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1 Robert Koch-Institut, Nordufer 20, D-13353, Berlin, Germany
2 Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
3 Friedrich-Schiller-University, Jena, Germany
4 Department of Microbial Pathogenicity Mechanisms, Lelbniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI), Beutenbergstraße 11a, D-07745 Jena, Germany
Correspondence
Bernhard Hube
bernard.hube{at}hki-jena.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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doa1 indicated that several genes associated with Ub-mediated proteolysis, including CDC48 and UBI4, are upregulated. These data suggest that DOA1 of C. albicans, like its orthologue in S. cerevisiae, is associated with Ub-mediated proteolysis and has multiple functions. However, some functions of CaDoa1 seem to be unique for C. albicans. These results support the hypothesis that Ub-mediated proteolysis plays an important role in the regulation of morphology in C. albicans.
The GEO accession number for the data reported in this paper is GSE 6905.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This includes knowledge about genes and factors associated with virulence attributes of C. albicans such as adhesion factors, the ability to alter cell morphology between yeast, pseudohyphal and hyphal forms, and the secretion of hydrolases (Calderone & Fonzi, 2001). For the latter, gene families have been discovered which encode secreted proteinases (Naglik et al., 2003), lipases (Hube et al., 2000) and phospholipases (Ghannoum, 2000). Genes encoding phospholipases B, C and D have been described and their impact on pathogenesis of C. albicans infections studied (Ghannoum, 2000; Hube et al., 2001; Knechtle et al., 2005; Kunze et al., 2005). However, although earlier reports demonstrated extracellular phospholipase A activity (Goyal & Khuller, 1992; Takahashi et al., 1991), no gene encoding a putative secretory phospholipase A has so far been identified.
With the increasing information gained about single virulence attributes and fitness factors, research has moved on to those factors which regulate the molecular and cellular processes associated with pathogenicity. One of the best examples of regulatory networks linked to processes essential for virulence of C. albicans is the regulation of morphogenesis (Kumamoto & Vinces, 2005a). Here, two major pathways, a MAP-kinase and a cAMP pathway, are of central importance for the initiation of hypha formation, but several other factors have also been identified to be crucial for hyphal morphogenesis. In particular, the key transcriptional factor of the cAMP pathway, Efg1, was shown to be essential not only for hypha formation under most conditions (Lo et al., 1997; Stoldt et al., 1997), but also for the expression of virulence attributes associated with dimorphism, such as the adhesion molecule Hwp1 or the secreted aspartic proteinases Sap4–6 (Felk et al., 2002; Kumamoto & Vinces, 2005b; Sundstrom, 2002).
Regulation of cellular processes in fungi has been studied extensively in the yeast Saccharomyces cerevisiae. In principle, regulation of eukaryotic cells occurs on a number of different levels including genomic, transcriptional and post-transcriptional (Castrillo & Oliver, 2006). One of the central post-translational mechanisms of regulation in eukaryotic cells is the controlled proteolysis of proteins such as transcription factors via ubiquitination. Protein ubiquitination is catalysed by ubiquitin (Ub) ligases in concert with Ub-conjugating enzymes, which facilitate the formation of an isopeptide bond between the C-terminus of Ub and a lysine side chain of a target protein (Sung et al., 1988). The specific biological signal mediated by a polyubiquitin chain is determined, in part, by chain topology, which is differentiated by the Ub lysine residue used for chain extension (Hofmann & Pickart, 2001; Pickart, 1997). Escort factors are involved in targeting ubiquitinated substrates to the proteosome (Rumpf & Jentsch, 2006). Furthermore, protein ubiquitination is reversible as Ub can be removed by deubiquitinating enzymes (Amerik et al., 2000). These regulatory processes require a network of factors to ensure a timely, precise and specific degradation of proteins with distinct cellular functions. In S. cerevisiae, it has been shown that one of the proteins associated with the escort factor Cdc48 is Doa1, which acts as an adaptor that possesses a novel Ub binding domain (Mullally et al., 2006).
