1 Institut für Mikrobiologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany
2 Jawaharlal Nehru University, New Delhi 110067, India
Correspondence
Joachim F. Ernst
Joachim.ernst{at}uni-duesseldorf.de
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ABSTRACT |
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Both authors contributed equally to this work.
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INTRODUCTION |
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In the yeast Saccharomyces cerevisiae the OLE1 gene product encodes a stearoyl-CoA desaturase (EC 1.14.99.5) located in membranes of the endoplasmic reticulum (ER), which transforms the CoA derivatives of palmitic and stearic acid into the corresponding 9 monounsaturated palmitoleic (C16 : 1) and oleic acid (C18 : 1) derivatives, respectively (Stukey et al., 1989
). The C16 : 1 derivative is the predominant unsaturated fatty acid in S. cerevisiae, while other fungi including Candida species also produce C18 : 2 (linoleic) and C18 : 3 (linolenic) fatty acids, which are not present in S. cerevisiae (reviewed by Mishra et al., 1992
). Fatty acid desaturases use electrons in cytochrome b5 (derived from NADH by NADH-dependent cytochrome b5 reductase) and molecular oxygen to form a double bond between C-atoms 9 and 10 of fatty acids. Remarkably, fungal fatty acid desaturases contain a cytochrome b5 domain as integral parts of their enzyme structures, while vertebrate enzymes use separate cytochrome b5 molecules (Mitchell & Martin, 1995
). The active site in the native protein comprises three histidine-rich sequences, which fold to form two iron-binding sites. Yeast Ole1p contains four putative transmembrane regions, which by forming two pairs of membrane-traversing regions could attach the desaturase to the ER membrane and leave most of the Ole1p sequences within the cytoplasm (Stukey et al., 1990
). OLE1 expression is regulated by fatty acids, oxygen and temperature. Saturated fatty acids induce a 1·6-fold increase in transcription, while unsaturated fatty acids repress OLE1 transcription up to 60-fold (McDonough et al., 1992
; Bossie & Martin, 1989
). At low temperatures and during oxygen limitation, OLE1 expression is induced (Kwast et al., 1998
; Nakagawa et al., 2002
). A deletion analysis of the OLE1 promoter identified a 111 bp fatty acid-regulated region (FAR) which is essential for transcription activation and repression by unsaturated fatty acids (Choi et al., 1996
). In addition, the low oxygen response promoter element (LORE) mediates oxygen repression of OLE1 (Nakagawa et al., 2001
; Vasconcelles et al., 2001
). Two genes encoding components of fatty acid transporters, FAA1 and FAA4, were found to be essential for unsaturated fatty acid-repression of OLE1 via FAR sequences (Faergeman et al., 2001
). Also, the acyl-CoA binding protein and the Ssn6Tup1 complex were shown to be involved in repression of OLE1 (Fujimori et al., 1997
). On the other hand, the Hap1 transcriptional activator (Choi et al., 1996
) and two transcription factors, Spt23 and Mga2p, which are initially synthesized as inactive ER-bound precursors, positively regulate OLE1 expression (Zhang et al., 1999
). Ole1p is a naturally short-lived enzyme and is degraded by ubiquitin/proteasome-dependent ER-associated degradation (Braun et al., 2002
). Lowering of Ole1p activity leads to a loss of mitochondrial inheritance (Stewart & Yaffe, 1991
) and disturbs the integrity of the nuclear membrane (Zhang et al., 1999
), although growth is not affected even if unsaturated fatty acid levels are reduced down to one-eighth of the normal level (Stukey et al., 1989
).
