PaCATB, a secreted catalase protecting Podospora anserina against exogenous oxidative stress

Cloning procedures and generation of P. anserina mutants

The generation of PaCatB over-expressing strains (PaCatB_OEx1-3) was performed by amplifying the open reading frame of PaCatB (plus ~ 500bps terminator region) by PCR using the oligonucleotides PaCatBEx1-1 (5‘-GGGGATCCAT GAAAAGGCTG CTAACG-3’) and PaCatBEx1-2 (5‘-CCAAGCTTAA AAGCTCACCG GCCAAC-3’), introducing Bam HI and Hin dIII restriction sites (underlined). The amplicon was cloned into the pExMthph backbone (BamHI / HindIII digested) resulting in the plasmid pPaCatBEx1. In this the PaCatB reading frame is under control of a strong constitutive metallothionein promoter [47]. The plasmid was used to transform P. anserina wild-type spheroplasts. Transformants were selected for hygromycin-resistance and verified by Southern blot analysis. Three different strains containing one additional copy of PaCatB were subsequently further analyzed (PaCatB_OEx1-3).

Deletion of PaCatB in wild-type strain s was performed according to a previously described method [48]. Briefly, small flanking regions of PaCatB were amplified using the 5‘-flank specific oligonucleotides PaCatBKO1-1 (5‘-CCGGTACCCC TTTGCCGGGG GGCGTG-3’) and PaCatBKO1-2 (5‘-CCCTGCAGCT GCTGCCGCTG CCGTGC-3’) introducing KpnI and Pst I restriction sites and the 3'-flank specific oligonucleotides PaCatBKO1-3 (5‘-GGACTAGTGG AAAAGGGAAT GGGTTC-3’) and PaCatBKO1-4 (5‘-GGGCGGCCGC ACTAATATAT ATACCG-3’) with Bcu I and Not I restriction sites. The fragments were cloned into the plasmid pKO4 [48, 49] next to the bifunctional resistance cassette consisting of a blasticidin resistance gene for selection in Escherichia coli and a phleomycin resistance cassette for selection in P. anserina. The resistance cassette with the flanking regions was excised by degistion with NotI and KpnI and transformed into the E. coli KS272 strain which contains plasmid pKOBEG [50] and the PaCatB gene containing cosmid 19B11 [51]. Homologous recombination between the flanks of the resistance cassette and cosmid 19B11 leads to generation of cosmid Δ19B11, containing the phleomycin-blasticidin cassette with large flanking genomic regions. The cosmid Δ19B11 was used to transform P. anserina wild-type spheroplasts. Transformants were selected by growth on phleomycin-containing medium. Successful deletion of PaCatB was indicated by phleomycin resistance and hygromycin sensitivity. The correct replacement of the PaCatB gene was verified by Southern blot analysis. The selected strain, lacking the PaCatB gene and harbouring the phleomycin gene instead, was termed ΔPaCatB.

Transformation of P. anserina.

Production, regeneration, and integrative transformation of P. anserina spheroplasts was performed as described [52, 53].

P. anserina strains and strain cultivation

In this study the P. anserina wild-type strain s [9], the newly generated PaCatB over-expressing strains (PaCatB_OEx1-3) and the deletion strain (ΔPaCatB) were used. All transgenic strains are in the genetic background of wild-type strain s. Strains were grown on standard cornmeal agar (BMM) at 27 °C under constant light [7]. For germination of spores BMM with 60 mM ammonium acetate was used and incubated at 27 °C in the dark for 3 days. All used strains were originating from mono-nucleate ascospores isolated from irregular asci. In order to obtain cultures of a defined age, mycelia pieces from cultures of freshly germinated ascospores were placed on one side of a Petri dish containing PASM (P. anserina synthetic medium) [18].

Growth rates under oxidative stress conditions

For incubation of strains with hydrogen peroxide, mycelia of monokaryotic isolates (wild-type, ΔPaCatB and PaCatB_OEx1-3: n=8) were grown on P. anserina synthetic medium (PASM) [26] containing different hydrogen peroxide concentrations (0-45 mM hydrogen peroxide) for 4 days. Plates were kept in the dark to protect hydrogen peroxide from degradation. Growth rates were assigned daily and calculated as growth rate per day.

