Community Research and Development Information Service - CORDIS



Project ID: 615585
Funded under: FP7-IDEAS-ERC
Country: Germany

Mid-Term Report Summary - MYCOTHIOLOME (Protein S-mycothiolation and real-time redox imaging in Corynebacterium diphtheriae during ROS stress and infection conditions)

Aim 1 : Publication 1 (In Revision in “Antioxidants & Redox signaling“):
Title: Monitoring global protein thiol-oxidation and S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress using “Voronoi redox treemaps”.

Mycothiol (MSH) is the major thiol-redox buffer in Actinomycetes and protects proteins by S-mycothiolation. We used shotgun proteomics, OxICAT and “Voronoi redox treemaps” to monitor protein S-mycothiolation and global thiol-oxidation levels in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation is accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues revealed that 857 Cys residues (78.1%) are <25% oxidized in control wild type cells indicating that most thiols are in their reduced state. Under NaOCl stress, 381 Cys residues (33.6%) showed >10% increased oxidation levels. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%) while only 21.9% Cys residues were >25% oxidized in the wild type. NaOCl stress resulted in a further oxidation increase in the mshC mutant with 255 Cys residues (30.9%) that showed >10% increased oxidations. Among the NaOCl-sensitive proteins are many Zn-redox switches, including the RseA and RshA anti-sigma factors and the Zur and NrdR repressors and their oxidation leads to an increased transcription of the SigH, SigE, Zur and NrdR regulons as determined by transcriptome sequencing. In conclusion, the combination of OxICAT, shotgun proteomics and redox treemaps demonstrates in a quantitative manner that MSH is important to maintain the reduced state of protein thiols and functions in thiol-protection by protein S-mycothiolation in M. smegmatis.

Aim 1 : Publication 2 (In preparation, To be submitted to “Scientific Reports“ in January 2017):
Title: “The glyceraldehyde 3-phosphate dehydrogenase GAPDH is redox-controlled by protein S-mycothiolation in Corynebacterium diphtheriae under oxidative stress”.

In C. diphtheriae, we quantified the level of thiol-oxidation for 695 Cys residues in 441 proteins under basal level and NaOCl stress conditions using the OxICAT approach. Under control condition, the majority of protein thiols (70%) are reduced and displayed <25% oxidation (481 Cys residues in 344 proteins). NaOCl stress resulted in a >20% increased oxidation of 492 Cys residues (71%), indicating that NaOCl stress strongly affects the thiol-redox balance of the majority of protein thiols in C. diphtheriae. Among the NaOCl-sensitive thiol-switches are known redox-sensitive antioxidant enzymes, such as the thiol peroxidase Tpx, the thioredoxin reductase TrxB, the AhpC homolog DirA and the mycothiol S-conjugate amidase Mca and many metabolic enzymes and transcription factors. This strong oxidation increase in C. diphtheriae might be related to the 20-fold lower MSH level compared to Mycobacteria which was revealed by thiol-metabolomics analysis. We further used shotgun proteomics to identify 26 S-mycothiolated proteins in C. diphtheriae under NaOCl stress. Among the S-mycothiolated proteins in C. diphtheriae, we discovered the glyceraldehyde dehydrogenase GapDH as main target for S-mycothiolation. The glycolytic Gap was observed as highly oxidized in our OxICAT approach and represents also the most abundant Cys protein in the proteome of C. diphtheriae. GapDH is S-mycothiolated at the active site Cys153 under hypochlorite stress that is known for its high reactivity towards H2O2 oxidation in many organisms. Treatment of Gap with increasing H2O2 and NaOCl concentrations in the presence of MSH resulted in S-mycothiolation and reversible inactivation of Gap in vitro. In the absence of MSH, high levels of H2O2 lead to irreversible inactivation of the Gap activity due to overoxidation of the active site. The S-mycothiolated GapDH was re-activated using both, the Trx and the Mrx1 pathway in vitro, while the overoxidized GapDH remained inactive. In summary, our results showed that S-mycothiolation of GapDH functions to protect the active site Cys against overoxidation and regulates its glycolytic activity.

Aim 1: Publication 3 (In press in Antioxidants & Redox signalling, PMID: 27967218 DOI: 10.1089/ars.2016.6897)
Title: "Protein S-bacillithiolation functions in thiol-protection and redox regulation of the glyceraldehyde-3-phosphate dehydrogenase Gap in Staphylococcus aureus under hypochlorite stress"

We used OxICAT and Voronoi redox treemaps to quantify hypochlorite-sensitive protein thiols in S. aureus USA300 and analyzed the role of BSH in protein S-bacillithiolation. The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in a >10% increased oxidation of 58 Cys residues (25.4 %) in the thiol-redox proteome. Among the highly oxidized NaOCl-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ and PpaC). The glyceraldehyde-3-phosphate dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing with 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by H2O2 or NaOCl in vitro. Treatment with H2O2 or NaOCl in the presence of BSH resulted in reversible Gap inactivation due to S-bacillithiolation, which could be regenerated by the bacilliredoxin Brx (SAUSA300_1321) in vitro. Molecular docking was used to model the S-bacillithiolated Gap active site suggesting that formation of the BSH mixed disulfide does not require major structural changes. In conclusion, we identified 58 novel NaOCl-sensitive proteins in the pathogen S. aureus that could play protective roles against the host immune defense and include the glycolytic Gap as major target for S-bacillithiolation. S-bacillithiolation of Gap did not require structural changes, but efficiently functions in redox regulation and protection of the active site against irreversible overoxidation in S. aureus.

Aim 3: Publication 4 (Work in progress, Publication submission scheduled for 2017)
Title: The role of the novel redox-sensing regulator MSMEG_4471 in the oxidative stress and antibiotics resistance of Mycobacteria.

The novel MarR-family transcriptional regulator MSMEG_4471 was identified as NaOCl sensitive thiol-switch in our OxICAT dataset and the single Cys58 showed very high oxidation increase of 42% under NaOCl stress. The gene for MSMEG_4471 is flanked by genes encoding an ABC transporters and multidrug-efflux pumps indicating that MSMEG_4471 could control these antibiotics resistance genes. The purified MSMEG_4471 protein was shown to bind to its own promoter region. The DNA-binding activity of MSMEG_4471 was reversibly inhibited by NaOCl treatment in vitro. DNA-binding assays using the MSMEG_4471C58S mutant further showed that Cys58 is essential for redox-sensing in vitro since DNA-binding is not affected by NaOCl stress. However, H2O2 stress did not inhibited the DNA-binding activity of MSMEG_4471 in vitro indicating that MSMEG_4471 is specific for NaOCl-redox sensing. Non-reducing SDS-PAGE analysis showed that MSMEG_4471 is oxidized to intermolecular disulphides under NaOCl stress. Phenotype analysis of the MSMEG_4471 deletion mutant are currently underway to analyse the function of the regulator in the oxidative stress and antibiotics resistance. First results revealed that the MSMEG_4471 deletion mutant is more resistant to NaOCl compared to the WT indicating that members of the MSMEG_4471 regulon confer specific protection against NaOCl. Transcriptional analysis using Northern blot analysis and RNA-seq are currently performed to identify the genes controlled by MSMEG_4471.

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