The human fungal pathogen Candida albicans is able to assume different growth forms, which appear to have different roles for hostcell interaction and virulence (reviewed by Ernst, 2000). At body temperatures and in the presence of inducing agents, a true hyphal form is induced, which may be involved in anchoring within and penetrating tissues, while at lower temperatures, in the absence of inducers, a unicellular yeast form is favoured (Joshi et al., 1993
; Sonneborn et al., 1999
). The yeast form, in special genetic backgrounds at low temperatures, may spontaneously form a rod-like appearance (opaque). Thick-walled chlamydospores appear in certain media, preferentially at lower temperatures. The striking dependence of temperature on the generation of morphological forms suggests a role of membrane fluidity in morphogenesis of C. albicans. Although some signalling pathways mediating the yeasthypha transition have been defined, no membrane sensors are yet known which mediate environmental cues and specifically could act as cellular thermometers. In this report, we characterize the OLE1 gene of C. albicans and show that it is essential for viability. We show for the first time in a fungal developmental system that levels of oleic acid in cellular lipids are critical for morphogenetic competence. Thus, a modest reduction in oleic acid does not affect growth in the yeast form, but effectively prevents the formation of hyphal filaments and chlamydospores in aerobic conditions. Our results indicate, however, that overall membrane fluidity is not directly responsible for the morphogenetic potential of C. albicans, but that oleic acid has a specific role to activate specific morphogenetic pathways.
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METHODS |
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S. cerevisiae Y0779 (ole1 : : LEU2 leu2,3-112 lys2-801 trp1-1 ura3-52), kindly provided by S. Jentsch, was cultured at 30 °C in SD medium supplemented with 0·2 % oleic acid in 0·2 % NP40 (Braun et al., 2002
). It was transformed by expression plasmids containing OLE1 (pSKM24) or OLE1-GFP (pSKM62) under transcriptional control of the C. albicans PCK1 promoter.
Chromosomal deletions of OLE1 and OLE2.
C. albicans sequence data were obtained from the Stanford Genome Technology Center website (http://www-sequence.stanford.edu/group/candida). The genomic region of OLE1 was isolated by PCR using DNA of strain CAI4. The entire OLE1 coding region was disrupted by the Ura-blaster method (Fonzi & Irwin, 1993). A cassette for disruption of OLE1 was constructed in several steps. First, sequences 5' and 3' of the OLE1 ORF were amplified by PCR and subcloned. A fragment (0·98 kb) of the 5' sequences flanking the start of the CaOLE1 ORF was amplified using primers Ole1disA and Ole1disB (5'-CTAGAGCTCGGATCCCACGGAACTAAAC-3'/5'-CTAGAGCTCGGATCCAGCAGCAATGGCATTC-3'; bold, regions of homology; italics, BamHI); similarly, 728 bp of the 3' untranslated sequences were amplified using primers Ole1disC and Ole1disD (5'-CTAGAGCTCTGCAGAAGGAAAAGCAATC-3'/5'-CTAGAGCTCTGCAGGCGACTACATACATAC-3'; bold, regions of homology; italics, PstI). PCR fragments were subcloned into pUC18, which resulted in plasmids pSKM8 and pSKM7, respectively. The BamHI fragment of pSKM8 was cloned into the BglII site of p5921. The PstI fragment of pSKM7 was inserted into the PstI site of the resulting plasmid pSKM55. A plasmid with OLE1 flanking sequences in the correct orientation was obtained (pSKM56). Its HindIII fragment containing the OLE1 disruption cassette was used to transform strain CAI4.
OLE2 was disrupted similarly by subcloning the regions flanking and partially including the ORF. The 876 bp 5' region was amplified by genomic PCR using primers 709 and 710 (5'-TATGGATCCAAAACTCCTGTAGATGG-3'/5'-TATGGATCCACATACAAGACTGC-3'; bold, regions of homology; italics, BamHI); the 870 bp 3' region was amplified by genomic PCR using primers 711 and 712 (5'-TATAGATCTGACTGCTGCGGTG-3'/5'-TATAGATCTGCCAACTTTTCTAATGC-3'; bold, regions of homology; italics, BglII). The BamHI fragment carrying the 5' region was subcloned into the BglII site of p5921, while the BglII fragment carrying the 3' region was subcloned into the BamHI site of the resulting vector, to construct pAP9a. The SacISphI fragment of pAP9a was used for sequential disruption of both OLE2 alleles (Fonzi & Irwin, 1993).
