The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans

Won Hee Jung and Lubomira I. Stateva

Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 1QD, UK

Correspondence
Lubomira I. Stateva
lubomira.stateva{at}umist.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The cAMP-dependent pathway, which regulates yeast-to-hypha morphogenesis in Candida albicans, is controlled by changes in cAMP levels determined by the processes of synthesis and hydrolysis. Both low- and high-affinity cAMP phosphodiesterases are encoded in the C. albicans genome. CaPDE2, encoding the high-affinity cAMP phosphodiesterase, has been cloned and shown to be toxic in Saccharomyces cerevisiae upon overexpression under pGAL1, but functional under the moderate pMET3. Deletion of CaPDE2 causes elevated cAMP levels and responsiveness to exogenous cAMP, higher sensitivity to heat shock, severe growth defects at 42 °C and highly reduced levels of EFG1 transcription. In vitro in hypha-inducing liquid medium CaPDE2, deletion prohibits normal hyphal, but not pseudohyphal growth. On solid medium capde2 mutants form aberrant hyphae, with fewer branches and almost no lateral buds, which are deficient in hypha-to-yeast reversion. The phenotypic defects of capde2 mutants show that the cAMP-dependent pathway plays specific roles in hyphal and pseudohyphal development, its regulatory role however, being greater in liquid than on solid medium in vitro. The increased expression of CaPDE2 after serum addition correlates well with a drop in cAMP levels following the initial rise in response to the hyphal inducer. These results suggest that Capde2p mediates a desensitization mechanism by lowering basal cAMP levels in response to environmental stimuli in C. albicans.


Abbreviations: CFW, calcofluor white; GlcNAc, N-acetylglucosamine; PDE, phosphodiesterase; PKA, protein kinase A


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Candida albicans, the most frequently isolated human fungal pathogen, grows in different morphological forms: hyphae, pseudohyphae and blastospores. Switching between yeast and hypha (a major contributing factor to its pathogenicity) is triggered in response to a range of environmental signals (Brown & Gow, 1999; Tripathi et al., 2002) and is regulated by several signalling pathways. The mitogen-activated protein kinase (MAPK) pathway regulates Cph1p (a homologue of Saccharomyces cerevisiae Ste12p) in response to nutritional signals (Csank et al., 1998). The pH-responsive pathway activates the Rim101 transcription factor (Davis et al., 2000) and matrix embedding/microaerophilic conditions induce filamentation through an, as yet, unidentified pathway involving Czf1p (Brown et al., 1999). Response to serum has been shown to involve two-component histidine kinases (Yamada-Okabe et al., 1999) and the Ras/cAMP-dependent pathway (Ernst, 2000), the latter also being associated with filamentation triggered by amino acid starvation (Tripathi et al., 2002).

The Ras/cAMP-dependent pathway operates via the secondary messenger cAMP (Borges-Walmsley & Walmsley, 2000). In budding yeast cAMP is synthesized by adenylate cyclase, Cdc35p (Kataoka et al., 1985). Ras (Toda et al., 1985), Gpa2p (Nakafuku et al., 1988) and Cap1p (Fedor-Chaiken et al., 1990; Field et al., 1990) regulate the adenylate cyclase. The role of cAMP is to activate protein kinase A (PKA), a heterotetramer consisting of two catalytic and two regulatory subunits, encoded redundantly by TPK1-3 and BCY1, respectively (Toda et al., 1987a, b). Active PKA stimulates enzymes involved in the utilization of storage carbohydrates, represses stress-activated genes, controls entry into stationary phase (Thevelein & de Winde, 1999) and promotes pseudohyphal morphogenesis (Pan & Heitman, 2002).

Morphological interconversions in other fungal species also depend on signalling through a cAMP-dependent pathway. In S. cerevisiae pseudohyphal morphogenesis depends on elevated cAMP levels. Similarly, high levels of cAMP are required for appressorium formation in the rice fungal pathogen Magnaporthe grisea. In contrast low cAMP levels are needed for the filamentous growth of the corn smut fungus Ustilago maydis. Whilst it has been generally accepted that hyphal development in C. albicans requires elevated cAMP levels, the reports on the exact pattern of changes are very controversial. Exogenously added cAMP and its analogue N6,O2-dibutyryl cAMP (dbcAMP) were shown to induce hyphal formation, as did the cAMP phosphodiesterase (PDE) inhibitors, theophylline and caffeine (Sabie & Gadd, 1992). Intracellular cAMP levels increased during the yeast-to-hypha transition and maximum levels coincided with maximum germ-tube formation (Niimi et al., 1980; Chattaway et al., 1981; Sabie & Gadd, 1992). In contrast the early stages of germ-tube formation were found to correlate with a transient decrease in intracellular levels of cAMP due to higher PDE activity (Egidy et al., 1989, 1990). Others have reported no fluctuations in cAMP levels (Sullivan et al., 1983). The exact pattern of cAMP changes still remains an unresolved issue, regardless of the fact that several components of the cAMP-dependent pathway have recently been characterized in C. albicans. The PKA isoforms Tpk1p and Tpk2p have both distinct and redundant roles in morphogenesis and growth (Sonneborn et al., 2000; Bockmühl et al., 2001). The adenylate cyclase CaCdc35p is essential for hyphal growth and virulence (Rocha et al., 2001). Cap1p and Ras are also required for bud-to-hypha transition, cAMP synthesis and virulence (Bahn & Sundstrom, 2001; Feng et al., 1999).

