Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 1QD, UK
Correspondence
Lubomira I. Stateva
lubomira.stateva{at}umist.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
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 KpnISacI 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 BamHIXbaI fragment was cloned into BamHI/XbaI-restricted pRS415 (Sikorski & Hieter, 1989
) to generate pRS415PDE2. The SacIISacI fragment from pMETtPKG3-3, containing the PGK1 terminator, was ligated into SacII/SacI-digested pRS415PDE2 to generate pRS415P2tPKG. Finally, the SmaIBamHI 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.
|
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 SacIPstI 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.
|
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.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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.
|
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).
|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 whiteopaque 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bahn, Y. S. & Sundstrom, P. (2001). CAP1, and adenylate cyclase-associated protein gene, regulates budhypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans. J Bacteriol 183, 32113223.
Bockmühl, D. P. & Ernst, J. F. (2001). A potential phosphorylation site for an A-type kinase in the Efg1 regulator contributes to hyphal morphogenesis of Candida albicans. Genetics 157, 15231530.
Bockmühl, D. P., Krishnamurthy, S. K., Gerads, M., Sonneborn, A. & Ernst, J. (2001). Distinct and redundant roles of the two protein kinase A isoforms Tpk1 and Tpk2 in morphogenesis and growth of Candida albicans. Mol Microbiol 42, 12431257.[CrossRef][Medline]
Borges-Walmsley, M. I. & Walmsley, A. (2000). cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity. Trends Microbiol 8, 133141.[CrossRef][Medline]
Brown, A. J. & Gow, N. A. (1999). Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol 7, 333338.[CrossRef][Medline]
Brown, D. H., Jr, Giusani, A. D., Chen, X. & Kumamoto, C. A. (1999). Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene. Mol Microbiol 34, 651662.[CrossRef][Medline]
Bullock, W. O., Fernandez, J. M. & Short, J. M. (1987). XL1-Blue a high-efficiency plasmid transforming recA Escherichia coli strain with -galactosidase selection. Biotechniques 5, 376378.
Charbonneau, H., Beier, N., Walsh, K. A. & Beavo, J. A. (1986). Identification of a conserved domain among cyclic nucleotide phosphodiesterases from diverse species. Proc Natl Acad Sci U S A 83, 93089312.[Abstract]
Chattaway, F. W., Wheeler, P. R. & O'Reilly, J. (1981). Involvement of adenosine 3' : 5'-cyclic monophosphate in the germination of blastospores of Candida albicans. J Gen Microbiol 123, 233240.[Medline]
Corbin, J. D., Beebe, S. J. & Blackmore, P. F. (1985). cAMP-dependent protein kinase activation lowers hepatocyte cAMP. J Biol Chem 260, 87318735.
Csank, C., Schröppel, K., Leberer, E., Harcus, D., Mohamed, O., Meloche, S., Thomas, D. Y. & Whiteway, M. (1998). Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and systemic candidiasis. Infect Immun 66, 27132721.
Daniel, J. (1996). Measuring the toxic effects of high gene dosage on yeast cells. Mol Gen Genet 253, 393396.[CrossRef][Medline]
Davis, D., Wilson, R. B. & Mitchel, A. P. (2000). RIM101-dependent and -independent pathways govern pH responses in Candida albicans. Mol Cell Biol 20, 971978.
Delbrück, S. & Ernst, J. F. (1993). Morphogenesis-independent regulation of actin transcript levels in the pathogenic yeast Candida albicans. Mol Microbiol 10, 859866.[Medline]
Egidy, G., Paveto, C., Passeron, S. & Galvagno, M. A. (1989). Relationship between cyclic adenosine 3' : 5'-monophosphate and germination in Candida albicans. Exp Mycol 13, 428432.
Egidy, G., Paveto, C., Passeron, S. & Galvagno, M. A. (1990). cAMP levels and in situ measurement of cAMP related enzymes during yeast-to-hyphae transition in Candida albicans. Cell Biol Int Rep 14, 5968.[Medline]
Ernst, J. F. (2000). Transcription factors in Candida albicans environmental control of morphogenesis. Microbiology 146, 17631774.
Fedor-Chaiken, M., Deschenes, R. J. & Broach, J. R. (1990). SRV2, a gene required for RAS activation of adenylate cyclase in yeast. Cell 61, 329340.[Medline]
Feng, Q., Summers, E., Guo, B. & Fink, G. (1999). Ras signaling is required for serum-induced hyphal differentiation in Candida albicans. J Bacteriol 181, 63396346.