Lifespan determination

Lifespan determination was performed with cultures originating mono-nucleate ascospores (wild-type: n= 66, ΔPaCatB: n=20, PaCatB_OEx1: n=29, PaCatB_OEx2: n=11, PaCatB_OEx3: n=14). Mycelial pieces from cultures of freshly germinated ascospores were placed on race tubes containing PASM [19]. Survival curves were calculated allowing the determination of the median lifespan.

Lifespan determination under oxidative stress

Lifespan determination under oxidative stress was performed as described above. The different isolates were placed on Petri dishes with PASM containing 0.75 mM hydrogen peroxide (wild-type: n=23, ΔPaCatB: n=27) or 3 mM hydrogen peroxide (wild-type and PaCatB_OEx1-3: n=40).

Isolation of secreted proteins

Mycelia from wild-type strain s, PaCatB_OEx1-3 or ΔPaCatB were allowed to overgrow the surface of cellophane foil occupied BMM plates. After an incubation of 3 days at 27 °C under constant light, the cultured mycelia were transferred into tubes of liquid media (150 ml CM) and were incubated for 4 days at 27 °C under constant light without shaking to prevent cell damages and consequential resultant contamination with total cell extract. The filtered liquid medium was enriched by filter tubes (Amicon Ultra-15, Ultracel-3k, Millipore, Schwalbach, Germany). Supernatants were mixed with 1:100 PIC (Protease Inhibitor Cocktail). The secreted proteins were precipitated by adding 2 volumes of ethanol and 2 volumes of acetone for 2 days at -20 °C. After centrifugation (30 min, 4 °C, 15000 rpm FA-45-24-11 fixed angle rotor) 1 ml desalinisation solution (2:1:1 water: ethanol: acetone) was added to the pellet. After 1 min of vortexing, the secreted proteins were incubated over night at -20 °C. This desalinisation procedure was repeated twice. Pellets were dried and resolved with 50 μl protein extraction puffer at room temperature.

Isolation of whole cell protein

For extraction of whole cell protein, mycelia from different P. anserina strains was allowed to overgrow a cellophane foil covered PASM surface for 4 days in the dark. Subsequently, harvested mycelia were pulverized in liquid nitrogen. The protein was isolated from the powder as described [20].

Two-dimensional gel electrophoresis and silver staining

Secreted protein probes were purified (ReadyPrep™ 2-D Cleanup Kit: Bio-Rad, Munich, Germany) and eluted in IEF buffer (7 M urea, 2 M thiourea, 0.4% dithiothreitol, 2% CHAPS and 0.5% ampholytes at pH 3-10). For the isoelectric focusing, protein (400 μg) was applied to IEF strips (17 cm, pH 3-10, Bio-Rad Munich, Germany). The electro-focusing program was limited to 50 μA per strip and started with 250V for 15 min. Voltage rose rapidly up at 10,000 V for 3 h. This was maintained till 60,000 Vh were obtained. The strips were equilibrated in Tris base (45 mM at pH 8.8, 6 M urea, 2% SDS, 30% glycerol and 2% dithiothreitol) for 10 min and then equilibrated for 10 min in the same buffer with 2.5% iodoacetamide instead of dithiothreitol. The strips were fixed on top of SDS separating polyacrylamide gels (10%) with a 0.5% agarose solution in SDS separation buffer with 0.00067% bromphenol blue. The gels were run at 6 mA/ gel for 3 h followed by 18 mA/ gel for 4 h. The current was set to 20 mA/ gel until the bromphenol blue flew out. The gels were silver stained as previously described [54].