Degradation of cAMP and the subsequent down-regulation of PKA, is catalysed by cAMP PDEs. PDE1 and PDE2 encode the low- and high-affinity cAMP PDEs, respectively, in S. cerevisiae (Sass et al., 1986; Nikawa et al., 1987). Higher sensitivity to heat shock and nutrient starvation has been reported for pde1 and pde2 mutants. Exogenous cAMP greatly affects cAMP levels in pde2 mutants, suggesting a role for Pde2p in breaking down exogenous cAMP (Wilson et al., 1993). Cell-wall-related phenotypes, such as lysis upon hypo-osmotic shock and enhanced transformability have also been reported for pde2 mutants, suggesting a role for Pde2p (and/or cAMP) in maintenance of cell-wall integrity in S. cerevisiae (Heale et al., 1994; Tomlin et al., 2000).

In this study, we provide genetic evidence that deletion of CaPDE2 hinders hyphal, but not pseudohyphal growth in liquid medium and causes formation of aberrant hyphae, with fewer branches and lateral buds, on solid medium in the presence of hyphal inducers. Our results show that the cAMP-dependent pathway plays specific roles in hyphal and pseudohyphal growth and suggest that a desensitization mechanism involving CaPde2p-catalysed cAMP hydrolysis is required for normal growth and development of hyphae in C. albicans.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids and media.
The strains are listed in Table 1. Standard yeast media with relevant supplements were used (Sherman et al., 1986). Ura- auxotrophs were selected on SD with 1 µg 5-fluoro-orotic acid ml-1 and 25 µg uridine ml-1. The medium for pH induction of hyphae was according to Lee et al. (1975); Spider and salt base N-acetylglucosamine (GlcNAc)-containing media were according to Liu et al. (1994) and Delbrück & Ernst (1993), respectively. All media were routinely prepared with uridine in order to use them for Ura- strains as well. The RPMI 1640 medium (Sigma) was buffered to pH 7·0. Minimal medium containing 0·67 % yeast nitrogen base (without amino acids) and 2 % Casamino acids was used for overexpression of EFG1 expressed under the promoter of PCK1. The defined minimal medium used for studying the effects of exogenous cAMP was prepared as described by Sabie & Gadd (1992). Bacterial strains were grown in LB and LB+ampicillin (50 µg ml-1) (Sambrook et al., 1989). The plasmids used and/or generated in this study are shown in Table 2.


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Table 1. Strains used in this study

 

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Table 2. Plasmids used in this study

 
DNA and RNA manipulation methods.
Bacterial plasmid DNA was isolated by the alkaline lysis method (Sambrook et al., 1989) or using the QIAprep Spin Miniprep Kit (Qiagen). All DNA fragments for cloning were gel-purified with the QIAquick Gel Extraction Kit (Qiagen). Yeast genomic DNA was isolated according to Adams et al. (1997). All DNA modifying enzymes were used according to the manufacturers' recommendations. The Amersham Pharmacia Biotech procedure was used for Southern analysis. DNA hybridization probes were labelled with [{alpha}-32P]dCTP (3000 Ci mmol-1; ICN Biomedical) using the Random Primed DNA Labelling Kit (Roche). Unincorporated radioactive nucleotides were separated using mini Quick Spin DNA columns (Boehringer Mannheim). Total RNA was isolated according to Schmitt et al. (1990) and used for Northern analysis as described previously (Warit et al., 1998). Sequencing was carried out in-house with ABI PRISM and the dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase.

Transformation.
E. coli XL-1 Blue was transformed by the calcium chloride method (Sambrook et al., 1989) or by electroporation with a Gene Pulser apparatus (Bio-Rad), C. albicans strains were transformed by the spheroplast method recommended for Pichia pastoris (Invitrogen) and S. cerevisiae strains by the DMSO-enhanced procedure (Hill et al., 1991).

Plasmids for heterologous expression, disruption and reintegration of CaPDE2.
CaPDE2 was PCR-amplified from SC5314 genomic DNA with primers SF1 and SR1 (Table 3) and cloned into BamHI/XbaI-restricted pYES2 to obtain pWHPDE2, in which CaPDE2 was fused to the S. cerevisiae GAL1 promoter. Alternatively a KpnI–SacI fragment from pMETtPGK3-3 (Table 2) containing the MET3 promoter was ligated to the KpnI/SacI-restricted pUC18 to generate plasmid pUC18pMtP. PCR-amplified CaPDE2 on a BamHI–XbaI fragment was cloned into BamHI/XbaI-restricted pRS415 (Sikorski & Hieter, 1989) to generate pRS415PDE2. The SacII–SacI fragment from pMETtPKG3-3, containing the PGK1 terminator, was ligated into SacII/SacI-digested pRS415PDE2 to generate pRS415P2tPKG. Finally, the SmaI–BamHI fragment containing the MET3 promoter was excised from pUC18pMtP and ligated into SmaI and BamHI sites of pRS415P2tPKG. On the resulting plasmid, pWHPDE2-M, the transcription of CaPDE2 was regulated by the MET3 promoter and the PGK1 terminator.


