Candida albicans Expresses an Unusual Cytoplasmic Manganese-containing Superoxide Dismutase ( SOD3 Gene Product) upon the Entry and during the Stationary Phase*

We report here that in addition to a cytoplasmic cop-per-zinc-containing superoxide dismutase (SOD) and a mitochondrial manganese-containing SOD, Candida albicans expresses a third SOD gene ( SOD3 ). The deduced amino acid sequence contains all of the motifs found in previously characterized manganese-containing SODs, except the presence of a mitochondrial transit peptide. Recombinant Sod3p expressed and purified from Escherichia coli is a homotetramer with a subunit mass of 25.4 kDa. Mass absorption spectrometry detected the presence of both iron and manganese in purified Sod3p but, as determined by metal replacement experiments, the enzyme displays activity only when bound to manganese. Overexpression of SOD3 was shown to rescue the hypersensitivity to redox cycling agents of a Saccharomyces cerevisiae mutant lacking the cytoplasmic cop-per-zinc-containing SOD. Northern blot analyses showed that the transcription of SOD3 is induced neither by the transition from the yeast to the mycelial form of C. albicans nor by drug-induced oxidative stress. In continuous cultures, the expression of SOD3 was strongly stimulated upon the entry and during the stationary phase, concomitantly with the repression of SOD1 Sod3 Vitro in Vivo Superoxide Dismutase Assays— Superoxide dismutase activity of Sod3pr vivo

Candida albicans is a lifelong commensal of the human gastrointestinal tract and vaginal mucosa, and among commensals, Candida is an alert opportunist. No ill effect normally results from colonization, but subtle defects in host defenses lead to infection. Its ability to be maintained as commensal implies that it has evolved to: (i) resist the host defense mechanisms and (ii) survive for prolonged periods of time in a nonreplicative form. Although it is generally accepted that different defense mechanisms are operating in systemic as compared with mucocutaneous candidiasis, phagocytes are key components in protection against both types of infections, pro-viding a first line of defense against superficial infections and dominating resistance to systemic infections (1). The fungicidal activity of neutrophils and macrophages involves the production of the superoxide anion (O 2 . ), the first intermediate in the sequential univalent reduction of dioxygen (O 2 ), and the primary reaction of the oxidative burst (2). The metabolic generation of reactive oxygen species (ROS) 1 is also known to affect the life span, and in fungi, mutants defective in the proper ROS detoxification have a poor viability during the stationary phase (3,4). This and the finding that C. albicans is more resistant to induced oxidative stresses than the nonpathogenic and closely related yeast Saccharomyces cerevisiae (5), prompted us to further investigate the components implicated in the C. albicans oxidant protection. Despite the existence of many nonenzymatic anti-oxidant compounds (i.e. lipid-soluble vitamin ␣-tocopherol, water-soluble vitamin ascorbate, glutathione), the most efficient way to eliminate ROS is through catalysis by antioxidant enzymes (i.e. superoxide dismutase, catalase, and glutathione peroxidase). The metalloenzyme superoxide dismutase (SOD) is the primary enzyme involved, and it disproportionates the O 2 . anion into O 2 and hydrogen peroxide (H 2 O 2 ) (6). SODs belong to four major classes according to the metal cofactor utilized; these are copper and zinc (Cu/ZnSOD), nickel (NiSOD), iron (FeSOD), and manganese (MnSOD). FeSODs and MnSODs are closely related enzymes, whereas Cu/ZnSODs and NiSODs define separate families of SODs. Typically, eukaryotes express both a cytoplasmic Cu/ZnSOD and a MnSOD that is targeted to the mitochondrial matrix (6). In C. albicans, the genes encoding the cytosolic Cu/ZnSOD (SOD1 gene product) and mitochondrial MnSOD (SOD2 gene product) were recently identified (7,8).
They were found to be orthologs of the well characterized S. cerevisiae SOD1 and SOD2 genes, respectively. S. cerevisiae sod1⌬ mutants grow poorly in air, are very sensitive to redox cycling agents, and die quickly in stationary phase, whereas sod2⌬ mutants are oxygen-sensitive when respiration is required (4,9). We report here the identification of a third C. albicans gene encoding a superoxide dismutase (SOD3). We show that the Sod3 enzyme utilizes manganese as cofactor, but, in contrast to most eukaryotic MnSODs, it is active within the cytosol rather than in the mitochondrial matrix. Expression studies suggest that C. albicans coordinates in the opposite direction the tran-scription of the SOD1 and SOD3 genes. Because the SOD3 gene is strongly induced upon and during the stationary phase, we propose that this gene may be part of a well orchestrated and distinct defense mechanism for survival in nutrient-limiting conditions.