Western blot analysis

10 μg secreted protein were fractionated by two-phase SDS-PAGE (6-18 % separating gels) according to standard protocol [25]. After electrophoresis, proteins were transferred to PVDF membranes (Immobilon Transfer Membranes, Millipore, Schwalbach, Germany). Blocking, antibody incubation and washing steps were performed according to the Odyssey Western blot analysis handbook (Li-Cor, Lincoln, NE, USA). As primary antibodies the following antibodies were used: Anti-PaCATB (1:15000 dilution) raised against a PaCATB specific synthetic peptide ([Ac]-CRYLGRFPVDEGAE-[OH] (New England Peptide, Gardner, USA). In all analyses, secondary antibodies conjugated with IRDye CW 800 (1:15000 dilution, Li-Cor, Lincoln, NE, USA) were used. The Odyssey infrared scanner (Li-Cor, Lincoln, NE, USA) was used for detection.

Qualitative ‘in-gel’ catalase activity assay

The ‘in-gel’ catalase stain was performed as described [55] with native gradient PAGE. Following the electrophoresis the gel was washed 3 times 15 min in water. After washing the gel it was incubated 10 min in a 0.003 % H₂O₂ solution. Subsequently, the gel was transferred into 30 ml freshly prepared solution containing 2 % potassium ferricyanide and 2 % ferric chloride. The gel tray was gently agitated over a light box until appearance of a green color in the gel. After rinsing the gel with water it was scanned for further analysis.

Quantitative photometric measurement of catalase activity

The photometric quantification of H₂O₂ degradation was performed in quartz glass cuvette (104-QS 0.500, Hellma, Müllheim, Germany) by measuring the absorption at 240 nm in intervals of 10 sec for a total of 10 min. The cuvette was loaded with 300 μl of a 0.01 M potassium phosphate buffer (pH 7.0) and 100μl of a 300 mM H₂O₂ solution dissolved in 0.01 M potassium phosphate buffer (pH 7.0) was added. After reaching a constant absorption, 100 μl of a 0.02 μg/μl protein solution, dissolved in 0.01 M potassium phosphate buffer (pH 7.0), was added. The percentage absorption decrease at 240 nm per time, representing the catalase activity, was calculated based on the amount of the linear slope after addition of the protein solution. All samples were measured as triplets in three independent experiments.

Identification of proteins separated by two-dimensional gel electrophoresis

Protein spots of interest were manually excised from gels and digested with trypsin (Promega Inc., Madison, WI, USA). The resulting peptide mixture was desalted and analyzed using a 4800 Plus MALDI TOF / TOF Analyzer (Applied Biosystems, Foster City, CA, USA) as described before [56]. From the raw data output, peak lists were generated by Data Explorer (Applied Biosystems, Foster City, CA, USA). MS and MS/MS peak lists were combined into search files and used to search protein databases using the Mascot search engine (Matrix Science Ltd, London, UK). Search parameters were as follows: Database: NCBInr 20110602 or UniProt Version 49; Taxonomy: all entries or fungi; Enzyme: trypsin; Allow up to 1 missed cleavage; Fixed modifications: none; Variable modifications: methionine oxidation; Peptide mass tolerance: 70 ppm; and Fragment mass tolerance: 500 ppm. Positively identified proteins have been assigned a significant Mascot score based on the probability that the observed match is a random event and protein scores greater than 56 are significant (P < 0.05).

Quantitative proteomics using iTRAQ labeling

Identification and quantitation of the secretome of juvenile and senescent P. anserina cultures have been achieved by iTRAQ labelling and nanoLC-MS/MS in combination with sample pre-fractionation by SCX (strong cathion exchange) following previously described protocols [57]. Briefly, proteins precipitated from growth medium have been reduced and alkylated and subsequently digested with trypsin endopeptidase. 100 μg of peptides derived from each sample were labeled with iTRAQ 114 and 117 according to manufacturer's instructions and mixed at 1:1 ratio. The mixed sample was separated by SCX into 6 fractions corresponding to 6 elution buffers with different ion force (25, 50, 75, 100, 200 and 500 mM KCl in 30% acetonitrile, pH 3). The iTRAQ labeled peptides from SCX fractions were analyzed using EASY nanoLC system (Proxeon, Denmark) equipped with in-house packed fused silica C18 analytical column (Reprosil, Dr. Maisch, 15 cm, 100 μm, I.D., 375 μm, O.D.). The gradient induced a linear increase of 0-32% acetonitrile in 0.1% formic acid over 50 minutes, at a flow rate of 250 nl/minute. Eluted peptides were sprayed into a LTQ-Orbitrap XL (Thermo Electron, Bremen, Germany) via a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). The mass spectrometer was operated in data-dependent mode automatically switching between MS, MS/MS CID (collision-induced dissociation) and MS/MS HCD (high-energy collision dissociation) modes using a 50000 threshold for ion selection.