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Table 3. Primers used in this study

 
The plasmid for the disruption of CaPDE2 was based on vector pMB7 (Fonzi & Irwin, 1993). The 4·0 kb SalI–BglII, hisG-URA3-hisG-containing fragment from pMB7 was ligated with the larger fragment produced by SalI and BglII restriction of plasmid pWHPDE2 (Table 2). The resulting plasmid, pWH-P2MB, was digested with HindIII and BstI1107 to generate the transforming 5·0 kb disrupting fragment, containing 428 bp 5' and 593 bp 3' CaPDE2 homologous sequences. For site-directed reintegration of CaPDE2 in strain WH2-RU, plasmid pWHP2-R was generated. A 2·7 kb fragment containing the CaPDE2 ORF, promoter and terminator regulatory sequences was PCR-amplified using SC5314 genomic DNA and primers PF1 and TR1 (Table 3). After restriction with XbaI and BamHI, the fragment was ligated into pUC18 digested with the same enzymes, resulting in plasmid pWH18P2-F. The 1·4 kb XbaI-derived, URA3-containing fragment from pDDB7 (Wilson et al., 2000) was ligated to XbaI-digested, CIP (calf intestinal phosphatase)-dephosphorylated pWH418P2-F. In this way pWH18P2-F-URA3 was obtained. The SacI–BglII 0·8 kb fragment from pWH18P2-F (3' CaPDE2 flanking region and putative terminator) was ligated into SacI/BamHI-restricted pWH18P2-F-URA3 to generate pWHP2-R. The 5·0 kb SacI–PstI fragment from pWHP2-R was used for transformation.

Construction of CaPDE2-deficient mutants.
The multi-step URA blaster technique (Fonzi & Irwin, 1993) was used. The deletion cassette obtained by HindIII and BstI1107 digestion of plasmid pWH-P2MB (see above) was transformed into strain CAI-4 (Table 1). Seven transformants were selected on SD without uridine and diagnostic PCR confirmed that six had 695 bp of internal CaPDE2 sequence in one of the alleles replaced by capde2 : : hisG-URA3-hisG. WH2-1U and WH2-7U (Table 1) were grown on SD medium containing 5-fluoro-orotic acid and uridine. Thirteen spontaneous uridine auxotrophs were isolated and the loss of URA3 was confirmed by diagnostic PCR. The remaining functional CaPDE2 allele in two of them (WH2-2 and WH2-8) was disrupted in a second round of transformation with the same deletion cassette (see Fig. 2a). Seventeen Ura+ colonies were obtained and diagnostic PCR indicated that a replacement of the second CaPDE2 allele with hisG-URA3-hisG had occurred in six of them. Two named WH2-3U and WH2-9U were used to generate the Ura- derivatives WH2-4 and WH2-10, respectively. Reintegration of one copy of CaPDE2 via homologous recombination of the 5·0 kb SacI–PstI fragment from pWHP2-R into the genome of WH2-4 (Table 1) yielded seven transformants. Diagnostic PCR using primers RF and QF3 (Table 3) showed that in five of the transformants the cassette had integrated into the proper location. One of them, named WH2-RU, was used further as the reconstituted wild-type control strain.



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Fig. 2. Disruption of CaPDE2 in C. albicans. (a) Genetic organization of the CaPDE2 locus. The positions of CaPDE2 ORF (black bar), hisG (chequered bar) and URA3 (hatched bar) are shown. (b) Southern analysis of genomic DNA digested with EcoRI and XbaI. Lanes: 1, CAI-4 (ura3); 2, WH2-1U (capde2/CaPDE2 URA3); 3, WH2-2 (capde2/CaPDE2 ura3); 4, WH2-3U (capde2/capde2 URA3); 5, WH2-4 (capde2/capde2 ura3); 6, WH2-RU (capde2/capde2 : : CaPDE2-URA3). The hybridization probe was the BglII–XbaI fragment from pWHPDE2 (Table 2).