To place OLE1 under control of the MET3 promoter we followed a previously described strategy (Care et al., 1999). First, a fragment of 624 bp corresponding to the 5' end of the OLE1 ORF was amplified by PCR using the primers OleBHI-Nterm (5'-CTTAAGCTTGGATCCCATGTGAGAAAACC-3') and OleStpBHI-HindIII (5'-CTTAAGCTTGGATCCTAAGATTGCTTTTCC-3'; bold, regions of homology; underlining, HindIII, BamHI) and the PCR fragment was cloned into pUC18, resulting in plasmid pSKM20. The BamHI fragment of pSKM20 was inserted into the single BamHI site of pCaDis, downstream of the MET3 promoter (Care et al., 1999
). The resulting plasmid pSKM25 was linearized by NcoI (which cuts within the OLE1 fragment) and was used to transform the heterozygous strain
O8.2 (OLE1/ole1
: : hisG).
Overexpression of OLE1 and OLE2.
To overexpress OLE1 in C. albicans we used pBI-1, a derivative of pRC2312 (Stoldt et al., 1997) containing the PCK1 promoter. The entire OLE1 coding region was amplified by genomic PCR using primers OLEHindIII-BHIATG (5'-CTTAAGCTTGGATCCACAATGACTACAGTTG-3') and OLEStpBHI-HindIII (5'-CTTAAGCTTGGATCCTAAGATTGCTTTTCC-3') (italics: HindIII, BamHI sites) and cloned into pUC18 to generate pSKM19. The 1·072 kb BamHI fragment of pSKM19 was inserted downstream of PCK1p into the BglII site of pBI-1, thereby generating OLE1-overexpression plasmid pSKM24. Similarly, the entire OLE2 ORF was amplified by genomic PCR using primers 707 and 708 (5'-TATGGATCCAGGCAAATAATATATCC-3'/5'-TATGGATCCATTAATCTGTTAAAGTAG-3'; italics, BamHI) and inserted, as a 1·58 kb fragment, into the BglII site of pBI-1 to generate the OLE2-overexpression vector pAP5.
For C-terminal tagging of OLE1 with green fluorescent protein (GFP), we generated a PCR fragment containing PCK1p-OLE1 with primers PCKp-HIII (5'-AGAAGCTTGGCTGCAGGTCGAC-3') and Ole1-GFP (5'-AATAAGCTTCCAGATTGCTTTTCCTTCTCC-3') (bold, regions of homology; underlined, HindIII), using pSKM24 as a template DNA and the resulting PCR fragment was cloned into pUC18 (pSKM58). The HindIII fragment of pSKM58 containing PCK1p-OLE1 without the stop codon was cloned into the single HindIII site in-frame with GFP into vector pRCGFP3 to generate pSKM62. To C-terminally tag OLE1 with a myc epitope, we used primers PCKp-HIII-BHI-SalI (5'-GTCGACGGATCCAAGCTTGGCTGCAGGTCGAC-3'; bold, regions of homology; underlined, SalI, BamHI, HindIII) and Ole1-Myc (5'-AATGGATCCTCACAAGTCTTCCTCGGAGATTAGCTTTTGTTCACCAGATTGCTTTTCCTTCTCC-3'; bold, regions of homology; underlined, BamHI; italics, myc epitope) to generate a PCR fragment, which was cloned into pUC18. The BamHI fragment of the resulting plasmid pSKM59 was cloned into the BglII site of pBI-1 to generate plasmid pSKM63.
Blotting procedures.
Total RNA was isolated from liquid cultures as described (Stoldt et al., 1997). The conditional strain
O7.2/25-2 was grown in SD medium without or with 0·25 mM methionine/cysteine. Following denaturing gel electrophoresis, RNA blots were probed with the 966 bp HindIIIEcoRI fragment of pSKM19, which corresponds with the 5' region of the OLE1 ORF, or with the 938 bp EcoRI fragment derived from the OLE2 ORF.
Immunoblottings for detection of Ole1p-myc fusions in crude extracts of transformants carrying pSKM63 were carried out as described (Weber et al., 2001).
Fatty acid analyses.
Lipids were extracted from cells disrupted by glass beads, as described (Daum et al., 1999), and subjected to methanolysis using BF3/methanol. Fatty acyl methyl esters were separated by GC on a Shimadzu GC-17A (version 3) gas chromatograph using a FS-CW-20M-0·25 polarity capillary column (0·25 mmx25 m; film thickness 0·25 mm) with a temperature gradient (160200 °C at 1 °C min1; hold at 200 °C for 20 min). Fatty acids were identified by comparisons to a commercial standard (RM-6; Supelco).