The data were processed described before with minor changes [57]. The raw data were analyzed using Proteome Discoverer, version 1.0 and an in-house MASCOT server (version 2.1) (Matrix Science Ltd., London, U.K.) for database searching through the Proteome Discoverer program. The data were searched against the Uniprot Podospora anserina sequence database (version 55). Trypsin was used as a cleavage enzyme with a maximum of 2 missed cleavages was allowed. Carbamidomethyl (C) was chosen as a fixed modification. As variable modifications, iTRAQ 4plex (K), iTRAQ 4plex (N-term) and Oxidation (M) were chosen. The data were searched with a peptide mass tolerance of 10 ppm and a fragment mass tolerance of 0.8 Da (CID) and 0.1 Da (HCD). Only proteins identified by at least 2 peptides and mascot peptide ion scores of at least 20 were considered. The quantitative results were normalized on protein median.

Identification of a secreted catalase which decreases in abundance during aging of P. anserina

In order to identify age-regulated proteins in the secretome of P. anserina, secreted proteins from juvenile and senescent wild-type cultures were isolated and analyzed by 2D SDS-PAGE and mass spectrometry (Figure 1). Along with secreted age-regulated enzymes like hydrolases, one laccase and proteins induced during incompatibility reactions, a catalase, named PaCATB, was identified. To confirm the expression changes observed by 2D SDS-PAGE isobaric tags (iTRAQ) combined with nano liquid chromatography and mass spectrometry (nanoLC-MS/MS) were used. Relative quantitation revealed that PaCATB abundance in secretome of senescent wild-type was 3.4 times decreased in comparison to secretome probe from juvenile cultures (Table 1).

Figure 1. Identification of age-regulated secreted proteins. For identification of age-regulated proteins, 400 μg secreted proteins of either juvenile or senescent wild-type were first separated by 2D SDS-PAGE using IPG strips of pH 3-10. The gels were silver stained and spots with differing abundance between juvenile and senescent probes were picked and analyzed by mass spectrometry. The table shows the proteins which were identified in spots 1-8. Different parameters clarifying protein identification by MS are indicated: accession number from UniProt database, mascot score (> 56 is significant at P < 0.05), % sequence coverage, no of matched peptides (MS), and no. of sequenced peptides (MS/MS).

The genome of P. anserina encodes five putative catalases

P. anserina encodes five putative catalases with different predicted localizations (http://podospora.igmors.u-psud.fr, http://wolfpsort.org). Two of them are small subunit catalases within the peroxisome (PaCATP1) or the cytosol (PaCATP2). In addition, a catalase-peroxidase (PaCAT2) is localized in the cytosol. Furthermore, two large putative catalases, PaCATA and PaCATB, are encoded by the P. anserina genome. PaCATA is predicted to be bound to the plasma membrane whereas PaCATB contains a putative secretion sequence. Homologues of PaCATB are known in various ascomycetes such as Neurospora crassa (Cat-3), Aspergillus nidulans (CatB) and Blumeria graminis (CatB) with a high similarity (e-value: 0; http://www.ncbi.nlm.nih.gov) (Figure 2). They all contain an N-terminal secretion signal, a highly conserved catalase domain and several glycosylation sites.

Figure 2. Schematic representation of PaCATB and different homologues. The schematic protein sequence of different PaCATB homologues is shown with their N-terminal secretion signal, glycolysation sites and catalase domain. Podospora anserina (Pa) PaCATB: Q9HDP5 (UniProt); Neurospora crassa (Nc) Cat3: Q9C169 (UniProt); Aspergillus nidulans (An) CatB: P78619 (UniProt); Blumeria graminis (Bg) CatB: AAl56982 (EMBL database). The amino acid similarity is indicated as percentage of the PaCATB amino acid sequence.