While searching for regulators which may be involved in phospholipase A activation in C. albicans, we identified a gene homologous to the mammalian PLAP (phospholipase A2-activating protein) (Clark et al., 1991) and the S. cerevisiae protein Doa1 (Ghislain et al., 1996; Hochstrasser & Varshavsky, 1990; Johnson et al., 1995; Mullally et al., 2006). The orthologue of Doa1 is involved in multiple cellular processes in C. albicans, including filamentous growth, diacylglycerol production, cell wall integrity, mitotic spindle formation, heavy metal tolerance, growth on non-fermentable carbon sources and haemolytic activity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cells were grown in YPD [2 % (w/v) glucose, 1 % (w/v) yeast extract, 2 % (w/v) bacto peptone] or SD [2 % (w/v) glucose, 0.5 % (w/v) (NH4)2SO4, 0.17 % (w/v) yeast nitrogen base without amino acids and ammonium sulphate; Difco]. For phenotype screening and expression studies the following media were used: M199 (Sigma), CAA medium (Leuker et al., 1997; Stoldt et al., 1997), Sabouraud glucose medium [2 % (w/v) glucose, 1 % (w/v) bacto peptone], YCB-BSA (Hube et al., 1994), blood agar {1 l bouillon [0.3 % (w/v) NaCl, 0.3 % (w/v) Na2HPO4.2H2O, 1 % (w/v) bacto peptone, 1.4 % (w/v) Lab-Lemco powder (Oxoid)], 15 g agar, 50 ml mutton blood}, Lee's medium pH 4.5 and 6.5 (Buffo et al., 1984), egg yolk agar (Fu et al., 1997), SLAD medium [6.7 g YNB l–1 (Difco) without amino acids and ammonium sulphate plus 0.05 mM (NH4)2SO4], Spider agar (Liu et al., 1994), YPD plus 5 % (v/v) fetal calf serum (FCS) (YSer) or SD containing cadmium sulphate, propranolol, butanol, caffeine, EDTA, LiCl, MnCl2, ZnCl2, CuCl2, NaCl, H2O2, hygromycin B, cycloheximide, tetracycline, benomyl, Congo Red, SDS, amphotericin B, amorolfine, cyclosporin A, calcofluor, ciclopirox, isoflurane, nocodazole or itraconazole at appropriate concentrations. For phenotypic screening of mutant strains a serial drop dilution test was used. Five microlitre suspensions of tenfold dilutions of cells were dropped onto the corresponding solid media.
For expression analysis, CAF2-1 was grown overnight at 30 °C in SD medium, diluted into fresh SD medium to give a concentration of 106 cells ml–1 and incubated at 37 °C to an OD600 of 0.6. The culture was then divided into 40 ml aliquots, centrifuged and the pellet resuspended in 40 ml of one of the media described above. After a given incubation period, cells were harvested for RNA extraction (see below). For induction of the PCK1 promoter we used media without glucose: SGlyc [SD with 2 % (v/v) glycerol instead of glucose] or SGlycAc [SD with 3 % (v/v) glycerol and 2 % (w/v) potassium acetate instead of glucose], CAA medium and succinate medium (B medium) (Leuker et al., 1997; Stoldt et al., 1997).
For cloning procedures, E. coli TOP10 cells (Invitrogen) were used as described by the manufacturer.
Microscopic investigations.
For viability staining, we used the LIVE/DEAD Yeast Viability Kit (Molecular Probes), according to the manufacturer's instructions. Briefly, 50 µl cells from a culture in late exponential phase were mixed with 1 ml sterile medium. Cells were harvested and resuspended in fresh medium at a cell density of 106–107 cells ml–1. The sample was mixed with the stain FUN1 (final concn 20 µM) and incubated for 30 min at 30 °C in the dark. Calcofluor white was used to stain fungal chitin and to discriminate between true hyphal cells and pseudohyphae. Stained cells were analysed with a fluorescence microscope.
Disruption of DOA1.
To disrupt DOA1 we produced the following plasmids. Primers DOA1-3 fwd, containing a SalI restriction site, and DOA1-4 rev with genomic DNA from strain SC5314 were used to amplify a 433 bp fragment from the 3' end of DOA1 (all primers are summarized in Table 1). The PCR product was cloned into pCR2.1-TOPO to give pDK-9. The DOA1 insert was released with HindIII and SalI and ligated into those sites of vector pMB7 to give pDK-10. Primers DOA1-1b fwd and DOA1-2a rev were used to amplify a 745 bp fragment of the 5' end of DOA1 and the PCR product was cloned into pCR2.1-TOPO to give pDK-11. Next, a HindIII and KpnI fragment of pDK-10 containing the 3' end of DOA1 and the hisG-URA-hisG cassette was ligated into pDK-11 digested with the same enzymes to give the final disruption cassette, pDK-12. The disruption cassette was used to transform C. albicans CAI-4 as described below. Integration of the disruption cassette at the correct locus, disruption of DOA1 and the genotypes of all other mutants, revertants and overexpressing strains described below were confirmed by PCR and Southern blot analysis. All strains produced in this study are summarized in Table 2.