EXPERIMENTAL PROCEDURES
Yeast and Bacterial Strains-C. albicans ATCC 32354 was used for the construction of the genomic library (10)  Media and Growth Conditions-The C. albicans blastospores were routinely cultured at 28°C in Iscove's modified Dulbecco's medium (IMDM) supplemented with glucose as described previously (11). For the Northern analyses performed with the yeast and mycelial forms of C. albicans, blastospores were first grown overnight at 28°C in the following media: 1% yeast extract, 2% peptone, and 2% glucose (YPD); YPD supplemented with 10% serum; IMDM, pH 6.5; and Lee's medium at either pH 4.5 or 6.5. The precultures were then diluted to an A 600 ϭ 0.1 and incubated for 16 h in the same media at either 28 or 37°C. For the SOD1 and SOD3 expression studies in continuous cultures, precultures of C. albicans blastospores grown in either YPD, YP supplemented with 2% galactose (YPGal), or 3% glycerol (YPG) were diluted to an A 600 ϭ 0.1 and incubated for 60 h at 30°C in the same media. For the expression of the C. albicans SOD genes in response to induced oxidative stresses, a preculture of C. albicans blastospores grown in YPD was diluted to an A 600 ϭ 0.1 and then further incubated for 24 h at 30°C in the same media. Aliquots were removed every 2 h and further incubated for 1 h at the same temperature in YPD or in YPD supplemented with either hydrogen peroxide (0.4 mM), menadione (0.5 mM), or paraquat (0.5 mM).
The S. cerevisiae strains were grown at 30°C in either YPD or, when transformed, in synthetic complete medium lacking uracil (SC-ura) as described (12). The E. coli strains were cultured in 2YT medium containing 0.2% glucose supplemented with antibiotics as required (13).
DNA Manipulations and Transformations-Screening of the C. albicans genomic library was done as described by Hanahan and Meselson (14) using a radioactive 397-bp EcoRV DNA fragment derived from plasmid p4E1 that was labeled as described previously (10).
For the 5Ј-rapid amplification of cDNA ends analysis, C. albicans blastospores grown in IMDM at mid-log phase were harvested by centrifugation (4000 ϫ g). Purification of total RNA was performed according to the procedure described by Kohrer and Domdey (15). The first strand cDNA synthesis was obtained by reverse transcription of the total RNA with the SuperScript II RNase H Ϫ reverse transcriptase (Life Technologies, Inc.) and the oligonucleotide R1SOD3 following the manufacturer's instructions. All of the oligonucleotides used in this study were purchased from Life Technologies, Inc., and their sequences are presented in Table I. The single-stranded cDNA was then tailed at the 3Ј-end by the terminal deoxynucleotidyl transferase (New England Biolabs Inc.) and dCTP according to the manufacturer's instructions. The resulting tailed cDNA was submitted to a first polymerase chain reaction (PCR) with the Taq DNA polymerase (Life Technologies, Inc.) and the oligonucleotides R1SOD3 and AAP as primers. A second round of PCR was performed with a 100-fold dilution of the amplicons generated during the first PCR, but this time using the oligonucleotides R2SOD3 and AUAP as primers. The thermal cycling conditions for both PCR were the following: 94°C for 2 min; then 94°C for 1 min and 72°C for 3 min for 40 cycles (72°C for 10 min at the last cycle); and 4°C until use. Direct automatic DNA sequencing of the resulting amplicon was done as described previously (10).
For the heterologous expression of SOD3 in E. coli and in S. cerevisiae, we constructed the plasmids pSOD3r and pVTSOD3, respectively. The full-length coding sequence of SOD3 was obtained by reverse transcription of total RNA essentially as described above, except that the Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) and the AP primer were used. The single-stranded cDNA obtained was next amplified by PCR using the oligonucleotides AUAP and FSOD3 as primers, and, following a 1000-fold dilution of the reaction mixture, a second round of PCR was performed using the oligonucleotides FSOD3 and RSOD3. In each case the thermal cycling conditions were: 94°C for 2 min; then 94°C for 1 min and 60°C for 1 min; 72°C for 2 min for 35 cycles (72°C/10 min at the last cycle); and 4°C until use. The resulting amplicon was then cut with BamHI and ligated to the BamHI-digested plasmids pQE30 (Qiagen) and pVT102U (16) to generate pSOD3r and pVTSOD3, respectively. In pSOD3r, a His 6 tag coding sequence provided by pQE30 flanks the 5Ј-end of the SOD3 coding sequence. The LacI repressor regulates expression of the chimeric gene in pSOD3r, whereas pVTSOD3 provides constitutive expression through the strong ADH promoter.