Verification of transgenic PaCatB P. anserina strains

In order to investigate the role of the putative secreted and age-regulated catalase PaCATB in lifespan control and ROS scavenging, we set out to generate strains in which PaCatB is deleted or over-expressed. One PaCatB deletion strain (ΔPaCatB) and three independent PaCatB over-expression strains (PaCatB_OEx1-3) containing one extra copy of PaCatB under the control of the strong constitutive metallothionein promoter [47] integrated at different sites in the genome of P. anserina were selected and subsequently analyzed.

First, PaCATB protein abundance and activity of the three PaCatB over-expression strains (PaCatB_OEx1-3) and the PaCatB deletion strain (ΔPaCatB) were verified (Figure 3 A-B). PaCATB abundance was investigated by Western blot analysis with secreted protein probes of PaCatB deletion strain and over-expression strains using a newly generated specific PaCATB antibody (Figure 3 A). PaCATB levels are increased in PaCatB over-expressors while PaCATB protein is undetectable in the PaCatB deletion strain (Figure 3 A).Consistently, no PaCATB activity was identified in secreted protein probes of the deletion strain while the PaCatB over-expressors showed an increased PaCATB activity in the ‘in-gel’ catalase activity assay (Figure 3 A). In order to verify the activity of PaCATB, we next measured the hydrogen peroxide decomposition in these transgenetic PaCatB strains (Figure 3 B). The deletion strain (ΔPaCatB) completely failed to decompose hydrogen peroxide while the three over-expressors (PaCatB_OEx1-3) decomposed hydrogen peroxide with a 2- to 6-fold higher rate compared to the wild-type (Figure 3 B).

Figure 3. Verification of PaCatB deletion and PaCatB over-expression strains. A. Probes of 10 μg secreted protein of wild-type, PaCatB deletion strain (ΔPaCatB) and the three independent PaCatB over-expression strains (PaCatB_OEx1-3) were analyzed by Western blot analysis and an ‘in-gel’ catalase activity assay (6-20 % separating gel). PaCATB was detected via a specific antibody against catalase B in P. anserina (Anti-PaCATB). The Coomassie stained PVDF membrane was used as a loading control of secreted protein probes. The activity of PaCATB is visualized as highlighted bands. Accession numbers: PaCATB: Q9HDP5 (UniProt). B Catalase activity was quantified by measuring the photometric hydrogen peroxide degradation of secreted protein probes of wild-type (n=2, p-value: p<0.01), PaCatB deletion strain (ΔPaCatB, n=9) and the three independent PaCatB over-expression strains (PaCatB_OEx1-3, n=9). The percentage absorption decrease at 240 nm per time represents the catalase activity. Error bars are ± SEM.

Impact of PaCATB on resistance against exogenous hydrogen peroxide stress

Next we tested the resistance of PaCatB deletion strain and PaCatB over-expression strains against hydrogen peroxide added to the growth medium in different concentrations (Figure 4). Compared to the wild-type strain, growth rates of the different PaCatB mutants did not differ on medium without hydrogen peroxide. However, on medium with additional exogenous hydrogen peroxide the PaCatB over-expressors (PaCatB_OEx1-3) displayed significant increased growth rates (Figure 4 A). These strains were able to grow on medium with high levels of hydrogen peroxide while the wild-type was not able to grow under such conditions. In contrast, the deletion strain (ΔPaCatB) was characterized by an increased sensitivity against low levels of exogenous hydrogen peroxide (Figure 4 B).

Figure 4. Analysis of hydrogen peroxide tolerance. The growth rate of wild-type, PaCatB deletion strain (ΔPaCatB) and the three independent PaCatB over-expression strains (PaCatB_OEx1-3) were investigated under oxidative stress. A Growth rates of PaCatB over-expression strains (n=8) were measured on PASM containing 0, 3, 6, 9, 12, 21, 27, 33, 39 and 45 mM hydrogen peroxide and were statistically significant for 3, 6 and 9 mM (PaCatB_OEx1-3: p<0.01), 12, 21, 27 and 33 mM (PaCatB_OEx1-3: p<0.01) and 39 mM hydrogen peroxide (PaCatB_OEx1-2: p<0.01).B Growth rates of PaCatB deletion strain (n=8) were measured on PASM containing 0, 0.375, 0.75, 1.5 and 3 mM hydrogen peroxide and were statistically significant for 0.75, 1.5 und 3 mM hydrogen peroxide (p-value: p<0.01). Error bars are ± SEM.