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) fusing the 5' end to the PCK promoter (pDK-22). The plasmid was used to transform doa1 and CAI-4.
Construction of a DOA1 mutant lacking the mellitin domain.
We amplified the DOA1 gene lacking an internal 75 bp fragment (containing the KVL-site) by inverse PCR (Long Expand Kit; Roche) by use of plasmid pDK-21 as a template and primers DOA1-k1a fwd and rev, containing KpnI sites. The linear fragment was digested with KpnI and religated to give pDK-26. The insert of pDK-26 was released with BglII and ligated into pBI-1 at the BglII site to give pDK-27. The plasmid was used to transform doa1.
Transformation of C. albicans.
To transform C. albicans, two modified lithium acetate protocols (Sanglard et al., 1997) and one electroporation protocol (De Backer et al., 1999) were used. Transformants were screened by PCR and Southern blot analysis as described previously (Felk et al., 2002).
RNA extraction and RT-PCR.
Liquid cultures were grown as described for each experiment, and cells were harvested and RNA isolated as described previously (Felk et al., 2002). For RT-PCR, 1.5 µg RNA was DNase-treated and cDNA was synthesized (Felk et al., 2002). Controls included cDNA synthesis without addition of reverse transcriptase and amplification of an intron-containing fragment of the EFB1 gene (Schaller et al., 1999) (with primers EFB1 fwd and EFB1 rev in Table 1
). To ensure that samples from the exponential phase of PCR amplification were examined, we used 20, 25, 30, 35 and 40 (or more) cycles. All RT-PCR experiments were done at least in duplicate with samples from two independent biological experiments. An additional internal standard control was the housekeeping gene ACT1, amplified with primers ACT1 fwd and ACT1 rev.
Microarray hybridization and analysis.
For transcript profiling, we used C. albicans microarrays (Eurogentec) containing 6039 ORFs. Arrays were designed as described at www.galarfungail.org/data.htm. Information about coding sequences and proteins was obtained from the CandidaDB database (http://genolist.pasteur.fr/CandidaDB/). RNA was reverse-transcribed into cDNA and labelled with Cy3 or Cy5 (dye swap) (Sigle et al., 2005). Labelled cDNA was hybridized to the C. albicans arrays as described by Sigle et al. (2005). For each experiment, triplicate arrays were used: two biological replicates and one dye swap. Hybridized slides were scanned with an Axon 4000B scanner at 10 µm resolution. Data were extracted using GenePix 4.1 software (Axon). An intensity-dependent data normalization (LOWESS) was performed using GeneSpring 6.0. The different sets of data were compared to each other by one-way analysis of variance (ANOVA) test with a P-value cut-off of <0.05 for genes that had an intensity in both channels higher than 100. Each gene that passed this test and showed at least 1.4-fold change in two arrays was defined as differentially expressed.
Systemic mouse infection.
C. albicans strains (doa1/doa1, CAF2-1) were used in a mouse model of systemic infection as described previously (Fradin et al., 2005).
Phospholipase assay.
To estimate phospholipase A-like activity in culture supernatants and soluble cell fractions, we used the NEFA C kit (WAKO) which measures free fatty acids after addition of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) as substrates. Samples were incubated at pH 6.0 and 7.5 with and without the addition of calcium ions. C. albicans cells were cultured in SD medium at 30 °C and used to inoculate Lee's medium (pH 4.5) at 25 °C. Selected media were inoculated with cells from this preculture at a density of 106 cells ml–1 and incubated for 15 h. One millilitre samples were taken for morphological analysis and to measure phospholipase activity. For the latter, samples were centrifuged for 5 min at 14 300 g and 800 µl of the supernatant was used to measure the extracellular phospholipase activity. The cells in the pellet were resuspended in 1 ml Tris/HCl (40 mM, pH 7.5) and lysed with glass beads (40 min). Cell debris was removed by centrifugation and soluble proteins were used to measure intracellular phospholipase activity. Phospholipase activity was measured according to the manufacturer's instructions with appropriate controls (e.g. medium only). Fatty acid concentrations were measured according to a standard curve based on 0.0, 0.2, 0.4 and 1.0 mM oleic acid. All samples were analysed in duplicates. Protein concentrations were measured with the Biuret-based BCA Protein Assay Reagent Kit (Pierce).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Takahashi et al., 1991), we have searched in one of the C. albicans genome databases (http://genolist.pasteur.fr/CandidaDB/) for putative phospholipase A genes. While no gene with significant similarity to phospholipase A genes was found, we identified a gene similar to the human gene encoding PLAP, known to be associated with the regulation of phospholipase A (Clark et al., 1991). This gene, IPF4477, is identical to CA4750 and orf19.4829 of assembly 19 (http://www-sequence.Stanford.edu/group/candida; http://genolist.pasteur.fr/CandidaDB/). CA4750 is a 2268 bp gene encoding a cytosolic protein (TargetP V1.0) (Emanuelsson et al., 2000) with 762 aa. Using the deduced protein sequence of CA4750 for a BLAST search at NCBI, we identified not only the gene encoding PLAP from mammals such as mice (GI 2507097/P 27612) and rats (GI 2007098/P 54319) (29 % identity), but also Doa1 (Zzz4, Ufd3) of S. cerevisiae with 35.5 % identity. Based on these results we renamed CA4750 as CaDOA1.