Field, J., Vojtek, A., Ballester, R. & 12 other authors (1990). Cloning and characterisation of CAP, the S. cerevisiae gene encoding the 70 kD adenylyl cyclase-associated protein. Cell 61, 319327.[Medline]
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717728.
Fradin, C., Kretschmar, M., Nichterlein, T., Gaillardin, C., d'Enfert, C. & Hube, B. (2003). Stage-specific gene expression of Candida albicans in human blood. Mol Microbiol 47, 15231543.[CrossRef][Medline]
Gettys, T. W., Blackmore, P. F., Redmon, J. B., Beebe, S. T. & Corbin, J. D. (1987). Short-term feedback regulation of cAMP by accelerated degradation in rat tissues. J Biol Chem 262, 333339.
Gillum, A. M., Tsay, E. Y. H. & Kirsch, D. R. (1984). Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198, 179182.[Medline]
Heale, S. M., Stateva, L. & Oliver, S. G. (1994). Introduction of YACs into intact yeast cells by a procedure which shows low levels of recombinagenicity and co-transformation. Nucleic Acid Res 23, 50115015.
Hill, J., Ian, K. A., Donald, G. & Griffiths, D. E. (1991). DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res 19, 5791.[Medline]
Hoyer, L. L., Cieslinski, L. B., McLaughlin, M. M., Torphy, T. J., Shatzman, A. R. & Livi, G. P. (1994). A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterisation of the recombinant enzyme. Microbiology 140, 15331542.[Abstract]
Kataoka, T., Broek, D. & Wigler, M. (1985). DNA sequence and characterisation of the S. cerevisiae gene encoding adenylate cyclase. Cell 43, 493505.[Medline]
Lee, K. L., Buckley, H. R. & Campbell, C. C. (1975). An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans. Sabouraudia 13, 148153.[Medline]
Liu, H., Köhler, J. & Fink, G. R. (1994). Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science 266, 17231725.[Medline]
Loten, E. G. (1978). Stimulation of a low Km phosphodiesterase from liver by insulin and glucagons. J Biol Chem 253, 746757.[Medline]
Ma, P., Wera, S., VanDijck, P. & Thevelein, J. M. (1999). The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signalling. Mol Biol Cell 10, 91104.
Nakafuku, M., Obara, T., Kaibuchi, K. & 7 other authors (1988). Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions. Proc Natl Acad Sci U S A 85, 13741378.[Abstract]
Namy, O., Duchateau-nguyen, G. & Rousset, J. P. (2002). Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae. Mol Microbiol 43, 641652.[CrossRef][Medline]
Niimi, M., Niimi, K., Tokunaga, J. & Nakayama, H. (1980). Changes in cyclic nucleotide levels and dimorphic transition in Candida albicans. J Bacteriol 142, 10101014.[Medline]
Nikawa, J., Sass, P. & Wigler, M. (1987). Cloning and characterisation of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae. Mol Cell Biol 7, 36293636.[Medline]
Pan, X. & Heitman, J. (2002). Protein Kinase A operates a molecular switch that governs yeast pseudohyphal differentiation. Mol Cell Biol 22, 39813993.
Rocha, C. R. C., Schröppel, K., Harcus, D., Marcil, A., Dignard, D., Taylor, B. N., Thomas, D. Y., Whiteway, M. & Leberer, E. (2001). Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell 12, 36313643.
Sabie, F. T. & Gadd, G. M. (1992). Effect of nucleosides and the relationship between cellular adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) and germ tube formation in Candida albicans. Mycopathologia 119, 147156.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sass, P., Field, J., Nikawa, J., Toda, T. & Wigler, M. (1986). Cloning and characterisation of the high-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 83, 93039307.[Abstract]
Schmitt, M. E., Brown, T. A. & Trumpower, B. L. (1990). A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18, 30913092.[Medline]
Sherman, F., Fink, G. R. & Hicks, J. B. (1986). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sikorski, R. S. & Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 1927.