 
Physiological tests.
For GAL1-regulated expression, strains were grown in SD with 2 % galactose at 30 °C following 3 h in the presence of 2 % glycerol. For MET3-regulated expression the conditions described by Warit et al. (2000) were used. Heat shock was performed by incubating strains at 50 °C in a water bath for 10, 20 or 30 min, followed by plating onto SD plates (with or without methionine) and incubating at 30 °C for 2–3 days. Comparing growth at 30 and 37 °C for S. cerevisiae strains, and 30 and 42 °C for C. albicans tested temperature sensitivity. Hyphal growth of C. albicans was induced as follows. Strains were grown in SD at 30 °C overnight and about 1·0x105 cells ml-1 were transferred to hypha-inducing liquid medium pre-warmed at 37 °C. For hyphae induction in the RPMI medium cells were grown overnight at 30 °C in RPMI 1640 and about 1·0x105 cells ml-1 were transferred to fresh RPMI 1640+10 % serum pre-warmed at 37 °C. Approximately 200 cells were spread onto appropriate plates for hyphal induction on solid medium. For induction by embedding, the same number of cells were added to liquefied Spider medium. Both liquid and solid cultures were incubated at 37 °C. Hyphal development was recorded microscopically. The effects of exogenous cAMP were determined as described by Sabie & Gadd (1992). Estimation of germ-tube formation was essentially as described by Sudbery (2001). Every experiment was done at least twice.

Extraction and determination of cAMP.
Intracellular cAMP concentrations were measured essentially as described by Rocha et al. (2001) with some modifications. For the time-course experiment strains were grown for 20 h in SD at 30 °C, diluted to 3·0x106 cells ml-1 in YPD with 10 % serum and incubated further at 37 °C. For cAMP determination in the presence of exogenous cAMP, strains were grown overnight in YPD at 30 °C, then diluted to 1·0x105 in fresh YPD with or without exogenous cAMP and incubated further at the same temperature for 12 h. Cells were harvested and cell pellets were immediately frozen in liquid nitrogen. For cAMP extraction, frozen cells were thawed, washed once (or four times when exogenous cAMP was present in the medium) with ice-cold water and finally resuspended in 1 ml cold water. Half of this suspension was used for dry weight, the remaining 500 µl was transferred to 2·0 ml centrifuge tubes containing 1·0 g acid-washed glass beads and 500 µl 10 % trichloroacetic acid. Cells were broken in a bead beater at 4 °C and centrifuged for 15 min at 12 000 r.p.m. at 4 °C. Supernatants were neutralized by five washes with water-saturated ether and, after freeze-drying, resuspended in 500 µl cAMP assay buffer. The concentration of cAMP was measured with the Correlate-EIA Cyclic AMP Enzyme Immunoassay Kit from Assay Designs, according to the manufacturer's instructions.

Miscellaneous.
For overexpression of EFG, the plasmid pBI-HAHYD, containing EFG1 under the control of the PCK1 promoter (Sonneborn et al., 2000) was transformed into WH2-4 (Table 4) and three transformants were tested. An Axiovert 200 (Zeiss) microscope equipped with a CoolSNAP HQ camera was used for observation of cell morphology in liquid medium at x630 magnification. Calcofluor white staining was carried out with 0·1 mg fluorescent brightener ml-1 (Sigma) and the images were converted to monochrome scale. A WILD M3Z microscope with a JVC video camera was used for observation of colonies on solid medium at x6 magnification.


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Table 4. Intracellular cAMP concentration

Data are presented as pmol (mg dry wt)-1±SD (based on two independent experiments).

 

   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CaPDE2 is functional in S. cerevisiae, but is toxic upon overexpression
Previous attempts to clone the C. albicans homologue of PDE2 by functional complementation have been unsuccessful (Hoyer et al., 1994; Taylor, 1999). However, a putative CaPDE2 ORF, with the conserved signature motif of a class I cAMP PDE (HDVGHPGTTNDF) (Charbonneau et al., 1986), 1716 bp long and with 28 % aa identity to ScPDE2, was revealed by the Candida genome sequence project (Tait et al., 1997). We amplified it by PCR from SC5314 (using the proof-reading Triple Master PCR system from Eppendorf) and sequenced it on both strands. Three nucleotide differences (C207T, A947C and A1023G) were found in comparison to the database sequence (http://genolist.pasteur.fr/CandidaDB) (accession no. AF527173), most likely the result of allele polymorphisms. Only one of these differences (A947C) is not synonymous and causes an amino acid substitution from K to T, but since it is outside the PDE signature, it is unlikely to be of functional significance.

Next we determined if the cloned CaPDE2 was able to complement the heat-shock sensitivity of the S. cerevisiae pde2 mutant (Fig. 1a). Strong overexpression of CaPDE2 using plasmid pWHPDE2 (Table 2 and Methods), was found to be toxic (data not shown). Unexpectedly, the transformants grown in the presence of glucose (repression conditions) were no longer sensitive to heat shock (data not shown). This implied that CaPDE2 was functional in S. cerevisiae. Moreover, considering the low transcription levels due to the leaky expression under the GAL1 promoter, CaPDE2 is probably not a highly expressed gene. Upon moderate expression using plasmid pWHPDE2-M, CaPDE2 complemented the heat-shock sensitivity (Fig. 1b). The complementation was due to pMET3- CaPDE2, since addition of 2 mM methionine restored sensitivity to heat shock (Fig. 1c). Therefore, CaPDE2 is functional in S. cerevisiae, but toxic upon overexpression, this being the most likely reason for earlier unsuccessful attempts to clone it by functional complementation.