Determination of membrane fluidity.
Cells grown to an OD600 value of 1 (or OD600 0·7 for the conditional strain) were washed and converted to spheroplasts by zymolase 20T treatment. Spheroplasts were washed thrice in 20 mM Tris/HCl pH 7·5/10 mM MgSO4/0·6 M sorbitol and resuspended in this buffer. An aqueous solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) was prepared by diluting a 2 mM solution in tetrahydrofuran into 50 ml of 20 mM Tris/HCl pH 7·5 and removing traces of tetrahydrofuran by flushing with nitrogen. DPH (2 µM) was added to spheroplasts (4x108 cells ml1) and incubated at 30 °C in a water bath shaker for 1 h. Fluorescence polarization was determined by excitation with vertically polarized monochromatic light (360 nm) and measurements of emission intensities at 426 nm using an analyser oriented parallel or perpendicular to the excitation light (Smriti et al., 1999; Kaur & Bachhawat, 1999
).
The degree of fluorescence polarization (P) was calculated according to the following formula
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GFP fluorescence microscopy.
Cells were used for fluorescence microscopy directly without fixation. Nuclei were stained by the addition of 10 µg 4',6-diamidino-2-phenylindole (DAPI) ml1 to the cell suspension. All cells were viewed using a Zeiss Axioplan 2 fluorescence microscope. Images were taken with a Quantix Digital CCD camera using METAMORPH software and processed in corel PHOTOPAINT 11.0.
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RESULTS |
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Using the above-mentioned procedure we also generated two independent lines of CAI4 derivatives, in which one or both OLE2 alleles were disrupted (data not shown). At the OLE2 locus strains cII and CIV are heterozygous (ole2/OLE2), while strains JA1 and JA2 are homozygous mutants (ole2/ole2). URA3-minus derivatives of strain JA1 were isolated by FOA selection (resulting strains JA1x and JA2x), which subsequently were transformed by the expression vector pAP5 to reconstitute OLE2. In Northern blottings using an OLE2 probe on total RNA of strain CAF2-1, we observed that OLE2 was expressed, generating a transcript of about 2 kb, but signal intensity was much lower compared to the OLE1 transcript (data not shown). Thus, OLE2 appears to be expressed at low levels, at least in the conditions used here.
Growth depends on OLE1 expression levels
The conditional strain O8.2/25-2 (MET3p-OLE1/ole1
: : hisG) was streaked on SD medium containing cysteine and/or methionine to repress OLE1 (Fig. 3
A). Growth was blocked completely at 2·5 mM cysteine, 0·25 mM methionine and 0·05 mM of a cysteine/methionine mixture, while the heterozygous strains
O7 and
O8 were not affected. Likewise, in liquid SD medium containing 2·5 mM methionine/cysteine, growth of the conditional strains was blocked completely, with a terminal phenotype of mostly unbudded cells that tended to aggregate (data not shown), suggesting a block in the G1 or G0 phase of the cell cycle. The threshold level, at which methionine/cysteine blocked growth, strongly depended on the type of media and growth conditions used: whereas cysteine/methionine at a concentration of 0·05 mM prevented growth on solid SD medium, it did not block growth in liquid SD medium up to 0·25 mM cysteine/methionine. Also, on Lee's medium, growth of the conditional strain was inhibited significantly only at methionine concentrations above 5 mM. This medium dependence may be due to different efficiencies of MET3p repression in different conditions. The addition of 0·5 mM oleic acid in 1 % Igepal CA-630 as solubilizer was able to restore growth of the conditional strain
O8.2/25-2 in OLE1-repressing conditions (Fig. 3B
). In contrast, the conditional control strain CA2d1m, which contains the essential SEC20 gene under control of MET3p (Weber et al., 2001
), could not be rescued by oleic acid. Thus, these results indicate that OLE1 is essential in C. albicans and suggest that it is involved in oleic acid biosynthesis.