Northern analyses and radiolabeling of the probes were performed as described previously (10). The probes for SOD1 and SOD3 were generated by PCR using oligonucleotides FSOD1-RSOD1 and FSOD3-RSOD3, respectively, along with cDNA clone as template (this study). Transformation of yeast using the lithium acetate salt was performed according to the rapid procedure described by Kaiser et al. (12). The CaCl 2 protocol was used for the transformation of E. coli (13).
Expression and Purification of the Sod3 Recombinant Protein-E. coli M15[REP4] cells transformed with pSOD3r were grown in 2YT medium supplemented with 100 g ml Ϫ1 ampicillin and 25 g ml Ϫ1 kanamycin to early log phase (A 600 of 0.6) at 37°C with vigorous shaking. The expression of SOD3r was then triggered either by the addition 1 mM isopropyl-D-thiogalactopyranoside (IPTG) followed by a further 5-h incubation (long induction) or by the addition of 0.1 mM EDTA, 1 mM MnCl 2 , and 0.1 mM IPTG and a 15-min incubation (short induction). All of the purification steps were then performed at 4°C. The cells were harvested at 4,000 ϫ g for 20 min and resuspended in 10 ml of 50 mM Tris-HCl buffer, pH 7.8, 300 mM NaCl, and 20 mM imidazole. They were lysed by three passages through a French pressure cell (Amicon) at 8000 p.s.i. The cell lysate was centrifuged at 10 000 ϫ g for 30 min, and the recombinant protein was purified with 5 ml of an 50% aqueous suspension of nickel-nitrilotriacetic acid resin has described by the manufacturer (The QIAexpressionist; Qiagen). The unbound material was removed by 10 washes with 10 ml of 50 mM Tris-HCl buffer, pH 7.8, 300 mM NaCl, and 20 mM imidazole. The bound recombinant Sod3 fusion protein was eluted with 2 ϫ 1 ml of 50 mM Tris-HCl buffer, pH 7.8, 300 mM NaCl, and 250 mM imidazole. The protein preparation was then dialyzed against 4 liters of 50 mM potassium phosphate, pH 7.8 for 16 h at 4°C and stored at 4°C or at Ϫ20°C for prolonged periods.
In Vitro and in Vivo Superoxide Dismutase Assays-Superoxide dismutase activity of the purified Sod3pr was assayed by the nitroblue tetrazolium method of Beauchamp and Fridovich (17). For the in vivo assay, 100 l of the indicated S. cerevisiae cultures grown overnight in SC-ura medium were mixed with 8 ml of molten top agar (0.7% w/v agar in SC-ura medium) maintained at 42°C. The top agar was rapidly overlaid onto SC-ura plates and allowed to solidify. Sterile filter discs were deposited on the plates, and 10 l of the indicated concentrations of either menadione or paraquat were spotted onto the filter discs. The plates were then incubated at 30°C for 48 h.
Metal Content and Reconstitution of SODs with Other Metals-The metal content of the purified Sod3pr enzyme was determined by atomic absorption spectrophotometry. The purified Sod3pr enzyme and commercial preparations of the E. coli FeSOD and MnSOD (Sigma Chemical Co.) used as controls were treated essentially as described by Gregory and Dapper (18) for metal removal and replacement.
Miscellaneous Methods-The molecular mass of the native enzyme was determined by gel filtration on a Superdex 75 HR 10/30 (Amersham Pharmacia Biotech) and eluted with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05 mM EDTA at a flow rate of 0.9 ml/min. The column was calibrated with the following gel filtration calibration molecular mass markers (Sigma): bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17.5 kDa), and vitamin B 12 (1.4 kDa). Subunit molecular mass was estimated by 12% SDS-PAGE in denaturing conditions as described previously (10). The protein concentration was measured by the method of Bradford (19).

RESULTS
Identification and Sequence Analysis of a Third SOD Gene from C. albicans-The CSA1 gene, which encodes an abundant mycelial surface antigen in C. albicans (11), was recently sequenced from a 7.4-kilobase genomic DNA fragment cloned into plasmid YEp24 (10). Further sequencing reactions of this plasmid (p4E1) revealed the presence of an incomplete open reading frame in the opposite direction relative to CSA1. This open reading frame encodes a putative protein with sequence similarity to members of the manganese-containing SOD enzyme family. Because the recently characterized C. albicans cytosolic Cu/ZnSOD (7) and mitochondrial MnSOD (8) were named Sod1p and Sod2p, respectively, we designated this novel gene and the corresponding enzyme as SOD3 and Sod3p.
