Identification and characterization of Cor33p, a novel protein implicated in tolerance towards oxidative stress in Candida albicans

Abstract

We applied two-dimensional gel electrophoresis to identify downstream effectors of CPH1 and EFG1 under hypha-inducing conditions in Candida albicans. Among the proteins that were expressed in wild-type cells but were strongly downregulated in a cph1Delta/efg1Delta double mutant in alpha-minimal essential medium at 37 degrees C, we could identify not-yet-characterized proteins, including Cor33-1p and Cor33-2p. The two proteins are almost identical (97% identity) and represent products of allelic isoforms of the same gene. Cor33p is highly similar to Cip1p from Candida sp. but lacks any significant homology to proteins from Saccharomyces cerevisiae. Strikingly, both proteins share homology with phenylcoumaran benzylic ether reductases and isoflavone reductases from plants. For other hypha-inducing media, like yeast-peptone-dextrose (YPD) plus serum at 37 degrees C, we could not detect any transcription for COR33 in wild-type cells, indicating that Cor33p is not hypha specific. In contrast, we found a strong induction for COR33 when cells were treated with 5 mM hydrogen peroxide. However, under oxidative conditions, transcription of COR33 was not dependent on EFG1, indicating that other regulatory factors are involved. In fact, upregulation depends on CAP1 at least, as transcript levels were clearly reduced in a Deltacap1 mutant strain under oxidative conditions. Unlike in wild-type cells, transcription of COR33 in a tsa1Delta mutant can be induced by treatment with 0.1 mM hydrogen peroxide. This suggests a functional link between COR33 and thiol-specific antioxidant-like proteins that are important in the oxidative-stress response in yeasts. Concordantly, cor33Delta deletion mutants show retarded growth on YPD plates supplemented with hydrogen peroxide, indicating that COR33 in general is implicated in conferring tolerance toward oxidative stress on Candida albicans.

FIG. 1.

Identification of Cor33-1p and Cor33-2p. Protein extracts from wild-type cells (A), as well as from a cph1Δ/efg1Δ mutant strain (B), cultured in α-MEM at 37°C to induce hyphal growth were separated by two-dimensional gel electrophoresis. The protein spots were visualized by silver staining, followed by detection of differentially expressed proteins by comparing the resulting spot patterns. The arrows indicate spots corresponding to Cor33-1p (1) and Cor33-2p (2), which are expressed in wild-type cells but are downregulated in the cph1Δ/efg1Δ mutant. Only sections of the complete two-dimensional gels are shown.

FIG. 2.

Alignment of the amino acid sequences of Cor33-1p and Cor33-2p. Peptide sequences derived from tryptic in-gel digests of Cor33-1p and Cor33-2p spots were used to run tblastn searches against assembly 19 of the Candida albicans genome database (http://www-sequence.stanford.edu/group/candida/index.html). The alignment of the amino acid sequences deduced from two open reading frames (orf19.113 and orf19.7761) exactly matching the peptide sequences are shown. Cor33-1p and Cor33-2p contain 299 amino acids lacking signal sequences at their N termini. Peptides derived from microsequencing are indicated by boldface letters; amino acids differing between the sequences are underlined. A consensus binding site for NAD(P)H (XGXXGXXGX) is indicated (+), and an acyl-coenzyme A dehydrogenase signature is highlighted by asterisks.

FIG. 3.