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In contrast to the results obtained for OLE1, both alleles of OLE2 could be deleted without any difficulty and the resulting mutants (e.g. strain JA2) grew as well as the wild-type in all media, in the absence of oleic acid. Furthermore, the CaOLE2 expression vector pAP5 was unable to reconstitute the S. cerevisiae ole1 mutant Y0779. Thus, these experiments provided no evidence for a function of the OLE2 gene product as a 9 stearoyl desaturase.
Hyphal morphogenesis depends on OLE1 expression levels
We examined, next, if partial repression of OLE1 expression in the conditional strain would still allow growth, but prevent hyphal morphogenesis. Common media used for induction of hypha formation already contain methionine and/or cysteine: Lee's medium contains 0·075 mM methionine (Lee et al., 1975), which in SD medium blocks growth of the conditional strain
O8.2/25-2 completely (Fig. 3A
); Spider and serum media contain complex sources of nitrogen likely to include methionine or cysteine. As stated above, growth of the conditional strain was not affected on Spider medium or on Lee's medium compared to the wild-type strain. In contrast, hypha formation on both media was completely blocked (Fig. 4
) and microscopy revealed that colonies consisted entirely of yeast-form cells. The importance of a sufficient dosage of wild-type OLE1 expression was confirmed by the OLE1/ole1 heterozygous strain, which had a reduced ability to form hyphae. A more complex phenotype was observed on serum medium, on which the conditional and the heterozygous strains produced hyphae, although their growth was retarded (presumably because of methionine/cysteine as well as traces of oleic acid in serum). The effect of lowered OLE1 expression on hyphal morphogenesis was confirmed in liquid SD medium containing 5 % serum and methionine/cysteine, although these experiments were hampered by the fact that both amino acids impaired hypha formation partially even in the control strain CAF2-1 (data not shown).
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Since these experiments had shown that low oleic acid levels could prevent morphogenesis, we also asked if increased levels would enhance hypha formation. Therefore, we pre-grew strain CAI4(pSKM24) in inducing SCAA medium to overexpress OLE1 by the PCK1 promoter. Although in this medium a small fraction of the transformant cells (about 5 %) formed elongated abnormal filaments resembling pseudohyphae, no typical true hyphae were detected. When 10 % horse serum was added and cells were incubated at 37 °C, they began to form true hyphae with similar kinetics as transformant cells which had been grown in PCK1p-repressing S4D medium and as control cells carrying empty vector pBI-1 (data not shown). Germ tubes were of identical lengths in all strains, suggesting that different Ole1p enzyme levels were not correlated with induction and elongation of hyphae. To confirm that the growth conditions had indeed led to an overproduction of Ole1p, we grew transformant CAI4(pSKM63), producing a myc-tagged version of Ole1p, in identical conditions, and by immunoblottings could verify that Ole1p production was increased in SCAA-grown cells five- to tenfold, as compared to S4D-grown cells (data not shown).
Thus, these results indicated that a minimal dosage of Ole1p and of oleic acid is crucial to allow efficient hypha formation in several but not all conditions known to induce hyphae in C. albicans. In contrast to these results, an ole2 deletion strain (JA2) was able to form true hyphae as was the wild-type in all conditions tested, suggesting that Ole2p does not have an essential role in hyphal morphogenesis.
Chlamydospore formation requires wild-type levels of OLE1 expression
Because of the requirement for OLE1 in hypha formation, we also considered the possibility that another morphogenetic event in C. albicans, chlamydospore formation, would be affected by OLE1 expression. Therefore, we streaked the conditional MET3p-OLE1 strain onto CMA containing a low level of methionine/cysteine and covered cells by a coverslip to generate microaerophilic conditions (Joshi et al., 1993; Sonneborn et al., 1999
). Following 5 days incubation at 25 °C, chlamydospores of all strains were visible in medium lacking methionine/cysteine, while only the wild-type and the heterozygous strain produced chlamydospores in the presence of methionine/cysteine (Fig. 5
). The conditional strain grew equally as well in this condition as the wild-type and heterozygous strains, but failed to form chlamydospores and this defect remained even after prolonged incubations. In addition to this defect, the conditional strain formed pseudohyphae that aggregated strongly, which was a phenotype not seen in the heterozygous and wild-type strains. Thus, wild-type expression levels of OLE1 and corresponding levels of oleic acid are necessary to allow chlamydospore formation in C. albicans. In contrast to these results, strains JA1 and JA2 were able to form chlamydospores at normal levels, indicating that Ole2p is not involved in chlamydospore formation.
