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. 2014 Feb 1;20(4):589-605.
doi: 10.1089/ars.2013.5423. Epub 2013 Sep 18.

Protein S-mycothiolation functions as redox-switch and thiol protection mechanism in Corynebacterium glutamicum under hypochlorite stress

Bui Khanh Chi  1 , Tobias Busche, Koen Van Laer, Katrin Bäsell, Dörte Becher, Lina Clermont, Gerd M Seibold, Marcus Persicke, Jörn Kalinowski, Joris Messens, Haike Antelmann
Affiliations

Protein S-mycothiolation functions as redox-switch and thiol protection mechanism in Corynebacterium glutamicum under hypochlorite stress

Bui Khanh Chi et al. Antioxid Redox Signal. .

Abstract

Aims: Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiol-redox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation.

Results: Transcriptomics revealed the strong upregulation of the disulfide stress σ(H) regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH.

Innovation and conclusion: We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Microarray intensity scatter plot (A) and hierarchical clustering analysis of the Corynebacterium glutamicum transcriptome after NaOCl stress indicates a σH-dependent disulfide stress response (B). (A) Ratio/intensity scatter plot of the C. glutamicum wild type transcriptome 30 min after NaOCl stress. Genes with at least twofold expression changes after NaOCl stress are marked with black squares, including σH-dependent genes that are labeled with circles. (B) Hierarchical clustering analysis of NaOCl and diamide induced gene expression profiles was performed using GENESIS (55). The transcriptome data sets included genes with at least twofold expression changes at 7.5 min after 2 mM diamide, 10 and 30 min after 180 μM NaOCl stress (Supplementary Tables S1–S3). Red indicates induction and green repression by NaOCl and diamide stress. The σH-regulon is blue-labeled and the redox-sensing QorR, ArsR1, CyeR, and RosR-regulons are red-labeled. The complete Heatmap is shown in Supplementary Figure S2. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 2.</b>
FIG. 2.
NaOCl-stress causes strongly increased reversible thiol-oxidations in the redox proteome of the C. glutamicum wild type. The redox proteome of C. glutamicum wild type at control conditions (green image) was overlaid with that of cells exposed to 180 μM NaOCl-stress (red image). For redox proteomics, reduced protein thiols in cell extracts were alkylated with IAM followed by disulfide reduction and labeling with BODIPY FL C1-IA. NaOCl-sensitive proteins with increased redox-ratios in NaOCl-treated cells are red-labeled and the identified S-mycothiolated proteins are marked with (*). The oxidized proteins were identified by MALDI-TOF-TOF MS/MS and are listed with their ratios of reversible thiol-oxidations versus protein amounts ratios in Supplementary Table S4. IAM, iodoacetamide; LC, liquid chromatography. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 3.</b>
FIG. 3.
Close-ups of NaOCl-sensitive proteins identified in the thiol-redox proteomes of C. glutamicum wild type (WT) and the mshC mutant (A) and monitoring protein S-mycothiolation by MSH Western blot analysis (B). (A) Examples for reversibly oxidized proteins in the redox proteomes of the wild type (WT) and the mshC mutant are shown after NaOCl treatment. Among these NaOCl-sensitive proteins, 16 proteins are labelled with (*) that have been identified as S-mycothiolated in the wild type using LC-MS/MS analysis (Table 1 and Supplementary Table S5, Supplementary Fig. S5). Overlay images represent the redox proteome (red) compared to the Coomassie-stained protein amount image (green) at control conditions (co) and 10 and 30 min after NaOCl exposure (1–3). Column 4 represents the overlay of the redox proteomes of control cells (green) versus the redox proteome after 10 min of NaOCl stress (red). The identified proteins and their fluorescence/protein quantity ratios are listed in Supplementary Table S4 and the complete redox proteomes of the wild type and the mshC mutant are given in Supplementary Figures S3–S4. (B) Nonreducing MSH-specific Western blot analysis indicates widespread S-mycothiolation in C. glutamicum wild type. The IAM-alkylated protein extracts of the wild type, mshC and malP mutants were harvested before (co) and 15 min after 180 μM NaOCl stress and subjected to nonreducing MSH-specific Western blot analysis. LC, liquid chromatography; MSH, mycothiol. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 4.</b>