The full-length sequence of this gene was completed with plasmid pSOD3, obtained through colony hybridization of a C. albicans genomic library with a radiolabeled 397-bp EcoRV fragment derived from p4E1 (Fig. 1). The complete 678-bp open reading frame is composed of two exons encoding, respectively, 35-and 171-amino acids segments separated by a 60-bp intron positioned between nucleotides 106 and 165. Typical fungal consensus sequences (20) described for the 5Ј ((g/G)TAHGTY) and 3Ј (MYA(G/g)) intron/exon boundaries, along with the splice signal for lariat formation (WRCTRAC) were identified in the intervening sequence (Fig. 1). Splicing of the intron at these proposed sites was verified by 5Ј-rapid amplification of cDNA ends analysis. Direct sequencing of the resulting amplicon gave the sequence 5Ј-CAAACACC-3Ј, hence confirming that nucleotides 105 and 166 were joined together in the cDNA. This analysis also identified the transcriptional start site that is located 9 bp upstream from the initiator ATG codon.
The deduced amino acid sequence of SOD3 is a 206-residue protein with a molecular mass of 22.7 kDa (Fig. 1). The consensus pattern DXWEHXXY (i.e. Asp 170 to Tyr 177 in Sod3p) and the four characteristic residues involved in metal binding (i.e. His 32 , His 81 , Asp 170 , and His 174 ) typical of the Mn/FeSOD family are present within Sod3p (21-23). Parker and Blake (21) identified critically positioned amino acids that allow iron-and manganese-containing SODs to be distinguished based on their primary structure. The amino acids Gly 75 , Gly 76 , Gln 155 , and Asp 156 found in Sod3p conform to the signature sequence for the MnSOD subfamily, suggesting that this third C. albicans SOD requires manganese at its catalytic site for activity.
At the amino acid level, Sod3p displays significant sequence identity with several members of the eukaryotic MnSODs and bacterial MnSOD and FeSOD but is most closely related to the C. albicans Sod2p (63% identity) and S. cerevisiae Sod2p (60% identity) (Fig. 2). In sharp contrast to the C. albicans Sod2p and S. cerevisiae Sod2p enzymes, however, as well as to most eukaryotic MnSOD enzymes characterized so far, Sod3p does not possess an amino-terminal extension of ϳ30 residues (transit peptide) that normally targets the enzyme to the mitochondria. It therefore predicts that the enzyme will be sequestrated within the cytosol.
Purified Sod3p Is a Tetrameric Enzyme That Requires Manganese for Activity-To biochemically characterize the C. albicans SOD3 gene product, its cDNA was cloned into the bacterial expression vector pQE30 (see "Experimental Procedures").
FIG. 1. Nucleotide and deduced amino acid sequences of the C. albicans SOD3 gene. Numbering, with the ϩ1 beginning at the coding region, is given for the gene and the amino acid sequences. The transcription start site is indicated by an arrow, and the stop codon is indicated by an asterisk. White and black dots indicate the typical fungal consensus sequences for the 5Ј and 3Ј intron/ exon boundaries, respectively. The bar underneath the sequence indicates the splice signal for lariat formation. The underlined and double underlined sequences indicate the polyadenylation signal and the putative TATA box, respectively. The consensus sequence and the residues involved in metal binding in the Fe/MnSOD family are underlined with a dashed line and circled, respectively. The residues that conform to the signature sequence of Parker and Blake (21) for the MnSOD subfamily are boxed.
The resulting plasmid (pSOD3r) expresses a histidine-tagged Sod3 enzyme, and a large amount of it (10 -100 mg/liter) was purified in its native form by a single affinity chromatography step on nickel-nitrilotriacetic acid resin. When analyzed by SDS-PAGE under denaturing conditions, the highly purified preparation migrated as a single band of 25.4 kDa, in agreement with the predicted molecular mass of 24.1 kDa for the His-tagged modified Sod3p subunit (Fig. 3, inset). The molecular mass of the native enzyme was also evaluated by gel filtration (Fig. 3). The elution profile showed a prominent, nearly symmetrical, peak of ϳ85 kDa and a minor peak of very low molecular mass. This is therefore consistent with a homotetrameric structure for native Sod3p, a result that further extends the similarity between the C. albicans Sod2 and Sod3 enzymes to their quaternary structure (8).
