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Protein S-mycothiolation and real-time redox imaging in Corynebacterium diphtheriae during ROS stress and infection conditions

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

Aim 1) In the ERC project “MYCOTHIOLOME” we investigated the physiological role of protein S-mycothiolations and other thiols-witches under oxidative stress in actinomycetes. Using shotgun LC-MS/MS analyses, 26 S-mycothiolated proteins were identified in C. diphtheriae under NaOCl stress, which is similar to the number of S-mycothiolated proteins described in C. glutamicum. Protein S-mycothiolation was more abundant in M. smegmatis due to the 20-fold higher MSH-content. In total, 58 S-mycothiolated proteins could be identified in M. smegmatis under NaOCl stress, which are involved in glycerol catabolism, glycolysis, the glyoxalate cycle, gluconeogenesis and biosyntheses of mycolic acids, fatty acids, amino acids, nucleotides, mycothiol, translation, antioxidant functions and transcriptional regulation. The redox proteomics approach OxICAT enabled the quantification of the redox state of 695 Cys in 441 proteins in C. diphtheriae under control and NaOCl stress. In total, 70% of protein thiols are in the reduced state with <25% oxidation in untreated cells. Under NaOCl stress, 492 Cys residues (71%) showed a 20-40 % increased oxidation, indicating that C. diphtheriae is very sensitive to thiol-oxidation under oxidative stress, probably due to its lower MSH content. The higher MSH levels in M. smegmatis resulted in a lower basal level oxidation and lower oxidation increase under NaOCl stress. Using OxICAT, the redox state for 1098 Cys was quantified in M. smegmatis. In total, 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation of 338 Cys residues (41.1%). Thus, there are significant differences in the level of thiol-oxidations and in the extents and targets of protein S-mycothiolation between low and high level MSH-producers as revealed by OxICAT and shotgun proteomics.
The identified NaOCl-sensitive proteins include 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 revealed by RNAseq. In conclusion, quantitative redox proteomics demonstrated that MSH is important to maintain the reduced state of protein thiols and functions in thiol-protection by protein S-mycothiolation in M. smegmatis. The highly oxidized conserved redox-switches could represent future drug-targets to combat live-threatening tuberculosis disease.

Aim 2) We designed a novel genome-encoded Mrx1-roGFP2 biosensor in C. glutamicum to measure the dynamic changes of the MSH redox potential (EMSH) during the growth, under oxidative stress and in different mutant backgrounds. The results showed that C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~-296 mV. Consistent with the H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants, which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Thus, the stably expressed Mrx1-roGFP2 biosensor was successful applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr, SigH, OxyR and KatA for the basal level EMSH and efficient H2O2 detoxification under oxidative stress.
Related genetically encoded biosensors were applied in S. aureus to detect BSH redox potential changes in the BSSB reductase deficient ypdA mutant and in response to redox-active antibiotics (e.g. AGXX®, allicin and lapachol). These biosensors can be applied to screen for ROS-producing antibiotics, which do not cause resistance mechanisms.

Aim 3) OxICAT analyses revealed metabolic enzymes that are conserved S-thiolated proteins in actinomycetes and firmicutes, such as the glyceraldehyde-3 phosphate dehydrogenase GapDH, which belongs to the most abundant S-mycothiolated and S-bacillithiolated proteins in C. diphtheriae and S. aureus. Therefore, we compared the kinetics of inactivation of GapDH by S-bacillithiolation and S-mycothiolation in both bacteria. Redox-regulation of S-mycothiolated GapDH in C. diphtheriae required the Mrx1 and Trx pathways, but the Mrx1 pathway was faster in de-mycothiolation compared to the Trx pathway. Furthermore, GapDH of S. aureus was faster inactivated by S-bacillithiolation compared to the overoxidation by H2O2 and NaOCl. These results revealed that S-bacillithiolation can efficiently protect the GapDH active site against overoxidation in vitro. In S. aureus, we further studied the regulation and function of the redox-sensitive aldehyde dehydrogenase AldA under NaOCl stress. AldA was identified as highly oxidized and S-bacillithiolated at its redox-sensitive active site Cys279 under NaOCl stress in S. aureus. Biochemical results showed that AldA has broad substrate specificity for oxidation of various aldehyde substrates. We hypothesize that AldA functions in detoxification of methylglyoxal that is elevated under NaOCl stress.

Further interesting thiol-switches are novel redox-sensing regulators that were identified using the OxICAT and RNAseq transcriptomics in M. smegmatis and S. aureus. Our OxICAT results in M. smegmatis under NaOCl stress revealed the 38% increased oxidation of Cys76 of the redox-sensitive anti-sigma factor RshA and 37% increased oxidation of the Cys67-Cys70-peptide of the anti-sigma factor RseA. These Cys residues are important for Zn binding and likely form an intramolecular disulfide under oxidative stress. Using RNA-Seq transcriptomics, we confirmed the strong up-regulation of the SigH and SigE regulons in M. smegmatis under NaOCl stress. This supports that RshA and RseA oxidation leads to up-regulation of their cognate SigH and SigE regulons.

In M. smegmatis, we have characterized the function and redox-sensing mechanisms of the MarR-type regulator HypS, which displayed 42% increased oxidation under NaOCl stress. RNA-seq transcriptomics and qRT-PCR analyses of the hypS mutant revealed that hypS is autoregulated and represses transcription of the co-transcribed hypO gene which encodes a multidrug efflux pump. DNA binding activity of HypS to the 8-5-8 bp inverted repeat sequence upstream of the hypSO operon was inhibited under NaOCl stress. However, the HypSC58S mutant protein was not impaired in DNA-binding under NaOCl stress in vitro, indicating an important role of Cys58 in redox sensing of NaOCl stress. HypS was shown to be inactivated by Cys58-Cys58' intersubunit disulfide formation under HOCl stress, resulting in derepression of hypO transcription. Phenotype results revealed that the HypS regulon confers resistance towards HOCl, rifampicin and erythromycin stress. Thus, HypS was identified as a redox-sensitive repressor that contributes to mycobacterial resistance towards HOCl stress and antibiotics. This study reveals a link between antibiotics and oxidative stress in M. smegmatis.

In addition to the MarR-type repressor HypS, we characterized the novel MarR-type regulator MhqR in S. aureus. MhqR senses quinones and quinone-like antimicrobials. MhqR was shown to negatively regulate the mhqRED operon which responds to MHQ, pyocyanin and ciprofloxacin. The MhqR repressor binds specifically to a 9-9bp inverted repeat (MhqR operator) upstream of the mhqRED operon and is inactivated by MHQ in vitro, which does not involve a thiol-based mechanism. In phenotypic assays, the mhqR deletion mutant was resistant to MHQ and quinone-like antimicrobial compounds. The mhqR mutant acquired an improved survival under lethal ROS stress and after long-term infections. These results provide a link between quinone and antimicrobial resistance via the MhqR regulon of S. aureus.