WD repeats
The most significant similarities between ScDoa1 and PLAP are based on WD repeats within these proteins. WD repeats are repetitive amino acid sequences with conserved positions for the amino acids GH (Gly-His) and WD (Trp-Asp). The consensus sequence for these domains is (X6–94-[GH-X23–41-WD])N4–8, according to Neer et al. (1994), with X describing the variable regions. In PLAP, four such WD repeats are found after a short N-terminal domain and are followed by a long C-terminal stretch (Fig. 1). Using the motif search function of InterPro (Falquet et al., 2002), seven WD repeats were identified in CaDoa1. These repeats were found directly at the N terminus and distributed over the first third of the protein (positions 3–40, 49–84, 87–123, 127–164, 166–204, 208–245 and 249–286).
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). Comparing mellitin sequences with the mellitin-like sequence of PLAP found by Clark et al. (1991), we identified a conserved motif ‘KVL’ in CaDoa1 (Fig. 1).
CaDOA1 is expressed in yeast and hyphal cells
To analyse under which conditions CaDOA1 is expressed in vitro, we used semiquantitative RT-PCR. We analysed the expression of DOA1 in media supporting the yeast growth form and media which induced hypha formation. Yeast cells from a preculture (Lee's medium, pH 4.5, at 25 °C, or SD medium) were used to inoculate media supporting either yeast or hyphal growth with samples taken at several time points (Fig. 2). After 3 h, up to 99 % of all cells had produced germ tubes in the hypha-inducing media (Fig. 2). Using either ACT1 or EFB1 as internal controls and several rounds of PCR to ensure that samples from the exponential phase of PCR amplification were examined, we detected DOA1 transcripts in all samples showing that this gene is constitutively expressed in yeast and hyphal cells under the conditions investigated. However, an increased expression of DOA1 was observed at late stages of yeast growth (Fig. 2).
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). A disruption cassette was constructed which contained a 745 bp fragment corresponding to position –274 to +471 bp relative to the ATG start codon of DOA1 and a 433 bp fragment corresponding to position +1181 to +1614 bp relative to the ATG flanking the hisG-URA3-hisG cassette. The disruption cassette was used to disrupt both alleles of DOA1. Integration into both alleles was confirmed by Southern analysis and PCR. Several independent isogenic mutants were produced to verify that no further mutational events occurred during the transformation steps and the 5-fluoro-orotic acid treatments. For all experiments with the doa1 mutant (DK2; Ura+) we used the wild-type strains SC5314 and/or CAF2-1 (one copy of URA3) as reference strains.
Mutants lacking DOA1 are filamentous and show reduced growth under multiple conditions
Mutants lacking DOA1 were analysed under a series of conditions. To analyse growth in liquid media, doa1 mutant (DK2) and wild-type cells (106 cells ml–1) from an SD preculture at 30 °C, which consisted predominantly of yeast cells, were inoculated into either SD or YPD medium at 37 °C. Microscopic investigation of cells grown under these conditions showed that wild-type cells grew in the yeast form, while doa1 mutant cells produced filaments (Fig. 3). By 4 h, 98 % of all wild-type cells grew as yeast forms in SD medium (96 % in YPD), while 92 % of doa1 cells had formed filaments (YPD, 48 %). Detailed microscopy, including staining with calcofluor white, showed that the filaments produced by the doa1 mutant were a mixture of pseudohyphae and true hyphae (Fig. 3).
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doa1 mutants, a heterozygote doa1 mutant (DK1, one copy of URA3 and DOA1) and the wild-type strain CAF2-1 (one copy of URA3).