A low level of dismutase activity was measured in initial experiments where Sod3p was purified from E. coli recombinants cultured in 2YT medium and induced for 5 h with IPTG (Table II). Consistent with its requirement for Mn 2ϩ at the catalytic site, a nearly 7-fold increase in specific activity was observed (ϳ2100 units/mg versus ϳ350 units/mg; Table II) when Sod3p was purified from E. coli recombinants cultured in a manganese-supplemented medium and induced for a shorter period with IPTG (15 min). Atomic absorption spectrometry performed on these two enzyme preparations, while indicating the presence of both Fe 2ϩ and Mn 2ϩ within Sod3p, also revealed a positive correlation between the manganese/iron ratio and the activity (the greatest manganese/iron ratio was found in the most active enzyme preparation). Finally, and in further support that Sod3p belongs to the MnSOD family, the activity of Sod3p was not affected by the presence of known inhibitors of either bacterial FeSODs (hydrogen peroxide) or Cu/ZnSODs (potassium cyanide). However, because neither enzyme preparation was filled exclusively with Fe 2ϩ ions, these results could not rule out the possibility that Sod3p is a member of the so-called cambialistic SODs. These enzymes represent a special group of Mn/FeSODs that are active, albeit at variable levels, with either metal ion present at their active site (24). We directly tested this possibility by first denaturing Sod3p in the presence of a chelator to generate apoSod3p and subsequently allowed the enzyme to refold in the presence of either metal cofactor (Table III). As expected, the apo form of Sod3p, as well as bacterial apoMnSOD and apoFeSOD used as controls, was totally inactive. When renatured in the presence of Fe 2ϩ , Sod3p displayed a very low background activity (ϳ130 units/mg). This activity was similar to, although somewhat lower than, that measured for the E. coli apoMnSOD reconstituted with Fe 2ϩ (ϳ310 units/mg). In contrast, when apoSod3p was refolded in the presence of Mn 2ϩ , the activity of the reconstituted enzyme was significantly higher (ϳ3900 units/mg) than the native enzymes purified from E. coli (ϳ350 and ϳ2000 units/mg; Table  II). Similarly, the E. coli apoMnSOD and apoFeSOD reconstituted with their corresponding metal cofactor were more active than the commercial preparations. Collectively, we concluded from these experiments that the C. albicans Sod3p is a bona fide manganese-containing SOD, active as a tetramer, that is predicted to reside rather unusually into the cytosolic compartment.
Sod3p Functionally Complements the Defects of the S. cerevisiae sod1⌬, but Not Sod2⌬ Mutant-The postulated cytosolic localization of Sod3p implies that its main function is to protect this cellular compartment against reactive oxygen radicals. To confirm this hypothesis and, in the positive, to evaluate whether the nature of the SOD expressed (i.e. Cu/ZnSOD versus MnSOD) might influence the efficiency by which the cell copes with induced oxidative stresses, the SOD3 cDNA was introduced into S. cerevisiae mutants lacking either the cytosolic Cu/ZnSOD (sod1⌬) or the mitochondrial MnSOD (sod2⌬) enzymes. Cell viability of the yeast transformants growing in the presence of either menadione or paraquat was then assayed (Fig. 4). These drugs are redox cycling agents that generate superoxide ions either within the cytosol (menadione) or in both the cytosolic and mitochondrial compartments (paraquat). Yeast killing was assessed by the presence of a clear zone ("halo") surrounding the filter disc containing the drug deposited on a Petri dish seeded with the indicated transformant. Wild type yeast, whether expressing (pVTSOD3) the SOD3 cDNA or not (pVT), was tolerant to both menadione and paraquat at moderate concentrations (10 and 40 mM, respectively). However, they both showed a marked decrease in cell viability in the presence of higher concentrations of these agents (50 and 400 mM, respectively). Because SOD3 was expressed from the multicopy plasmid pVT under the control of the strong constitutive ADH promoter, this suggests that under these conditions other proteins involved in the oxidative stress response become limiting. As described previously (25), sod1⌬ mutant yeast carrying the vector alone (pVT) were hypersensitive to both drugs. Overexpression of the C. albicans SOD3 cDNA (pVT-SOD3) restored the menadione and paraquat sensitivities of sod1⌬ yeast to wild type levels. In contrast, constitutive expres-

FIG. 3. Gel filtration chromatography and SDS-PAGE analysis of the Sod3r enzyme purified from E. coli transformants. The
Sod3r enzyme expressed from the M15 transformants during a long induction period (5 h) with IPTG was purified by affinity chromatography with nickel-nitrilotriacetic acid resin as described under "Experimental Procedures." An aliquot of the purified material (100 g) was applied to a Superdex 75 HR 10/30 fast protein liquid chromatography column and eluted at a flow rate of 0.9 ml/min. The arrows indicate the molecular mass (kDa) of the calibration markers. Inset, the indicated amounts of purified Sod3r enzyme were resolved by 12% SDS-PAGE and stained with Coomassie Blue.

FIG. 2. Alignment of the amino-terminal amino acid sequences of the C. albicans and S. cerevisiae
MnSODs. The C. albicans Sod3 (CaSod3) and the C. albicans and S. cerevisiae Sod2 (CaSod2 and ScSod2, respectively) enzymes were aligned using the ClustalW multiple sequence alignment program. The Boxshade program was used for visualizing the results. Regions of sequence identity and similarity are in black and gray boxes, respectively. Residues in italic type indicate the transit mitochondrial peptide sequence in CaSod2 and ScSod2. The Sod2p amino acid sequences were obtained from GenBank TM , and their accession numbers are: CaSod2, AAB86583; ScSod2, CAA26092.
sion of the C. albicans SOD3 cDNA did not alleviate the known paraquat hypersensitivity of sod2⌬ yeast (26). These data are thus consistent with the predicted localization of Sod3p. They also indicate that the presence of either a MnSOD or a Cu/ ZnSOD within the cytosol similarly protects the cell upon induced oxidative stresses.