Transcriptional regulation of COR33. (A) Northern blot analysis using total RNAs of wild-type cells (lanes 1, 5, and 9), cph1Δ (lanes 2, 6, and 10), efg1Δ (lanes 3, 7, and 11), and cph1Δ/efg1Δ (lanes 4, 8, and 12) mutant strains cultured in α-MEM at 37°C (lanes 1 to 4), in YPD at 30°C (lanes 5 to 8), or in YPD plus 10% serum at 37°C (lanes 9 to 12). The blots were tested using probes specific for COR33 or ACT1. (B) Northern blot analysis using total RNAs from wild-type cells cultured in YPD at 30°C for 8 h (lane 1), in α-MEM at 37°C for 8 h (lane 2), in YPD plus 0.5 mM cadmium chloride at 30°C for 8 h (lane 3), or in YPD plus 5 mM hydrogen peroxide at 30°C for 1 hour (lane 4). The blots were tested using probes specific for COR33 or ACT1. (C) Time course experiment following transcription of COR33 and EFG1 at different time points after induction of C. albicans wild-type cells with 5 mM hydrogen peroxide in YPD at 30°C. The cells were harvested for the isolation of total RNA after 0 (lane 1), 30 (lane 2), 60 (lane 3), 120 (lane 4), or 240 min (lane 5). The blots were tested using probes specific for COR33 or EFG1. (D) Northern blot analysis of wild-type (wt) cells (lane 1) and an efg1Δ mutant (lane 2) cultured in YPD plus 5 mM hydrogen peroxide for 1 hour at 30°C. The blots were tested using probes specific for COR33 or ACT1. (E) Northern blot analysis of wild-type cells (lane 1) and a cap1Δ mutant CJD21-PMK (lane 2); a corresponding cap1Δ/CAP1 revertant strain, CDJ21-CAP1 (lane 3); and a cap1Δ/CAP1-TR mutant strain expressing a hyperactive form of CAP1, CDJ21-CAP1-TR (lane 4), cultured in YPD plus 5 mM hydrogen peroxide for 1 hour at 30°C. The blots were tested using probes specific for COR33 or ACT1.

FIG. 4.

Induction of COR33 transcription in a tsa1Δ mutant. Northern blot analysis using total RNAs isolated from wild-type cells (lanes 1 and 2), from a tsa1Δ mutant strain (lanes 3 and 4), and from the corresponding tsa1Δ/TSA1 revertant strain either grown in YPD at 30°C for 40 min (lanes 2, 4, and 6) or induced by the addition of 0.1 mM hydrogen peroxide to the medium for the same period of time (lanes 1, 3, and 5). The blots were tested using probes specific for COR33, TSA1, or ACT1.

FIG. 5.

Construction of a cor33Δ deletion mutant. The serial deletion of both COR33 alleles was carried out by using the FLP recombinase approach. (A) Schematic of the general principle. Two flanking regions, FR1 and FR2, that are adjacent to the COR33 open reading frames in the C. albicans genome were cloned at the 5′ and the 3′ ends of the FLP recombinase DNA cassette containing the FLP recombinase gene under the control of a SAP2 promoter, as well as a URA3 marker. After transformation of the DNA cassette, the COR33 alleles were replaced by homologous recombination. Induction of the SAP2 promoter led to expression of the FLP recombinase with subsequent excision of the DNA cassette via the FRT sites. The arrows indicate restriction sites for EcoRV used for the Southern blot analysis to confirm the genomic integration events. The asterisk indicates one EcoRV site that is specific for the genomic locus of COR33-1 and that allows discrimination between the COR33-1 and COR33-2 loci by Southern blot analysis. (B) Southern blot analysis of different COR33 transformants: COR33-1/COR33-2 (strain SC5314; lane 1), COR33-1/cor33-2::FLP (strain RSC10; lane 2), COR33-1/cor33-2::FRT (strain RSC11; lane 3), cor33-1::FLP/cor33-2::FRT (strain RSC12; lane 4), and cor33-1::FRT/cor33-2::FRT (strain RS22; lane 5).

FIG. 6.

Plate assay to determine tolerance toward oxidative, as well as cadmium, stress. (A) C. albicans wild-type cells (lane 1), the cor33Δ deletion mutant (lane 2), and the corresponding cor33Δ/COR33 revertant strain (lane 3) were plated in 10-fold serial dilutions onto either YPD plates supplemented with 5 mM hydrogen peroxide (right plate) or YPD plates without any supplements as a control (left plate). The plates were cultured at 30°C for 1 day. (B) C. albicans wild-type cells (lane 1), the cor33Δ deletion mutant (lane 2), and the corresponding cor33Δ/COR33 revertant strain (lane 3) were plated in 10-fold serial dilutions onto either YPD plates supplemented with 0.5 mM cadmium chloride (right plate) or YPD plates without any supplements as a control (left plate). The plates were cultured at 30°C for 1 day.