All attempts to generate a CaPLC1 null mutant failed and disruption of the second allele was only possible in mutants where the chromosomal CaPLC1 region had been duplicated, suggesting that CaPLC1 is an essential gene. This is further supported by the fact that the PI-Plc-specific inhibitor ET-18 completely blocks growth of C. albicans at higher concentrations. Thus, inhibition of CaPLC1 in C. albicans and perhaps other Candida species appears to be a possible therapeutic strategy.
Although PLC1 of S. cerevisiae has been shown to be essential in certain genetic backgrounds, other studies showed successful disruption of PLC1 in other strains. In an attempt to further show that CaPLC1 is in fact essential, we used the MET3 promoter of C. albicans, which has previously been used to study essential genes such as URA3 (Care et al., 1999). In our hands, addition of 2·5 mM methionine and cysteine repressed the MET3 promoter; however, transcript levels of CaPLC1 were still detectable and the Caplc1/pMET3-CaPLC1 mutant was still viable. Since the level of CaPLC1 transcripts in wild-type strains was also very low, we reasoned that the low level of transcription of CaPLC1 in the controllable mutant was sufficient for survival. Low transcript levels for PLC1 in wild-type cells had also been reported in S. cerevisiae (Flick & Thorner, 1993
) and Bennett et al. (1998)
were unable to detect CaPLC1 transcripts in C. albicans using Northern blot analysis. Therefore, the MET3 promoter seems to be too leaky to reduce transcription below an essential level for CaPLC1 and possibly other essential genes with low basal transcription levels in wild-type cells of C. albicans.
To our knowledge, until the present study, there has been no protocol described that would generally provide unambiguous evidence that a particular gene is essential for C. albicans. Similar to the approach of a conditional mutant using the MET3 promoter (Care et al., 1999), Roemer et al. (2003)
set out to define C. albicans essential genes by deleting one allele and fusing a second allele to a tetracycline-repressible promoter, creating a GRACE (gene replacement and conditional expression) strain. However, in addition to the creation of conditional mutants Davis et al. (2002)
used another indirect approach to identify essential genes by using the UAU1 cassette, which permits selection of homozygous mutants from heterozygotes (Enloe et al., 2000
).
Disruption of PLC1 in S. cerevisiae in a non-lethal genetic background revealed phenotypic responses such as defective utilization of carbon sources other than glucose, osmosensitivity and temperature sensitivity (Flick & Thorner, 1993; Payne & Fitzgerald-Hayes, 1993
). The latter phenotype could be partially restored with exogenous Ca2+ ions, suggesting a role of PLC1 in Ca2+-signal transduction via IP3. The phenotypes of the conditional Caplc1/pMET3-CaPLC1 mutant of C. albicans were generally comparable to those observed for the PLC1 mutant from S. cerevisiae, suggesting that CaPLC1 may have functions similar to PLC1 of S. cerevisiae. Growth of Caplc1/pMET3-CaPLC1 of C. albicans under repressed conditions included reduced growth (1) on media containing 5 % (w/v) sorbitol or 1 M NaCl (causing increased osmotic stress), (2) on media with galactose as the sole source of carbon instead of glucose, (3) at elevated (43°) temperature or (4) at low (18 °C) temperature. Furthermore, reduced growth on media with arginine as sole nitrogen source suggests that CaPLC1 may, like its orthologue in S. cerevisiae, be necessary for the production of IP messengers that modulate distinct nuclear processes via regulation of the ArgRMcm1 complex (Odom et al., 2000
). A putative nuclear localization signal (NLS) (Odom et al., 2000
; Payne & Fitzgerald-Hayes, 1993
) rich in basic amino acids within the X and Y domain supports the view that CaPlc1p in C. albicans may in fact act in the nucleus. In addition to a putative role in nuclear mRNA export (Odom et al., 2000
; York et al., 1999
), sensitivity to nocodazole of the Caplc1/pMET3-CaPLC1 mutant may point to a role of CaPlc1p in chromosome segregation, since mutants lacking Plc1p in S. cerevisiae showed chromosome mis-segregation when exposed to this drug (Lin et al., 2000
). Therefore, CaPlc1p from C. albicans, like the Plc1p counterpart in S. cerevisiae, seems to be involved in multiple cellular processes. This may explain why a genome-wide transcriptional profiling of the Caplc1/pMET3-CaPLC1 mutant did not produce a clear expression pattern reflecting distinct transcriptional responses to heat treatment. The observation that CaPLC1 seems to be an essential gene in C. albicans cannot simply be explained by the fact that CaPlc1p produces two essential second messengers, IP3 and DAG, as these molecules can also be produced by other enzymes (e.g. by Pld1p or CaPlc2p or CaPlc3p). Therefore, it must be concluded that CaPlc1p is the only enzyme in C. albicans which can produce IP3 and DAG under certain conditions at distinct cellular locations, or that CaPlc1p has further functions that cannot be compensated by other enzymes.