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The conditional mutant O7.2/25-2 and the control strain CAF2-1 were pre-grown in SD medium and then inoculated into SD medium containing low amounts of methionine and cysteine (0·25 mM). At these levels, the conditional strain downregulated OLE1 to an extent to prevent hypha formation, but to still allow growth. Following about three cell doublings, we isolated lipids and generated fatty acid methyl esters, which were analysed by GC. Representative results indicate that levels of C18 : 1 (oleic) acids were reduced in the conditional strain, while the C18 : 0 precursor was strongly increased relative to amounts in the control strain (Table 2
). On the other hand, levels of C18 : 2 and C18 : 3 acids were identical in the control and conditional strains, suggesting that lowering of OLE1 expression primarily affects the C18 : 0 to C18 : 1 conversion. We also note that the levels of C16 : 0 and C16 : 1 acids were diminished in the conditional strain. In contrast, fatty acid composition in the ole2 deletion strain JA2 appeared similar to the control strain CAF2-1 (Table 2
).
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Membrane fluidity measurements
It appeared possible that lowering of membrane oleic acid would lead to decreased membrane fluidity. Alternatively, we speculated that cells would cope with alterations in oleic acid levels by compensatory alterations in membrane lipids and/or proteins, which would maintain membrane fluidity at a relatively constant level. To decide between these alternatives, we measured membrane fluidity in strains with altered OLE1 expression levels, by detection of the mobility of the fluorochrome 1,6-diphenyl-1,3,5-hexatriene, using fluorescence polarization measurements. Results were expressed as P-values, which at low values indicate high fluorochrome mobility, i.e. high fluidity, whereas elevated values indicate decreased membrane fluidity.
Partial repression of MET3p-OLE1 in the conditional strain O7.2/25-2 led to increased P-values, indicating a decrease in fluidity, as expected for a decrease in oleic acid (Table 2
). On the other hand, deletion of OLE2 also decreased fluidity, although levels of unsaturated fatty acids were very similar in the ole2 mutant and the control strain. Furthermore, membrane fluidity was unaltered in a strain with an OLE2-overexpression vector and fluidity was lowered rather than increased in a transformant carrying an OLE1-overexpression plasmid relative to the control. Thus, we did not detect any correlation between fatty acid composition and membrane fluidity. A complicating factor in these studies was the fact that membrane fluidity depended on the type of growth medium, because CAF2-1 control cells grown in SCAA and SD medium had different fluidities (P-values of 0·145 and 0·131, respectively); furthermore, membrane fluidity in the control CAI4-transformant was unusually low (P=0·184).
Since strains with different levels of overall membrane fluidity were obtained, we tested a possible correlation between membrane fluidity and the ability to form hyphae. In spite of the significantly enhanced membrane rigidity of the OLE1-overexpression strain, it was able, upon addition of 10 % serum, to form hyphae with about equal kinetics compared to control cells. The different rigidities of the control strains grown in SCAA medium or SD medium also did not alter the ability of cells to form hyphae. Furthermore, the addition of the fluidizer benzyl alcohol (Horvath et al., 1998) to wild-type cells did not change the response to hypha-inducing agents (data not shown). Thus, overall membrane fluidity per se does not appear to be a crucial factor to establish morphogenetic competence, whereas levels of individual membrane components such as oleic acid, which may occur in membrane subdomains or rafts, may be relevant.