FIG. 4.
Metabolic pathways for glycolysis, biosynthesis of methionine, thiamine, GMP, MSH, and glycogen metabolism that include the identified S-mycothiolated metabolic enzymes. The enzymatic metabolic pathways were derived from the genome sequence of C. glutamicum or from previous publications (37) with the identified S-mycothiolated or reversibly oxidized proteins labeled with colors (S-mycothiolated proteins are dark gray; reversibly oxidized proteins are white-gray shadowed; both reversibly oxidized and S-mycothiolated proteins are light gray). The selected S-mycothiolated metabolic enzymes include MetE, SerA, Hom (Met biosynthesis); Fba, Pta (glycolysis); MalP (glycogen utilization); Ino-1 (MSH biosynthesis); ThiD1, ThiD2 (thiamine biosynthesis); GuaB1, GuaB2 (GMP biosynthesis). Further proteins with Cys-SSM sites are involved in translation (Tuf, PheT, RpsC, RpsF, RpsM, RplM) and antioxidant functions (Tpx, Gpx, MsrA) that are not shown here. Cys, cysteine; GMP, guanosine 5′-phosphate.
<b>FIG. 5.</b>
FIG. 5.
Mutants lacking MSH, σH and MalP are sensitive to NaOCl stress in growth assays. Growth phenotype assays of C. glutamicum wild type (WT) in comparison to the ΔmshC (A), ΔsigH (B) and ΔmalP (C) mutant strains that were treated with 180 μM NaOCl at an OD500 of 8 and growth was monitored. The survival assay of the ΔmalP mutant (D) after 180 μM NaOCl stress indicates a stronger killing effect of NaOCl in the mutant. OD500, optical density at 500 nm.
<b>FIG. 6.</b>
FIG. 6.
Effects of NaOCl stress on the uptake of [14C]-labelled glucose (A) and intracellular glycogen content in C. glutamicum wild type (B). (A) The glucose uptake rates were determined after NaOCl stress as in previous studies (34). (B) The intracellular glycogen content was measured in untreated C. glutamicum wild type cells (Δ) and in cells treated with NaOCl (▪) or NaCl (○) stress. The time point 0 indicates the time of NaOCl or NaCl addition. Glycogen contents and glucose uptake rates were determined in triplicates from 3 biological replicate experiments.
<b>FIG. 7.</b>
FIG. 7.
Purified Tpx is active as peroxidase, inactivated by S-mycothiolation and can be reactivated by Mrx1 in vitro. (A) Progress curves for the consumption of NADPH are shown. The electron transfer pathway of Tpx with Trx, TrxR and NADPH was reconstructed with 20 μM (□) or 100 μM (▴)H2O2 as substrate for Tpx. A control sample (○) without H2O2was included. (B) An identical sample but with Tpx omitted showed no peroxidase activity. (C)The electron transfer pathway was reconstructed for S-mycothiolated Tpx showing no peroxidase activity. (D–F) The Mrx1 electron transfer pathway composed of Mrx1, MSH, Mtr and NADPH was reconstructed and reduced (▿) or S-mycothiolated (▴) Tpx was added as substrate. A control sample without Tpx (○) was included. In (D) 50 μM of reduced (▿) or S-mycothiolated (▴) Tpx was used and in (E) 75 μM of reduced (▿) or S-mycothiolated (▴) Tpx was used. (F) Mrx1 was omitted in the above pathways using 50 μM reduced (▿) or S-mycothiolated (▴) Tpx. In the absence of Mrx1, no electron transfer was observed. (G, H) Mass spectrometry revealed complete reduction of Tpx-SSM by Mrx1. The Spectral counts for the S-mycothiolated (G) and NEM-alkylated (H) Cys60-, Cys81- and Cys94-peptides of Tpx are calculated using the Scaffold software after different times of Mrx1 reduction (see Supplementary Tables S6A–C). H2O2, hydrogen peroxide; Mrx1, mycoredoxin1; NEM, N-ethylmaleimide; Trx, thioredoxin; TrxR, thioredoxin reductase.
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