In their natural habitat or during infections, bacteria are frequently exposed to reactive oxygen species (ROS) and reactive chloride species (RCS), which cause an oxidative stress response and the induction of antioxidant defense mechanisms. ROS and HOCl can damage all macromolecules of the cell, including proteins, nucleic acids and lipids. To cope with ROS and to restore the reduced state of the cytoplasm, bacteria produce low molecular weight (LMW) thiols as important antioxidants and scavengers of ROS. Gram-negative bacteria and eukaryotes utilize glutathione as major LMW thiol. However, Gram-positive firmicutes and actinomycetes do not encode the enzymes for GSH biosynthesis and instead produce alternative LMW thiol, such as bacillithiol (BSH) and mycothiol (MSH), respectively. The various functions of the LMW thiol BSH in Bacillus subtilis and Staphylococcus aureus are summarized in chapter 1. Under oxidative stress, the LMW thiols GSH, BSH and MSH were shown to form post-translational modifications with protein thiols, termed as protein S-glutathionylations, S-bacillithiolations and S-mycothiolations, respectively. Protein S-thiolations protect protein thiols from irreversible overoxidation to Cys sulfonic acids and function in redox regulation of proteins. In S. aureus, about 57 proteins were previously found S-bacillithiolated under HOCl stress including GapDH as major target. The reduction of S-bacillithiolated proteins is catalyzed by bacilliredoxins (Brx) which are regenerated by BSH and the NADPH-dependent BSSB reductase (YpdA) in the Brx/BSH/YpdA redox pathway as described in chapter 2. Using NADPH-coupled electron transfer assays I showed that YpdA acts as BSSB reductase which depends on the redox-active Cys14. I further revealed that the Brx/BSH/YpdA pathway can catalyze de-bacillithiolation of S-bacillithiolated GapDH in vitro. Interestingly, YpdA was shown to be involved in detoxification of S-thioallylated BSH, termed as allylmercaptobacillithiol (BSSA), under allicin stress which is presented in chapter 3. BrxA catalyzed reduction of S-thioallylated GapDH to regenerate in part GapDH activity. Thus, YpdA and Brx function to restore the pool of reduced LMW thiols and protein thiols in S. aureus under allicin stress. In eukaryotes, glutaredoxins have been fused to redox-sensitive GFP2 (Grx-roGFP2) to measure dynamic changes in the GSH redox potential at high spatio-temporal resolution. In actinomycetes, related mycoredoxins have been used to construct Mrx1-roGFP2 biosensors for measurements of the MSH redox potential in Mycobacterium tuberculosis (Mtb), revealing heterogeneity of the MSH redox potential (EMSH) during macrophage infections and in antibiotics resistant Mtb isolates. An overview of redox biosensor applications in pathogenic bacteria under oxidative stress and infections is presented in chapter 4. Most of these redox biosensors are expressed ectopically on plasmids, resulting in different expression levels of roGFP2 fusions. The first main goal of this PhD thesis was to construct a stable integrated Mrx1-roGFP2 biosensor for quantification of EMSH changes in Corynebacterium glutamicum, which is described in chapter 5. The Mrx1-roGFP2 biosensor was integrated in the genomes of C. glutamicum wild type and mutants lacking redox regulators and antioxidant enzymes to measure EMSH changes during the growth and under oxidative stress. Biosensor measurements revealed that C. glutamicum wild type cells maintain a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of -296 mV. Due to its H2O2 resistant phenotype, Mrx1-roGFP2 responds weakly to 20-40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response of Mrx1-roGFP2 in mshA, mtr, sigH, oxyR, mpx, tpx and katA mutants which are compromised in redox-signaling and the antioxidant defense. While the probe was constitutively oxidized in the mshA and mtr mutants, a small oxidative shift in basal EMSH was observed in the ∆sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification system 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. Further live imaging experiments using confocal laser scanning microscopy documented the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stable integrated Mrx1-roGFP2 biosensor was successfully applied as novel redox tool to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutant backgrounds revealing major roles of MSH, SigH and KatA for intracellular EMSH. We were further interested to identify novel thiol-based redox regulators that sense HOCl via thiol-oxidation in actinomycetes and confer protection under oxidative stress. Previous redox proteomics studies identified the novel MarR-type regulator MSMEG_4471 (HypS) as highly oxidized under HOCl stress. As second main goal of this PhD thesis, I have characterized the function and redox-regulatory mechanism of HypS in Mycobacterium smegmatis which is described in chapter 6. 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 HypR regulon confers resistance towards HOCl, rifamipicin and erythromycin stress. Thus, HypS was identified as a novel redox-sensitive repressor that contributes to mycobacterial resistance towards HOCl stress and antibiotics. In summary, the results of my PhD thesis contributed to a deeper understanding of the impact of redox regulators and antioxidant enzymes towards MSH homeostasis under basal growth conditions and oxidative stress in actinomycetes. I further characterized a novel thiol-based redox regulator that confers resistance to HOCl and antibiotics and could be a future drug target to fight life-threatening tuberculosis infections.