The most striking phenotype on solid media was filamentous growth as already shown under liquid conditions. Colonies of doa1 mutants grew mostly as filaments in media which normally do not induce hypha formation (Fig. 4). The hyperfilamentous phenotype was often associated with a crumpled colony surface. This includes colonies for which macroscopic production of filaments was not obvious. For example, wild-type growth on YPD at 30 or 37 °C appeared as smooth, creamy colonies and contained few hyphae, but doa1 colonies had a crumpled surface with filamentous cells which could be detached from the agar surface as a firm mass (Fig. 4c). As shown in Fig. 4(c), cells from CAF2-1 colonies consisted of yeast cells only, while cells from the mutant colonies were associated with each other as a network of mainly filaments and few yeast cells. Under conditions which induce hypha formation in wild-type cells (e.g. serum or spider medium), the mutant formed filaments more rapidly and mostly longer than in the wild-type. Growth at 25 and 40 °C under the same conditions did not alter these phenotypes.
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doa1 mutants to several compounds (Fig. 4). For example, the addition of propranolol strongly reduced growth of the doa1 mutants. This compound is known to inhibit the lipid phosphate phosphohydrolases that convert phosphatidic acid (PA) into diacylglycerol (DAG) and hence hypha formation in C. albicans (Baker et al., 2002). Furthermore, the metabolic inhibitor caffeine and the chelator EDTA caused severe growth inhibition of doa1 mutants. The inhibition caused by EDTA (more pronounced at 30 °C) was reversible by addition of either MnCl2 (15 mM), ZnCl2 (25, 50 µM) or CuCl2 (50, 100 µM), with MnCl2 being the most efficient and CuCl2 the least efficient salt for reversing the effect (not shown). An increased susceptibility was also observed with antifungal agents such as itraconazole. Similar results were obtained with nocodazole, which causes disassembly of mitotic spindles. Since it had been shown that S. cerevisiae mutants lacking DOA1 were sensitive to volatile anaesthetic agents such as isoflurane (Keil et al., 1996), we also investigated the isoflurane sensitivity of the doa1 mutants from C. albicans and found they grew slower as compared to wild-type cells. The observed phenotypes varied with the temperature of incubation. For example, higher concentrations of CdSO4 (120 µM) or butanol (4 %) were necessary at 30 °C compared with 30 µM CdSO4 or 2 % butanol at 37 °C to cause growth inhibition of the doa1 mutants (not shown). Use of non-fermentable carbon sources such as glycerol or acetate instead of glucose led to reduced growth of the doa1 mutant at 37 °C, but not at 30 °C (not shown). To investigate the haemolytic activity of doa1 mutants, we also inoculated blood agar plates. At 37 °C we observed strongly reduced growth and greyish and brown colonies with a clearing zone around colonies on this type of agar. In contrast, the doa1 mutants showed hyperfilamentous growth (as compared with the wild-type) with no or only reduced clear zones around the colonies at 30 °C. On YCB-BSA agar, which can be used to test extracellular proteolytic activity, we observed an earlier and stronger production of filaments for the doa1 mutants at 30 °C compared with 37 °C and with the wild-type under the same temperatures. Phenotypes of doa1 mutants are summarized in Table 3.
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doa1doa1 mutant showed filamentation under conditions which normally favour yeast growth, we investigated the morphology of cells in each sample. Both wild-type (CAF2-1) and doa1 mutant (DK2) cells grew in the yeast form in Lee's medium at pH 4.5 at 25 °C (not shown). However, growth in SD medium at 30 and 37 °C caused at least moderate filamentation of the doa1 mutant (2–4 %; not shown). Generally, the phospholipase activity was reduced below that of wild-type in SD medium. Extracellular phospholipase activity was reduced by 68.4±3.5 % at pH 6.0 and 68.5±2.8 % at pH 7.5 at 30 °C. At 37 °C the activity was further decreased (78.5±1.0 % at pH 6.0 and 74.9±5.2 % at pH 7.5). Intracellular phospholipase activity was reduced by 56.1±4.1 % at pH 6.0 and 49.4±1.9 % at pH 7.5 at 30 °C. At 37 °C the activity was less reduced (25.4±7.3 % at pH 6.0 and 25.4±7.3 % at pH 7.5).