C. albicans SOD1 and SOD3 Genes Are Divergently Expressed in Nutrient-limiting Conditions and upon Induced Oxidative Stress-The existence in C. albicans of two cytoplasmic SOD enzymes that, based on functional assays in S. cerevisiae mutants, appear to have redundant roles in the detoxification of ROS prompted us to investigate the expression of the corresponding genes under various growth conditions. An important aspect of C. albicans physiology is its dimorphic nature, being able to grow as and to switch from a yeast or a mycelial form. Numerous factors, including the temperature, the pH level, the growth phase, and the presence of serum are known to favor or trigger the transition from the yeast to the hyphal form. To test whether the expression of SOD1 or SOD3 is associated with a particular morphological form, we performed a series of Northern hybridizations with total RNA prepared from C. albicans growing in various culture media at 28 and 37°C. As a control for the temperature shift, C. albicans were also grown in YPD medium where the yeast form was either the exclusive (28°C) or the predominant (37°C) species observed (Fig. 5B). In this medium SOD3 was expressed at both temperatures, whereas the transcription of SOD1 was largely repressed (Fig. 5A). When cultured in YPD supplemented with serum (YPDS), the opposite was observed. The C. albicans SOD1 gene was strongly expressed at both 28 and 37°C, whereas the SOD3 mRNA was not detected at 28°C and present albeit at reduced level at 37°C when compared with that observed in YPD. Because hyphal structures were present at 37°C exclusively (Fig. 5B), this provided a first indication that the growth conditions (i.e. nature of the culture medium) rather than the transition from the yeast to the mycelial phase per se greatly influenced the expression of these two C. albicans SOD genes. This was further supported by the expression profile observed in Lee's medium. In this medium, C. albicans rapidly switched from the yeast to the mycelial form upon raising the temperature from 28 to 37°C under both acidic (pH 4.5) and nearly neutral (pH 6.5) conditions (Fig. 5B). Yet, only the SOD1 gene was expressed under all conditions (Fig. 5A). For C. albicans grown in IMDM pH 6.5 medium, only the SOD1 mRNA was detected at 28°C (predominantly the yeast form). The transition from the yeast to the mycelial form induced by the temperature shift (37°C) was accompanied by a slight induction in the transcription of SOD3, a sharp decrease in the SOD1 mRNA level, and the accumulation of a novel mRNA species hybridizing with the SOD1 probe. The nature of this mRNA, larger in size than the SOD1 mRNA, is at present not clear but could be derived from the transcriptional induction of a SOD1 homologous gene whose existence was revealed by the completed genome sequence of C. albicans. 2 More importantly, our data indicated that neither of the SOD genes is specifically regulated by the dimorphism. Because the SOD1 and SOD3 genes were rarely expressed or repressed together, it also suggested that their transcriptional regulation may be oppositely coordinated. To further substantiate this hypothesis, we studied their expression profile in different metabolic conditions as well as following various oxidative stresses.
Northern hybridizations performed with total RNA extracted at different time points in glucose-grown (YPD) C.  a The native C. albicans Sod3r enzyme expressed in E. coli and the commercial E. coli MnSOD and FeSOD were denatured to generate the Apo forms (e.g. ApoSod3r) and reconstituted with either Fe 2ϩ or Mn 2ϩ .
b The specific activity shown is the average of triplicate samples except for the ApoMnSOD ϩ Mn 2ϩ sample (duplicate). In all cases the standard deviation did not exceed 10% of the value. c NDA, no detectable activity.
FIG. 4. Heterologous expression of the C. albicans SOD3 gene in S. cerevisiae. S. cerevisiae wild type cells (WT) and mutants lacking either the copper-zinc superoxide dismutase (sod1⌬) or the manganese superoxide dismutase (sod2⌬) expressing (pVTSOD3) or not (pVT) the C. albicans SOD3 cDNA were assayed for hypersensitivity to the indicated concentrations of menadione and paraquat (see "Experimental Procedures"). Yeast killing was assessed by the presence of a clear zone surrounding the disc.
albicans cultures detected the presence of SOD1 mRNA at the earliest time point considered (2 h), but its abundance was maximal after 8 h of growth (Fig. 6B). As the cells entered the slow growth phase (by ϳ12 h of growth; Fig. 6A), the expression of SOD1 was gradually repressed, and no SOD1 mRNA could be detected beyond 16 h of growth. Concomitant to this repression of SOD1, the transcription of SOD3 was induced at 12 h and maintained as the cells entered the stationary phase. This profile of expression was not specific to glucose-grown cultures because nearly superimposable patterns of expression were observed when C. albicans was grown in an alternative fermenting (galactose) or a nonfermenting (glycerol) carbon source (Fig. 6). Hence nutrient limiting conditions rather than the respiratory chain seem to regulate both the transcriptional repression of SOD1 and the induction of SOD3.