Incubation of C. albicans wild-type strains under conditions which repress the pMET3 promoter (addition of 2·5 mM methionine and cysteine) strongly inhibits the yeast-to-hyphal transition in media which are otherwise known to induce hyphal formation (such as media containing serum). Similarly, Shepherd et al. (1980) showed that higher concentrations of cysteine (>5 µM) inhibit germ tube formation. Therefore, the
Caplc1/pMET3-CaPLC1 conditional mutant was not suitable to investigate the effect of CaPLC1 on hyphal formation. In order to investigate the impact of reduced levels of CaPLC1 on hyphal formation, we analysed the growth of the heterozygous
Caplc1/CaPLC1 mutant in comparison with the CaPLC1/CaPLC1 wild-type strain under hyphal-inducing conditions. Similarly, Uhl et al. (2003)
showed that that heterozygous mutants produced in a large-scale loss-of-function genetic screen had defects in filamentous growth due to gene dosage effects.
Ansari et al. (1999) showed that Plc1p interacts with the receptor-like membrane protein Gpr1p as a component of the nitrogen-signalling pathway for formation of pseudohyphae in S. cerevisiae. Furthermore, Gpr1p is associated with the regulator protein Gpa2 at the initial steps of signal transduction (Ansari et al., 1999
). Based on these results it was suggested that Plc1p acts in two filamentation pathways in S. cerevisiae, the cAMP- and the Ras2-controlled (MAPK) pathway. The cAMP and the MAPK pathways in C. albicans are known to be associated with the yeast-to-hyphal transition. In fact, mutants of C. albicans lacking either one copy of CaPLC1, or both CaPLC2 and CaPLC3, had reduced abilities to produce hyphae on certain media (e.g. Spider medium). The fact that the
Caplc1/CaPLC1 heterozygote had reduced ability to grow on serum agar plates may be related to modified Ca2+-mediated signal transduction and increased sensitivity to serum (Sanglard et al., 2003
). However, addition of external Ca2+ ions at several concentrations did not alter the phenotype of the heterozygous mutant (not shown). Sanchez-Martinez & Perez-Martin (2002)
showed that mutants lacking GPA2 of C. albicans also failed to produce hyphae on Spider medium and concluded that Gpa2p acts in the MAPK pathway. However, a direct interaction between Gpa2 and Gpr1 or any of the proteins CaPlc13p with proteins of the MAPK pathway in C. albicans has yet not been shown.
The exact role of CaPLC2 and CaPLC3 of C. albicans remains unclear. The function of both genes must be clearly different from CaPLC1, as neither CaPLC2 nor CaPLC3 can compensate for the loss of CaPLC1. The fact that these two genes are unique for C. albicans and do not exist in S. cerevisiae may suggest a role in adaptation to the human host. However, both genes are dispensable for virulence in one model of infection (this study) and for interaction with macrophages (Knechtle et al., 2005). It also remains a mystery why C. albicans provides two almost identical copies of this unusual type of PLC gene in eukaryotes. One possible explanation would be a differential regulation of CaPLC2 and CaPLC3 under different conditions, as has been shown for other gene families in C. albicans (Fradin & Hube, 2003
). However, although the promoter regions of both genes are very different, the expression pattern was similar under all conditions tested and both genes seem to be constitutively expressed in vitro. It is possible that CaPLC2 and CaPLC3 are differentially expressed under certain conditions in vivo and that the expression under these conditions is crucial for survival of C. albicans.
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ACKNOWLEDGEMENTS |
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Received 19 July 2005;
accepted 1 August 2005.
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