Reintroduction of DOA1 into doa1 restored wild-type phenotypes
To fulfil Koch's postulates (Falkow, 1988), we reintroduced a native DOA1 copy into the doa1 mutant (DK2) and investigated the phenotype of the retransformant (DK3) in comparison with the doa1 mutant on selected media. For this we fused the DOA1 gene with the controllable PCK1 promoter from C. albicans into the plasmid pBI-1 (Leuker et al., 1997; Stoldt et al., 1997). Genes controlled by the PCK1 promoter are expressed under low concentrations of glucose. A PCR fragment containing the entire DOA1 ORF and 200 bp of the untranslated 3' region was cloned into pBI-1 to produce the retransformation plasmid pDK-22. The URA3-negative doa1 mutant was transformed with pDK-22. To produce a control strain, we also transformed the URA3-negative doa1 mutant with the empty plasmid pBI-1. Transformants with the PCK1-driven DOA1 gene (DK3) and the mutant with the empty vector (DK5) were tested on media known to induce the PCK1 promoter and containing compounds previously shown to inhibit growth of the doa1 mutants. These included CAA medium, succinate medium (B medium), SGlycAc medium and CAA medium with isoflurane or itraconazole (1 µg ml–1) at 30 and 37 °C.
As shown in Fig. 5, extrachromosomal expression of DOA1 reversed the altered growth phenotypes of DOA1 disruption on all media. To show further that the observed phenotypes of the doa1 mutants were in fact due to the disruption of DOA1, we also cloned DOA1 including its own promoter and 3' untranslated region into the vector CIp10 (Murad et al., 2000) (to give pDK-24 and pDK-25) and inserted the gene into the RPS1 locus of C. albicans (DK8). When compared to strains carrying the empty plasmid CIp10 (DK7), transformants carrying pDK-25 (DK8) reversed the phenotype observed for doa1 mutants into wild-type phenotypes (not shown).
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investigated the role of the mellitin-like region of the PLAP protein for the activation of phospholipase A2. To investigate the functional role of the mellitin-like region downstream of the WD repeats we deleted a small region containing the KVL motif in the DOA1 gene sequence used to rescue the wild-type phenotype in the doa1 mutant. This was accomplished using plasmid pDK-21 as a template for an inverse PCR with primers DOA1-k1a fwd and rev. The resulting fragment lacking base pairs +1801 to +1875 after the ATG start codon of DOA1 (representing the amino acids of CaDoa1 shown in Fig. 1b) was fused to the PCK1 promoter in pBI-1 (pDK-27). Transformants (DK6) were analysed on PCK1-inducing media such as CAA, SGlycAc and blood agar at 30 and 37 °C in serial drop dilution tests. As shown in Fig. 6, the PCK promoter-driven DOA1 gene lacking the 75 bp fragment of the mellitin-like region was not able to fully restore the phenotypes of the doa1 mutant on CAA (wild-type and retransformant have smooth surfaces, mutants have wrinkled surfaces) and blood agar (wild-type and retransformant produce smooth colony edges, mutants produce hyphae at the edge of the colony), suggesting that this region is essential for full function of CaDoa1.
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DOA1 transcription under selected growth conditions
Phenotypic analysis of doa1 mutants showed that disruption of DOA1 caused multiple growth effects under distinct conditions. Since we reasoned that DOA1 might be regulated under the conditions which caused growth defects for doa1 mutants, we investigated the transcriptional levels under selected conditions, such as SD medium containing 60 µM CdSO4 or SGlyc medium at 37 °C, as compared with mRNA levels in SD medium at 37 °C in wild-type cells. Furthermore, we monitored the transcript level in SD medium at 42 °C. Only this heat-shock condition caused an increased transcriptional level of DOA1 (Fig. 7).
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doa1doa1 mutant (DK2) as compared to the wild-type strain CAF2-1 using microarrays. Cells from the doa1 mutant and the wild-type strain CAF2-1 were precultured in SD at 30 °C, and 106 cells ml–1 were inoculated in SD medium at 37 °C for 7 h before RNA was isolated. Transcriptional array data confirmed the expression data obtained with RT-PCR. Including the genes investigated by RT-PCR, 96 genes were significantly differentially expressed in both strains (P<0.05, t-test). Genes were 11.1- to 1.4-fold differentially expressed. Only two genes were identified as downregulated (both 2.1-fold) (Table 4). Fig. 8 shows the functional categories to which the 96 differentially expressed genes could be associated. One-third of all the genes (36 genes) encode proteins of unknown function (Fig. 8). Beside this group, the largest group of genes is associated with protein degradation, mostly linked to the proteosome, Ub or Ub-independent proteolysis. Other functional groups were transcription and replication, transport, fat metabolism and energy, amino acid metabolism, stress and oxidative stress. Despite the fact that the mutant produced mainly pseudohypha and few true hypha, we identified the true hypha-associated gene HWP1 as being upregulated. For selected genes (HWP1, RBT6, ACB1, PRE10, PRE6, SMT3, IPF3262 and IPF3262) we again used RT-PCR to verify the microarray data (not shown).