Sonneborn, A., Bockmühl, D. P., Gerads, M., Kurpanek, K., Sanglard, D. & Ernst, J. (2000). Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans. Mol Microbiol 35, 386396.[CrossRef][Medline]
Srikantha, T., Tsai, L. K., Daniels, K. & Soll, D. (2000). EFG1 null mutants of Candida albicans switch but cannot express the complete phenotype of white-phase budding cells. J Bacteriol 182, 15801591.
Stoldt, V. R., Sonneborn, A., Leuker, C. E. & Ernst, J. (1997). Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi. EMBO J 16, 19821991.
Sudbery, P. E. (2001). The germ tubes of Candida albicans hyphae and pseudohyphae show different patterns of septin ring localisation. Mol Microbiol 41, 1931.[CrossRef][Medline]
Sullivan, P. A., Yin, C. Y., Mollow, C. & Templeton, M. D. (1983). An analysis of the metabolism and cell wall composition of Candida albicans during germ-tube formation. Can J Microbiol 29, 15141525.[Medline]
Tait, E., Simon, M. C., King, S., Brown, A. J., Gow, N. A. R. & Shaw, D. (1997). A Candida albicans genome project: cosmid contigs, physical mapping, and gene isolation. Fungal Genet Biol 21, 308314.[CrossRef][Medline]
Taylor, J. (1999). Searches for cyclic-AMP phosphodiesterase genes from Candida albicans. PhD thesis, UMIST.
Thevelein, J. M. & deWinde, J. H. (1999). Novel sensing mechanism and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol 33, 904918.[CrossRef][Medline]
Toda, T., Uno, I., Ishikawa, T. & 7 other authors (1985). In yeast, Ras proteins are controlling elements of adenylate cyclase. Cell 40, 2736.[Medline]
Toda, T., Cameron, S., Sass, P. & Wigler, M. (1987a). Three different genes in the yeast Saccharomyces cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell 50, 277287.[Medline]
Toda, T., Cameron, S., Sass, P., Zoller, M., Scott, J. D., McMullen, B., Hurwitz, M., Krebs, E. G. & Wigler, M. (1987b). Cloning and characterisation of BCY1, a locus encoding the regulatory subunit of the cAMP-dependent protein kinase in yeast. Mol Cell Biol 7, 13711377.[Medline]
Tomlin, G. C., Hamilton, G. E., Gardner, D. C. J., Walmsley, R. M., Stateva, L. I. & Oliver, S. G. (2000). Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis. Microbiology 146, 21332146.
Tripathi, G., Wiltshire, C., Macaskill, S., Tournu, H., Budge, S. & Brown, A. (2002). Gcn4 co-ordinates morphogenetic and metabolic responses to amino acid starvation in Candida albicans. EMBO J 21, 54485456.
Warit, S., Walmsley, R. & Stateva, L. (1998). Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae. Microbiology 144, 24172426.[Abstract]
Warit, S., Zhang, N., Short, A., Walmsley, R., Oliver, S. & Stateva, L. (2000). Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species. Mol Microbiol 36, 11561166.[CrossRef][Medline]
Wilson, R. B., Renault, G., Jacquet, M. & Tatchell, K. (1993). The PDE2 gene of Saccharomyces cerevisiae is allelic to rca1 and encodes a phosphodiesterase, which protects the cell from extracellular cAMP. FEBS Lett 325, 191195.[CrossRef][Medline]
Wilson, R. B., Davis, D., Enloe, B. M. & Mitchell, A. P. (2000). A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions. Yeast 16, 6570.[CrossRef][Medline]
Winston, F., Dollard, C. & Ricupero-Hovasse, S. L. (1995). Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11, 5355.[Medline]
Yamada-Okabe, T., Mio, T., Ono, N., Kashima, Y., Matsui, M., Arisawa, M. & Yamada-Okabe, H. (1999). Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans. J Bacteriol 181, 72437247.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103119.[CrossRef][Medline]
Zhang, N., Gardner, D., Oliver, S. G. & Stateva, L. (1999). Genetically controlled cell lysis in the yeast Saccharomyces cerevisiae. Biotechnol Bioeng 64, 607615.[CrossRef][Medline]
Zinman, B. & Hollenberg, C. H. (1974). Effect of insulin and lipolytic agents on rat adipocyte low Km cyclic adenosine 3' : 5'-monophosphate phosphodiesterse. J Biol Chem 249, 21822187.
Received 27 May 2003;
revised 27 June 2003;
accepted 30 June 2003.