In many organisms, including the yeast S. cerevisiae (27,28), the gene encoding the cytoplasmic SOD is transcriptionally stimulated by treatment with various oxidative agents such as hydrogen peroxide, menadione, and paraquat. The expression of the SOD1 and SOD3 genes in response to oxidative stresses was studied with C. albicans cells taken at different time points in the growth phase (Fig. 7). Control samples taken at 4, 8, 18, and 22 h of growth in glucose medium (YPD) were representative of that observed previously (Fig. 6B); SOD1 mRNA was the only species detected at 4 h, both SOD1 and SOD3 mRNAs were present after 8 h, and only the SOD3 mRNA was observed at later time points (18 and 22 h). In all treated samples (i.e. hydrogen peroxide, menadione, and paraquat), and irrespective of the age of the culture, the oxidative stress stimulated the transcription of SOD1 (Fig. 7). At later time points (18 and 22 h), this was accompanied by the transcriptional repression of SOD3. Remarkably, the decrease in SOD3 mRNA paralleled in magnitude the relative increase in SOD1 mRNA. These results, along with the expression profiles determined in continuous cultures, thus provided further evidence that C. albicans coordinates in the opposite direction the expression of the SOD1 and SOD3 genes. DISCUSSION C. albicans is a successful commensal organism found in the mouth and gastrointestinal tract of 30 -50% of the population. It is believed that its ability to survive within the host environment lies on the expression of specific factors that distinguish it from its closely related yeast species, the fermenting yeast S. cerevisiae. To protect against the damaging effect of oxygen radicals, most eukaryotes express a cytoplasmic Cu/ ZnSOD and a mitochondrial MnSOD that convert the superoxide anion into hydrogen peroxide. In addition to these ubiquitous enzymes, encoded by the SOD1 (7) and SOD2 (8) genes, respectively, we report here that C. albicans also expresses an atypical manganese-containing SOD enzyme (Sod3p).
Several lines of evidence support the classification of Sod3p as a member of the MnSOD family as follow: (i) the primary structure of Sod3p conforms to the signature sequence proposed by Parker and Blake (21) for MnSODs; (ii) the activity of the purified recombinant enzyme was not modified by the presence of known inhibitors of FeSODs; but (iii) the activity of the purified recombinant enzyme was stimulated by an increase content of Mn 2ϩ at the active site; and finally (iv) in vitro reconstituted recombinant Sod3p with Fe 2ϩ at the metal-binding site was totally inactive but displayed maximal activity with Mn 2ϩ . However, in contrast to most MnSODs described so far, Sod3p does not possess an amino-terminal peptide enabling mitochondrial import and was shown to correct the sensitivity of an S. cerevisiae sod1⌬ mutant to menadione and paraquat. Previous studies on the heterologous expression of the bacterial cytosolic FeSOD or MnSOD into S. cerevisiae showed that the ability to suppress the paraquat sensitivity of yeast sod1⌬ and sod2⌬ mutants was directly linked to the intracellular localization of the enzyme (26, 29 -31). We therefore conclude that the C. albicans Sod3p is a manganese-containing enzyme that is active in the cytosol.
What could be the advantage for C. albicans to express, into the same cellular compartment, both a Cu/ZnSOD and a Mn-SOD? The simplest hypothesis is that the presence of both enzymes may confer an increased resistance to oxidative stresses. However, because wild type yeast, expressing or not expressing SOD3 (pVT versus pVTSOD3), and sod1⌬ yeast transformed with pVTSOD3 have nearly the same sensitivities to elevated concentrations of menadione and paraquat (Fig. 4); this argues against this hypothesis. Furthermore, although these drugs stimulated the transcription of SOD1, it concomitantly repressed the expression of SOD3, suggesting that C. albicans modulates the expression of these genes to maintain rather than to increase its supply in cytoplasmic SOD (see below).