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doa1doa1 mutant, no significant differences were observed in terms of mouse survival and organ burden compared with wild-type strain CAF2-1. Cell morphologies in the inoculum suspension comprised only yeast forms and short pseudohyphae. The cells in the kidneys of moribund animals were filamentous, as were those in the kidneys of animals infected with wild-type C. albicans. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and Doa1 in S. cerevisiae (Tzermia et al., 1994). Due to the high level of similarity to ScDOA1, we named this gene (Ca)DOA1. Although DOA1 was transcribed under all conditions investigated in yeast and pseudohyphal/filamentous forms of C. albicans, this gene is not essential for growth as mutants lacking DOA1 were viable. It is not clear whether DOA1 has a direct influence on phospholipase activity; however, doa1 mutants had reduced phospholipase activity.
The Doa1 amino acid sequence showed strong similarities to members of the WD-repeat protein family. These proteins have diverse cellular functions, such as regulation of signal transduction, transcription, pre-mRNA processing or cytoskeleton assembly (Smith et al., 1999). Typically, WD-repeat proteins do not have catalytic functions, but are involved in multi-protein complexes in direct interaction with other proteins via the WD repeats (Neer et al., 1994). However, some WD-repeat proteins are fused to a functional domain and have catalytic activities, for example the mammalian protein PLAP (Neer et al., 1994). In PLAP, the functional domain contains a mellitin-like sequence (Clark et al., 1991) which is used to activate phospholipase A2. A similar sequence exists in CaDoa1 as shown in this study. We showed that this putatively catalytic domain is essential for full function in C. albicans. Therefore, it may be possible that Doa1 in C. albicans has a catalytic function. The protein with the highest overall similarity to CaDoa1 is ScDoa1. Similar to CaDoa1, the second and seventh WD repeat of ScDoa1 (Keil et al., 1996) differ in the consensus sequence, confirming relatively high similarity between these two orthologues. However, the most similar regions between ScDoa1 and the mammalian PLAP are beyond the WD repeats in the C-terminal part, but exclude the catalytic mellitin-like sequence of PLAP. In contrast, CaDoa1 and PLAP are more similar within the mellitin-like sequence.
ScDoa1 has been shown to play a key role during Ub-dependent protein degradation, regulation of the cellular Ub concentration and DNA damage response (Johnson et al., 1995; Lis & Romesberg, 2006). In two hybrid assays, ScDoa1 bound to Cdc48 and other factors via the C terminus (Decottignies et al., 2004; Ghislain et al., 1996; Johnson et al., 1995; Keil et al., 1996; Seigneurin-Berny et al., 2001). The human orthologue of the chaperone Cdc48 is a central element in a major Ub-associated escort pathway (Rumpf & Jentsch, 2006). Recently, Mullally et al. (2006) showed that ScDoa1 is a Cdc48 adaptor that possesses a novel Ub binding domain. A number of phenotypes and the transcript profiles of mutants lacking CaDoa1 suggest that this orthologue in C. albicans has both distinct and similar functions and may also be involved in ubiquitination.
Deletion of ScDOA1 caused resistance to isoflurane and other anaesthetics, and increased sensitivity to cadmium (Keil et al., 1996; Wolfe et al., 1998). The doa1 mutant of C. albicans is moderately more sensitive to both isoflurane and cadmium. The mode of action of isoflurane on fungal cells is unclear; however, an influence of isoflurane on membrane lipids and proteins, and on their role in membrane integrity has been proposed (Keil et al., 1996). In contrast, Wolfe et al. (1998) postulated a possible direct or indirect correlation with Ub metabolism. Likewise, sensitivity to cadmium can be linked to Ub-mediated proteolysis. Cadmium reacts with thiol groups of proteins and can substitute Zn2+ ions. These modified proteins are likely to be substrates for proteolytic degradation via the proteosome. For example, mutants lacking Ub-conjugating proteins such as Ubc1, Ubc4 and Ubc7, or subunits of the proteosome such as Pre1 are hypersensitive to cadmium in S. cerevisiae (Jungmann et al., 1993).
Microarray analyses of the doa1 mutant further hint at a role of Doa1 in the Ub degradation pathway. Several genes upregulated in the mutant are linked with Ub degradation. Of particular interest are UBI4 and CDC48.