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Fig. 1. CaPDE2 expressed under the MET3 promoter complements the heat-shock sensitivity of S. cerevisiae pde2 mutants. Heat-shock sensitivity of S. cerevisiae pde2 mutant DJ28 (a); DJ28 transformed with pWHPDE2-M after growth on SD medium without (b) and with methionine (c).

 
Initial characterization of CaPDE2 mutants
In the current analysis, isogenic strains were used with different CaPDE2 relevant genotypes (at least two independent isolates of each type) (Table 1 and Methods). All had one copy of URA3 at the same locus, thus eliminating the possible ‘URA3 effect (Fig. 2a). One of them, WH2-RU, also contained the wild-type CaPDE2 reintegrated into its authentic chromosomal location. This strain was used throughout this investigation as a control.

Southern blot analysis (Fig. 2b) demonstrated no genome rearrangements in the CaPDE2 chromosomal locus. The figure shows the results of one representative strain of every relevant genotype; however, identical results were produced for independently isolated mutants (data not shown). Northern analysis confirmed the deletion of CaPDE2 in the homozygous mutant WH2-3U (Fig. 3) and subsequent restoration of transcription by homologous reintroduction of CaPDE2 in the reconstituted strain WH2-RU. Interestingly, this experiment also demonstrated an upsurge in CaPDE2 expression 15 and 30 min after serum addition in all strains except the capde2 homozygous mutant. This observation suggested that an early phase in hyphal development might require down-regulation of the cAMP-dependent pathway.



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Fig. 3. Time-course Northern analysis of CaPDE2 expression levels during serum-induced hyphal transition. Strains were grown in SD at 30 °C overnight and about 1·0x105 cells ml-1 were transferred to liquid YPD+10 % serum pre-warmed at 37 °C. Aliquots were withdrawn from the cultures at the indicated time points and used for RNA isolation. The 695 bp BglII–SalI fragment from pWHPDE2 (Table 2) was used as probe for CaPDE2. The 687 bp probe for 18S RNA was generated by PCR using primers recommended by Bahn & Sundstrom, (2001).

 
The reconstituted strain WH2-RU and the reference strain CAF2-1 had identical doubling times of 2 h (in SD) and 1 h 30 min (in YPD) at 30 °C, with that of the homozygous mutant longer only in SD (2 h 30 min). Higher sensitivity to heat shock at 50 °C and growth inhibition at 42 °C were observed for the homozygous mutant in comparison to the control and the reference strains WH2-RU and CAF2-1 (data not shown). Following this experiment an S. cerevisiae pde2 mutant was tested and also found to have a growth defect at 37 °C (data not shown). Deletion of CaPDE2 also resulted in elevated levels of cAMP, which increased significantly in response to exogenous cAMP in comparison to the reference strain (Table 4).

Deletion of CaPDE2 precludes development of hyphae but not pseudohyphae upon addition of hyphal inducers in liquid medium
In YPD and SD at 30 °C all strains grew in the yeast form, the cells of the homozygous mutant were, however, enlarged, forming chains (about 5 % of the cell population) reminiscent of pseudohyphae in YPD. This capde2 mutant phenotype was even stronger in RPMI 1640 medium at 30 °C, where after 20 h the whole homozygous mutant population was growing as pseudohyphae. Following addition of 10 % serum to SD (data not shown) and YPD, the reconstituted strain (WH2-RU) had completed the transition after 75 min (Fig. 4a) and subsequently developed normal mycelium (Fig. 4b), as did the standard reference strain CAF2-1. In contrast, less than 15 % of the homozygous capde2 cells had responded to serum, and only pseudohyphae and some abnormal round yeast cells were visible 5 h post serum addition. A similar phenotype was apparent in serum containing RPMI 1640 (Fig. 4c), in Lee medium at pH 6·5 and Spider medium at 37 °C (data not shown). The homozygous mutant did not respond at all to the addition of GlcNAc (data not shown).



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Fig. 4. Phenotypic response to 10 % serum in liquid medium. Strains were grown in SD at 30 °C overnight and about 1·0x105 cells ml-1 were transferred to liquid YPD+10 % serum pre-warmed at 37 °C (a and b). Alternatively, cells were grown in RPMI 1640 at 30 °C overnight and 1·0x105 cells ml-1 were transferred to RPMI 1640+10 % serum pre-warmed at 37 °C (c). (a) Cells with germ tubes as a percentage of the total (±SD from two independent experiments), and images taken after 5 h in (b) YPD+10 % serum and (c) RPMI 1640+10 % serum. (a) Open circle, WH2-3U (capde2); filled circle, CAF2 (WT); filled triangle, WH2-RU (capde2/capde2 : : CaPDE2-URA3). (b, c) Bars, 50 µm.