The expression of the SOD1 and SOD3 genes under various growth conditions rather suggested that C. albicans, by coor-dinating inversely the transcription of these genes, ensures a constant presence of superoxide dismutase activity within the cytosol. In this hypothesis the differential expression of SOD1 and SOD3 would be dictated by the availability of the metal ions (Cu 2ϩ , Zn 2ϩ , and Mn 2ϩ ) and/or the need for the presence of a specific metal scavenger. The S. cerevisiae SOD1 gene was shown to be essential for the stationary phase survival (4). In C. albicans, SOD1 is repressed concomitantly to the induction of SOD3 upon the entry and during the stationary phase (Fig.  6), indicating that in C. albicans, it is the expression of an usual cytosolic MnSOD that confers antioxidant protection during these growth phases. Based on this and previous studies, several lines of evidence suggest that this switch from SOD1 to SOD3 expression reflects: (i) the requirement for increased protection against both ROS and copper toxicity during the stationary phase and (ii) the difference between S. cerevisiae and C. albicans in the mechanisms involved in the homeostatic control of copper (see below).
Consistent with the requirement for an added protection against the deleterious effect of copper in stationary phase cells, a recent genome-wide expression profiling study conducted in S. cerevisiae revealed that the entry into the stationary phase was accompanied by a strong induction of the genes involved in copper resistance (e.g. ACE1 and CUP1) and by the down-regulation of the genes implicated in copper uptake (e.g. CTR1 and CTR3) (28). Recent works performed with the filamentous yeast Podospora anserina provided direct evidence that copper is implicated in the aging process (32,33). A mutant (grisea) defective in copper uptake survived nearly twice as long as wild type P. anserina in continuous cultures. Furthermore, a similar extension in life span was observed when wild type P. anserina was cultured in copper-depleted media. In C. albicans, an added protection against copper toxicity during the stationary phase provides an explanation for the repression of the SOD1 gene and the induction of an alternate cytosolic SOD activity.
In S. cerevisiae the main defense mechanism against copper toxicity is the expression of copper scavengers such as the metalothionein encoded by CUP1 and the Sod1 enzyme, which was shown to suppress the copper toxicity phenotype of a cup1⌬ mutant (34). Consistent with this role of Sod1p in copper buffering, its gene is induced by the Ace1p copper sensor, a DNA transactivator (25). In contrast, despite possessing an ortholog of CUP1 (CaCUP1), the main defense mechanism of C. albicans against copper appears to rely predominantly on the coppertransporting P-type ATPase encoded by the CaCRP1/CaCRD1 gene (35,36). This plasma membrane transporter, not present in S. cerevisiae, was shown to extrude the cytosolic copper ions into the medium, hence leading to depletion of this ion from the cytosolic compartment. Under these conditions, the expression of the Cu/ZnSOD encoded by SOD1 would therefore be inappropriate. The expression of both CaCUP1 and CaCRP1/ CaCRD1 was found to be stimulated by copper and proposed to involve an Ace1p-like DNA transactivator. Unlike CaCUP1 and CaCRP1/CaCRD1 and in support of this hypothesis, no Ace1-responsive elements are present in the promoter region of C. albicans SOD1 (not shown).
Although most eukaryotic MnSODs are targeted to the mitochondria, cytoplasmic manganese-containing SODs have been found in few organisms, namely in unicellular green algae (37) and in various species of filamentous fungi (38). Interestingly, unicellular green algae (39) and filamentous fungi (40,41), including C. albicans (42), are also known to possess an alternative respiratory pathway in which the mitochondrial cytochrome c oxidase, requiring copper as cofactor, is replaced by an alternate oxidase (Aox) using iron as cofactor. In the P. FIG. 7. Expression of the SOD1 and SOD3 genes in response to induced oxidative stresses. Northern blot analysis of SOD1 and SOD3 transcripts was carried out with RNA samples prepared from C. albicans cultures taken at the indicated time (in hours) of growth in YPD, followed by a 1-h incubation in the absence (lanes C) or presence of either hydrogen peroxide (lanes H), menadione (lanes M), or paraquat (lanes P) as described under "Experimental Procedures." The ethidium bromide-stained 25 and 18 S ribosomal RNAs are also shown as loading control.
anserine grisea mutant or in wild type yeast grown in copperdepleted media, the increase life span is associated with the induction of this alternative respiratory pathway that decreases the generation of oxygen radicals (33). This suggests that organisms adapted to grow in the absence of copper have similarly evolved to express an alternate cytosolic SOD to replace the ubiquitous Cu/ZnSOD. The ability to survive for prolonged periods of time in a dormant state is what we would expect for a successful commensal organism like C. albicans. We have shown here that during the stationary phase, C. albicans expresses an unusual cytoplasmic MnSOD instead of the ubiquitous eukaryotic Cu/ ZnSOD. The expression of the C. albicans SOD3 gene may be part of a well orchestrated mechanism involving the homeostatic control of copper ions to survive, in a nonreplicative form, into the host environment, and future studies should be directed to validate this hypothesis.