Overexpression of ScUBI4 complements the defects of doa1 in S. cerevisiae (Johnson et al., 1995) and the orthologue of UBI4 from C. albicans can complement the defects of ubi4 in S. cerevisiae (Roig & Gozalbo, 2003). C. albicans wild-type cells upregulate UBI4 during thermal stress and starvation, and mutants lacking Ubi4 have a moderately increased sensitivity to heat shock (Roig & Gozalbo, 2003). However, ubi4 mutants of C. albicans have a clear morphological dysfunction as increased formation of hyphal and pseudohyphal cells was observed (Roig & Gozalbo, 2003). The same expression pattern and phenotypes were shown for doa1 in this study.
As mentioned above, Mullally et al. (2006) suggest that ScDoa1 acts as an adaptor between Cdc48 and Ub. CDC48 is upregulated in the doa1 mutant of C. albicans and, due to the high similarities between ScDoa1 and CaDoa1, it is likely that CaDoa1 has similar functions within the Ub degradation pathway. In this context, it is worth mentioning that the Ub binding domain in ScDoa1, the PFU domain (Mullally et al., 2006), is also conserved in CaDoa1 (aa 354–448).
However, in addition to the similar features, there must be differences in function since the most obvious phenotype of the doa1 mutant of C. albicans was filamentous growth under conditions that normally support yeast morphology. These filaments were a mixture of true hyphae and pseudohyphae as indicated by the location of septa within the filaments (Sudbery, 2001). Surprisingly, we observed that the known hypha-specific gene HWP1 was upregulated in the doa1 mutant compared to wild-type cells, even though the mutant cells grew mostly as pseudohyphae at the time of sampling.
Filamentous phenotypes have been observed in a number of mutants, such as strains lacking SSN6 (Hwang et al., 2003), SPT3 (Laprade et al., 2002), UBI4 (Roig & Gozalbo, 2003), RAD6 (Leng et al., 2000), CDC4 (Atir-Lande et al., 2005) and GRR1 (Butler et al., 2006) in C. albicans, with the last four genes putatively linked to the Ub degradation pathway. Therefore, ubiquitination seems to be an important and previously underestimated regulator of morphology in C. albicans.
In addition to the filamentous morphology, we observed further phenotypes for the doa1 mutant of C. albicans, which were not described for the corresponding mutant in S. cerevisiae. For example, Ghislain et al. (1996) did not observe reduced growth for mutants lacking Doa1 (Ufd3) in S. cerevisiae when cells were exposed to caffeine (inhibitor of cAMP phosphodiesterase), while growth of the doa1 mutant of C. albicans was dramatically reduced when exposed to caffeine. Furthermore, propranolol, an inhibitor of phosphatidate phosphohydrolase, the enzyme which can convert PA to DAG, strongly reduced growth of doa1. DAG, which can also be produced by phosphatidate phosphohydrolase-independent pathways, is an essential molecule for certain molecular processes (Kearns et al., 1997). It was suggested to be a regulator of morphology in C. albicans as mutants lacking phospholipase D, which cannot produce PA from PC, have reduced abilities to produce hyphae (Hube et al., 2001). Since propranolol had such a strong effect on growth, it must be concluded that other pathways of DAG production, such as PLC-mediated activity, are blocked or inhibited in the doa1 mutant.
Several observations, including increased susceptibility to SDS, amphotericin B, amorolfine or itraconazole, suggest a defect of the doa1 mutant in cell surface integrity. These defects could also be responsible for the reduced haemolytic activity observed on blood agar. Furthermore, cell membrane or cell wall defects may have caused reduced protection against extracellular exposure to inhibitors, such as cycloheximide and hygromycin B, which cause increased growth inhibition in the doa1 mutant. The view that disruption of DOA1 caused a defect in membrane integrity was supported by the observation that genes associated with sterol metabolism, such as SCS7 or ACB1 (Faergeman et al., 2004; Gaigg et al., 2001; Jensen-Pergakes et al., 2001; Swain et al., 2002; Yang et al., 1996), or oxidative or nitrogen stress, such as TTR1, TRR1 (Fradin et al., 2005) or YHB1 (Hromatka et al., 2005), were upregulated in the mutant.
Although the doa1 mutant had severe growth defects under a number of conditions and was filamentous, the virulence in a mouse model of systemic infections was not attenuated. Since both yeast and filamentous cell forms seem to be essential for full virulence in C. albicans (Kumamoto & Vinces, 2005a, b) it must be concluded that other factors compensate the loss of function of Doa1 in vivo.
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Edited by: J. Pla
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