Modulation of PaCATB abundance affects lifespan at increased exogenous hydrogen peroxide stress

In order to address the impact of the modulation of PaCATB abundance and activity on aging we next determined the lifespan of the different strains under different oxidative growth conditions. On standard medium without added hydrogen peroxide the PaCatB deletion and PaCatB over-expressing strains and the wild-type are characterized by similar lifespans (Figure 5 A). In contrast, clear differences were observed when strains were grown on media with increased hydrogen peroxide (Figure 5 B and C). On hydrogen peroxide supplemented medium the PaCatB over-expression strains showed an increased median lifespan (+35 %) compared to the wild-type (Figure 5 B). In contrast, PaCatB deletion resulted in a highly significant reduced median lifespan (-49 %) (Figure 5 C).

Figure 5. Determination of median lifespan under normal and oxidative stress condition. A. Median lifespan under normal standard condition (PASM without hydrogen peroxide) was analyzed for the wild-type (n=66), ΔPaCatB (n=20), PaCatB_OEx1 (n=29), PaCatB_OEx2 (n=11) and PaCatB_OEx3 (n=14). Neither median lifespan of ΔPaCatB (17.67 d) nor median lifespan of PaCatB over-expression strains (PaCatB_OEx1:18.23 d, PaCatB_OEx2:18.25 d and PaCatB_OEx3:18.5 d) were differentially to the median lifespan of the wild-type (18.48 d). P values were determined using the Superior Performing Statistical Software (SPSS) 16 (SPSS Incorporated). B Median lifespan under oxidative stress (PASM containing 3 mM hydrogen peroxide) was analyzed from the wild-type (n=40), PaCatB_OEx1-3 (n=40). The median lifespan for wild-type was 7.2 d, PaCatB_OEx1:10 d, PaCatB_OEx2:9.72 d and PaCatB_OEx3:9.93 d. Lifespan was 35 % significant increased (p-value: p<0.001). C Median lifespan under oxidative stress (PASM containing 0.75 mM hydrogen peroxide) was analyzed from the wild-type (n=23) and ΔPaCatB (n=27). The median lifespan for wild-type was 13.12 d and for ΔPaCatB 6.7 d. Lifespan was 49 % significant reduced (p-value: p<0.001).

Induction of PaCATB activity as a response to hydrogen peroxide mediated stress

To determine the induction of PaCATB activity and thereby an active mechanism of protection against hydrogen peroxide, catalase activity of whole protein extracts from different strains were analyzed (Figure 6).

Figure 6. Examination of catalase activity during oxidative stress conditions. Whole cell protein was extracted from mycelia grown on PASM for four days in the dark on media containing different hydrogen peroxide concentrations (0, 0.75, 3 mM H₂O₂). 40 μg protein of wild-type strain, deletion strain (ΔPaCatB) and an over-expressing strain (PaCatB_OEx3) were analyzed by an ‘in-gel’ catalase activity assay (10 % separating gel). Catalase activities differing from PaCATB are marked as PaCATX. A Coomassie stained gel loaded with 40 μg of the same protein aliquot was used as a loading control.

These strains were grown on media containing different amounts of hydrogen peroxide (0, 0.75 and 3 mM). When grown on media with added hydrogen peroxide, the wild-type strain exhibits an increased catalase B activity compared to the control without oxidative stress. While the wild-type shows an induction of PaCATB activity by hydrogen peroxide stress, the deletion strain proofs that the remaining catalases of P. anserina (Figure 6: PaCATX) have no compensatory effects. In the over-expressors no obvious up-regulation of catalase activity can be detected. This is consistent with the transcriptional regulation of the extra PaCatB copy which is under the control of a strong constitutive promoter. In these strains, the hydrogen peroxide regulated CATB activity of the endogenous gene most likely is overlaid by the activity of the protein encoded by the transgene.