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DISCUSSION |
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Complete repression of OLE1 prevented growth, indicating that Ole1p and its product oleic acid provide essential functions. We presume that oleic acid is also needed for specific functions, such as for oleic acid-dependent membrane sensors, and that it has a role in regulating overall and localized membrane fluidity. Low levels of OLE1 expression did not impair growth of the yeast form, but significantly blocked hyphal morphogenesis on solid and in liquid induction media. A specific set of signalling pathways leading to hyphal morphogenesis is known to be required in most induction conditions in the presence of oxygen (Ernst, 2000), while in embedded hypoxic conditions an alternative signalling pathway, which is downregulated in wild-type cells, is operative (Brown et al., 1999
; Sonneborn et al., 1999
). Because low OLE1 expression permitted hypha formation in embedded/hypoxic conditions but prevented filamentation in aerobic conditions, it appears likely that threshold levels of oleic acid are required specifically for the function of the aerobic pathways. Thus, oleic acid does not appear to have a general role in filament formation, for example for late events in hyphal development, but it is implicated in early events of activation of specific signalling pathways. Conceivably, because the biosynthesis of oleic acid requires oxygen, it could be a signalling molecule activating (yet unknown) membrane sensors transmitting external cues to internal pathways operative in aerobic conditions.
It is known that elevated temperatures increase membrane fluidity, while lowered temperatures decrease fluidity. Because elevated temperatures are a decisive environmental cue to trigger hyphal morphogenesis in C. albicans, we speculated that the state of its membrane fluidity could act as a cellular thermometer signalling directly to morphogenetic pathways. It has indeed been reported that stress responses and in particular the heat-shock responses in yeasts are activated by membrane perturbations (Carratu et al., 1996; Moskvina et al., 1999
). Lowering OLE1 expression in C. albicans indeed led to decreased membrane fluidity, as expected for a direct role of membrane fluidity in adjusting the set point of temperature induction of hyphal growth. However, further findings provide arguments against a direct correlation between membrane fluidity and morphogenesis: (1) elevated temperatures did not restore hypha formation in the MET3p-OLE1 conditional strain, (2) no correlation between membrane fluidity and hyphal induction was detected, (3) benzyl alcohol, a membrane fluidizer, did not increase hypha formation, and (4) overexpression of OLE1 led to a slightly increased level of oleic acid, but strongly increased membrane rigidity, which nevertheless did not interfere with hypha formation. Increased rigidity in the latter experiments may be due to compensatory increases in other membrane components, for example an increase in ergosterol levels, which may maintain the physical state of membranes constant. According to a similar principle, it has been described that the composition of the C. albicans cell wall is subject to compensatory alterations (Kapteyn et al., 2000
). Furthermore, a genome-wide transcriptional profiling of genes induced during hyphae induction recently revealed that a heat shock alone is not able to induce hyphae-specific genes and hyphal morphogenesis (Nantel et al., 2002
). Thus, we favour a model in which stress responses and induction of morphogenesis do not share the same dependence on membrane fluidity. We rather postulate that levels of oleic acid have a direct effect on specific components of the hyphal induction machinery.
In addition to the defect in hyphal morphogenesis, the development of chlamydospores was blocked at low OLE1 expression levels. Although the functions of the thick-walled chlamydospores in the biology and virulence of C. albicans are currently unclear, it is evident that their development requires a specific morphogenetic pathway, which depends on oleic acid. The finding of a threshold level of oleic acid for morphogenetic events in C. albicans suggests that the reason for defective phenotypes in several morphogenetic mutants of C. albicans may be an impaired lipid and/or fatty acid and/or oleic acid metabolism. In agreement with this notion, in S. cerevisiae the Tup1 regulator represses transcription of OLE1 (Fujimori et al., 1997) and C. albicans tup1 mutants grow in a pseudohyphal form (Braun & Johnson, 1997
), while we observed that OLE1 overexpression favours an abnormal pseudohyphal growth form, although only in a small fraction of cells. Our results further suggest that Ole1p may be a suitable target for future antifungal agents. Because OLE1 is essential, it is likely that potential Ole1 inhibitors will prevent cell growth and lead to a rapid loss of viability. This would be an advantage compared to azole inhibitors of ergosterol biosynthesis, which do not kill fungal pathogens. Even at low doses of inhibitors, which reduce but do not eliminate Ole1 function, hyphal morphogenesis and consequently virulence of C. albicans would be blocked. A major structural difference between mammalian and fungal Ole1 proteins is the presence of an integral cytochrome b5 domain in fungal desaturases, which may allow the development of selective inhibitors.
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ACKNOWLEDGEMENTS |
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Received 13 January 2004;
revised 16 February 2004;
accepted 23 February 2004.
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