 
These results suggested that CaPDE2 deletion inhibited hyphal, but not pseudohyphal growth and development. Experimental conditions which discriminate the formation of the two (Sudbery, 2001) were used to test this hypothesis. In YPD at 35 °C (pseudohypha-only inducing conditions) capde2 homozygous mutant (WH2-3U) formed normal pseudohyphae (Fig. 5a), as confirmed by calcofluor white (CFW) staining (data not shown). Upon addition of 10 % serum (hypha-only inducing conditions) the mutant formed highly abnormal opaque-looking cells (Fig. 5b). These results provided further support for our hypothesis that hyphal, but not pseudohyphal growth and development in liquid medium requires down-regulation of the cAMP-dependent pathway.



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Fig. 5. Differential induction of pseudohyphae and hyphae. Strains were grown in SD at 30 °C overnight and about 1·0x105 cells ml-1 were transferred to (a) liquid YPD pre-warmed at 35 °C (pseudohypha-only inducing conditions) or (b) YPD+10 % serum pre-warmed at 35 °C (hypha-only inducing conditions). The images were taken after 5 h incubation at 35 °C. Black arrowheads indicate pseudohyphal constrictions. Bars, 10 µm.

 
Exogenous cAMP induces low levels of pseudohypha formation in Candida strains of the reference genetic background
The phenotypes of the capde2 mutant in liquid medium seem to contradict previous reports on the effects of exogenous cAMP on hyphae formation in Candida (Sabie & Gadd, 1992). In our hands, however, the addition of exogenous cAMP at 100 µM did not induce hyphae as previously reported. Under the exact experimental conditions described by Sabie & Gadd (1992) all wild-type strains formed pseudohyphae only (Fig. 6) after 24 h. Their percentage, however, was nowhere near 50 % as reported by Sabie & Gadd (1992), the only exception being the capde2 homozygous mutant, where it reached 42 % in the presence of exogenous cAMP (Table 5).



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Fig. 6. Effects of exogenously added cAMP. Strains were grown in minimal defined medium as described by Sabie & Gadd (1992) at 25 °C for 2 days. About 2·0x106 cells ml-1 were transferred to fresh same medium with or without 100 µM exogenous cAMP. The images (with and without CFW staining) of strains SC5314 (a), CAF2 (b) and capde2 mutant WH2-3U (c) were taken after 24 h incubation at 25 °C. Black and white arrows indicate constrictions and septa, respectively.

 

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Table 5. Percentage of pseudohyphae in defined minimal medium at 25 °C (±SD based on two independent experiments)

 
Deletion of CaPDE2 distorts the pattern of changes in cAMP levels during serum-induced germ-tube formation in liquid medium
Previous reports on cAMP levels during yeast-to-hypha transition (Egidy et al., 1989, 1990; Sabie & Gadd, 1992; Bahn & Sundstrom, 2001) are difficult to compare because of differences in strains and indeed experimental conditions. We measured changes in cAMP levels in response to serum with our set of strains (Fig. 7). The early peak in cAMP levels was followed by a gradual reduction, the timing of which appears to correlate well with the rise in CaPDE2 expression levels. However, several noticeable differences were observed in the homozygous mutant. Its cAMP levels were considerably higher at the start (nearly twofold) and at the peak (nearly fourfold). After a steep decrease they remained higher throughout the course of the experiment. These measurements were performed three times and the mean values are presented in Fig. 7.



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Fig. 7. Changes in cAMP levels in response to serum. The strains were grown for 20 h in SD at 30 °C, diluted to 3·0x106 cells ml-1 in YPD+10 % serum pre-warmed at 37 °C and incubated further at 37 °C. At the indicated time points aliquots were taken and used for cAMP extraction and measurement as described in Methods. Bars indicate SD. Open circle, WH2-3U (capde2); filled circle, CAF2 (WT); filled triangle, WH2-RU (capde2/capde2 : : CaPDE2-URA3).

 
capde2 mutants form aberrant hyphae with fewer branches and lateral buds on solid medium and are defective in hypha-to-yeast reversion
CaPDE2 deletion enhanced levels of filamentation on yeast-growth-promoting solid medium as shown by the percentage of wrinkled colonies observed (Table 6). After 5 days on YPD+10 % serum the colonies of the homozygous mutant were highly wrinkled, but without protruding hyphae (Fig. 8a) and upon closer microscopic observation their filaments were found to contain less branches and lateral buds (Fig. 8b). Similarly abnormal hyphae were formed by the homozygous mutant on Spider medium at 37 °C, upon embedding at 30 °C and upon addition of GlcNAc. Moreover on Spider medium at 37 °C the growth of the homozygous mutant was highly invasive (data not shown).


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Table 6. Colony morphologies on yeast-growth-promoting solid medium

 


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Fig. 8. Phenotypic responses to serum on solid YPD. (a) Colony morphologies in YPD+10 % serum at 37 °C after 5 days and (b) the appearance of the filaments.

 
These results suggested that once converted to hyphae the capde2 cells could not efficiently revert to the yeast form on solid medium. To test this hypothesis, hyphae induced by GlcNAc (Fig. 9a) were excised from the plate, transferred to an SD plate and incubated at 30 °C for up to 5 days. Wild-type hyphae reverted quickly to the yeast form as shown by the smooth round appearance of the colonies, but the capde2 mutant cells remained in the aberrant hyphal form (Fig. 9b).



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Fig. 9. Hypha-to-yeast reversion of GlcNAc-induced hyphae. Strains were grown in liquid SD at 30 °C overnight. Approximately 200 cells were transferred to solid salt base medium containing GlcNAc (Delbrück & Ernst, 1993) and incubated at 37 °C. Seven days later hyphae were excised from the edges of the colonies, transferred to solid SD and incubated at 30 °C for up to 5 days. The images represent the edges of the colonies formed in the presence of GlcNAc at 37 °C (a) and after 5 days on SD medium at 30 °C (b).

 
capde2 mutants have highly reduced levels of EFG1 transcription during yeast-to-hypha transition
It has been reported that a reduced level of EFG1 expression induces cell elongation and pseudohyphal filamentation and prohibits formation of true hyphae (Stoldt et al., 1997). Moreover, efg1 knockout mutants have been filamentous when grown under embedded conditions at 25 and 37 °C (C. Kumamoto, personal communication). Since deletion of CaPDE2 caused similar phenotypes, we tested the expression levels of EFG1 in the current set of strains. Two hybridization products homologous to the EFG1 probe were observed in the yeast form of WH2-RU (reconstituted strain); however, only the high molecular species was detected in the homozygous mutant (Fig. 10). At all time points, up to 75 min after serum addition, the low-molecular-mass EFG1 homologous mRNA was detected in all strains except the capde2 mutant, where there was hardly any positive hybridization signal. The plasmid pBI-HAHYD, containing EFG1 under the control of the PCK1 promoter (Sonneborn et al., 2000), was transformed into WH2-4 (ura3 capde2 homozygous mutant); however, overexpression of EFG1 did not suppress the capde2 mutant phenotype in liquid medium (data not shown).



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Fig. 10. Northern analysis of EFG1 expression levels during serum-induced hypha transition. Strains were grown in SD at 30 °C overnight and about 1·0x105 cells ml-1 were transferred to liquid YPD+10 % serum pre-warmed at 37 °C. Aliquots were withdrawn from the cultures at the indicated time points and used for RNA isolation. The 1568 bp EcoRI–BglII fragment from pBI-HYHYD (Table 2) was used as probe for EFG1. The 687 bp probe for 18S RNA was generated by PCR using primers recommended by Bahn & Sundstrom, (2001).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isogenic mutants with CaPDE2-relevant genotypes have been generated and used in the current phenotypic analysis. They all contain one copy of URA3 at the CaPDE2 chromosomal locus and have not undergone any genome rearrangements as a result of the transformation (Fig. 2). The reconstituted strain, which also contains the wild-type CaPDE2 reintegrated at its authentic site, together with one copy of URA3 at the same locus, was used as a control throughout. At least two independent transformants for every representative genotype were analysed, and since no colony variations were observed (data not shown) the phenotypic differences can indeed be attributed to the changes affecting CaPDE2. With one exception (Table 6), the phenotypes of our heterozygous mutants and the reconstituted control strain were indistinguishable. We also included the reference strain CAF2-1 as a standard as its phenotypes under most of the experimental conditions employed in our studies have been reported.

Deletion of CaPDE2 renders Candida strains sensitive to heat shock (at 50 °C), inhibits growth at 42 °C and increases levels of intracellular cAMP, especially in response to exogenous cAMP (Table 4). In a parallel investigation we have demonstrated that capde2 mutants have a range of cell-wall-related phenotypes, such as sensitivity to SDS, CFW, caffeine and the antifungals amphotericin B and itraconazole (W. H. Jung & L. I. Stateva, unpublished). Similar phenotypes have been described for S. cerevisiae pde2 mutants. In response to hyphal inducers, the homozygous capde2 mutants were able to produce normal pseudohyphae, but not hyphae, in liquid medium (Fig. 4 and Fig. 5). On solid medium they formed aberrant hyphae, with fewer lateral buds and side chains (Fig. 8b and data not shown), which could not revert back to the yeast form (Fig. 9). The most likely explanation for this phenotype could be the higher cAMP levels which impair the ability of the cells to sense nutrient depletion. Taken together these results suggest that transitions between different morphological forms are dependent on Capde2p-mediated changes in cAMP levels. Elevated cAMP levels caused by CaPDE2 deletion (Fig. 7), are prohibitive for hyphal but not pseudohyphal growth in liquid medium (Fig. 4, Fig. 5 and data not shown). Other regulatory pathways contribute to hyphal growth on solid medium; however, CaPde2p-mediated changes in cAMP levels are, apparently, required for the hypha-to-yeast transition. Contrary to published results (Sabie & Gadd, 1992) we found that exogenous cAMP caused formation of pseudohyphae only (Fig. 6 and Table 5). These discrepancies could be due to differences in strain background or due to the fact that previously the distinction between hyphae and pseudohyphae was not as clearly defined. However, in the CAI-4 genetic background the effects of exogenous cAMP are consistent with the data from the capde2 mutant phenotypic analysis.

Previously reduced levels of EFG1, a downstream target of the cAMP-dependent pathway which plays a significant role in hypha and chlamydospore formation and in the white–opaque switch event (Srikantha et al., 2000), have been shown to suppress hypha, but not pseudohypha formation (Stoldt et al., 1997). In the current investigation very low EFG transcript levels were observed (Fig. 10) in the capde2 mutant. Our results suggest that EFG1 expression is cAMP-dependent and provide further evidence for its different roles as a regulator of hyphal and pseudohyphal morphogenesis in C. albicans. The failure of EFG1 overexpression to complement capde2 phenotypes is probably due to it being expressed under a heterologous promoter. EFG1 has been shown to autoregulate itself (Stoldt et al., 1997; Bockmühl & Ernst, 2001). Moreover, expression of EFG1 under a heterologous promoter was accompanied by a disappearance of the authentic EFG1 transcript (Stoldt et al., 1997).

A recent report has shown that in S. cerevisiae the concentration of Pde2p is limiting (Namy et al., 2002). The low expression levels of CaPDE2 as suggested by the functional activity of the GAL1-CaPDE2 leaky expression on glucose medium (data not shown) and the results in Fig. 3 suggest the same is true for C. albicans. Furthermore, our results and those of others imply that small increases in cAMP levels can have a profound effect. Deletion of the two copies of CaPDE2 increased cAMP levels by 40 % (Table 4) after 12 h and 100 % after 20 h growth (Fig. 7, zero time point). Small changes in cAMP levels have been observed in S. cerevisiae pde2 mutants; however, they increase upon addition of exogenous cAMP (Wilson et al., 1993), as they did in C. albicans. The increased expression of CaPDE2 (Fig. 3) in response to serum could therefore represent a need for a higher concentration of Capde2p to respond to the changes in intracellular cAMP levels (Fig. 7). Interestingly, a transient increase in CaPDE2 expression levels was recently reported in a microarray study of human-blood-induced changes in gene expression in C. albicans (Fradin et al., 2003). In most mammalian tissues maximum responses have been triggered by up to twofold increases in cyclic nucleotides (Corbin et al., 1985), and as our results show, C. albicans operates within similar ranges (Fig. 7). Moreover, the increased expression of CaPDE2 (Fig. 3; Fradin et al., 2003) might constitute a feedback mechanism for lowering basal cAMP levels. One such feedback system involving the high-affinity cAMP PDE has already been proven in mammalian cells (Zinman & Hollenberg, 1974; Loten, 1978; Gettys et al., 1987), where cAMP hydrolysis plays an important role in terminating the hormonal stimulus and is one of several mechanisms inducing desensitization in the target cells. A similar mechanism is apparent in Candida as illustrated in the current investigation. The low-affinity cAMP PDE CaPde1p, whose S. cerevisiae orthologue Pde1p is involved in control of glucose-induced cAMP signalling (Ma et al., 1999), is the obvious enzyme responsible for hydrolysis of cAMP in the capde2 homozygous mutant. However, its mode of regulation and activity significantly distorts the pattern of cAMP changes upon serum-induced hyphal transition in C. albicans (Fig. 7). CaPDE1 has been cloned, but no capde1 mutants have yet been constructed (Hoyer et al., 1994). Analysing such mutants might help to elucidate the more specific roles of the low-affinity cAMP PDE in C. albicans. Interestingly, nuclear localization of Pde2p in S. cerevisiae was recently demonstrated (Namy et al., 2002). This has prompted the speculation that cAMP exerts its effects by a gradient in its concentration, being higher around the membrane and lower around the nucleus (Namy et al., 2002), and that the high-affinity cAMP PDE plays a significant role in this process. We are currently investigating whether a similar mechanism operates in C. albicans as well.

Our data show that cAMP-mediated signalling in response to hyphal inducers in vitro is medium-specific. Medium-dependent morphological defects have been reported for ras1/ras1 Candida mutants (Feng et al., 1999) and positive specific roles in filament formation in different environments have also been demonstrated for both Tpk isoforms. Moreover, although filamentation is stimulated upon overexpression of Tpk isoforms, normal hyphal development is not. This is further supported by our current findings, which show that down-regulation of the cAMP-dependent pathway (PKA) is required for hyphal development in liquid medium and for hypha-to-yeast reversion on solid medium, and that this down-regulation requires functional CaPde2p. It will be of great interest to determine what effects CaPDE2 deletion will have in vivo.


   ACKNOWLEDGEMENTS
 
The authors would like to thank A. Brown, J. Ernst, N. Zhang and D. Jones for strains and plasmids, P. Hopkins and J. Miyan for help with the microscopy, C. Kumamoto and M. Jacquet for helpful discussions, and N. Gow and R. Walmsley for critical reading of the manuscript. This work was supported by a BBSRC grant to L. S.


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Received 27 May 2003; revised 27 June 2003; accepted 30 June 2003.