Low molecular weight (LMW) thiols function as thiol-redox buffers to maintain the reduced state of the cytoplasm. The best studied LMW thiol is the tripeptide glutathione (GSH) present in all eukaryotes and Gram-negative bacteria. Firmicutes bacteria, including Bacillus and Staphylococcus species utilize the redox buffer bacillithiol (BSH) while Actinomycetes produce the related redox buffer mycothiol (MSH). In eukaryotes, proteins are post-translationally modified to S-glutathionylated proteins under conditions of oxidative stress. S-glutathionylation has emerged as major redox-regulatory mechanism in eukaryotes and protects active site cysteine residues against overoxidation to sulfonic acids. First studies identified S-glutathionylated proteins also in Gram-negative bacteria. Advances in mass spectrometry have further facilitated the identification of protein S-bacillithiolations and S-mycothiolation as BSH- and MSH-mixed protein disulfides formed under oxidative stress in Firmicutes and Actinomycetes, respectively. In Bacillus subtilis, protein S-bacillithiolation controls the activities of the redox-sensing OhrR repressor and the methionine synthase MetE in vivo. In Corynebacterium glutamicum, protein S-mycothiolation was more widespread and affected the functions of the maltodextrin phosphorylase MalP and thiol peroxidase (Tpx). In addition, novel bacilliredoxins (Brx) and mycoredoxins (Mrx1) were shown to function similar to glutaredoxins in the reduction of BSH- and MSH-mixed protein disulfides. Here we review the current knowledge about the functions of the bacterial thiol-redox buffers glutathione, bacillithiol, and mycothiol and the role of protein S-thiolation in redox regulation and thiol protection in model and pathogenic bacteria.
Aims: Bacillithiol (BSH) is the major low-molecular-weight thiol of the human pathogen Staphylococcus aureus. In this study, 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.Results: The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in >10% increased oxidation of 58 Cys residues (25.4%) in the thiol redox proteome. Among the highly oxidized sodium hypochlorite (NaOCl)-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ, and PpaC). The glyceraldehyde-3-phosphate (G3P) dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by hydrogen peroxide (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.Conclusion and Innovation: Using OxICAT analyses, 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. Antioxid. Redox Signal. 28, 410–430.
Significance: Since the discovery and structural characterization of bacillithiol (BSH), the biochemical functions of BSH-biosynthesis enzymes (BshA/B/C) and BSH-dependent detoxification enzymes (FosB, Bst, GlxA/B) have been explored in Bacillus and Staphylococcus species. It was shown that BSH plays an important role in detoxification of reactive oxygen and electrophilic species, alkylating agents, toxins, and antibiotics.Recent Advances: More recently, new functions of BSH were discovered in metal homeostasis (Zn buffering, Fe-sulfur cluster, and copper homeostasis) and virulence control in Staphylococcus aureus. Unexpectedly, strains of the S. aureus NCTC8325 lineage were identified as natural BSH-deficient mutants. Modern mass spectrometry-based approaches have revealed the global reach of protein S-bacillithiolation in Firmicutes as an important regulatory redox modification under hypochlorite stress. S-bacillithiolation of OhrR, MetE, and glyceraldehyde-3-phosphate dehydrogenase (Gap) functions, analogous to S-glutathionylation, as both a redox-regulatory device and in thiol protection under oxidative stress.Critical Issues: Although the functions of the bacilliredoxin (Brx) pathways in the reversal of S-bacillithiolations have been recently addressed, significantly more work is needed to establish the complete Brx reduction pathway, including the major enzyme(s), for reduction of oxidized BSH (BSSB) and the targets of Brx action in vivo.Future Directions: Despite the large number of identified S-bacillithiolated proteins, the physiological relevance of this redox modification was shown for only selected targets and should be a subject of future studies. In addition, many more BSH-dependent detoxification enzymes are evident from previous studies, although their roles and biochemical mechanisms require further study. This review of BSH research also pin-points these missing gaps for future research. Antioxid. Redox Signal. 28, 445–462.
Aims: Bacillithiol (BSH) is utilized as a major thiol-redox buffer in the human pathogen Staphylococcus aureus. Under oxidative stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolation, which can be reversed by bacilliredoxins (Brx). In eukaryotes, glutaredoxin-fused roGFP2 biosensors have been applied for dynamic live imaging of the glutathione redox potential. Here, we have constructed a genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus.Results: The Brx-roGFP2 biosensor showed a specific and rapid response to low levels of bacillithiol disulfide (BSSB) in vitro that required the active-site Cys of Brx. Dynamic live imaging in two methicillin-resistant S. aureus (MRSA) USA300 and COL strains revealed fast and dynamic responses of the Brx-roGFP2 biosensor under hypochlorite and hydrogen peroxide (H2O2) stress and constitutive oxidation of the probe in different BSH-deficient mutants. Furthermore, we found that the Brx-roGFP2 expression level and the dynamic range are higher in S. aureus COL compared with the USA300 strain. In phagocytosis assays with THP-1 macrophages, the biosensor was 87% oxidized in S. aureus COL. However, no changes in the BSH redox potential were measured after treatment with different antibiotics classes, indicating that antibiotics do not cause oxidative stress in S. aureus.Conclusion and Innovation: This Brx-roGFP2 biosensor catalyzes specific equilibration between the BSH and roGFP2 redox couples and can be applied for dynamic live imaging of redox changes in S. aureus and other BSH-producing Firmicutes. Antioxid. Redox Signal. 26, 835–848.
Multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) pose an increasing health burden and demand alternative antimicrobials to treat bacterial infections. The surface coating AGXX® is a novel broad-spectrum antimicrobial composed of two transition metals, silver and ruthenium that can be electroplated on various surfaces, such as medical devices and implants. AGXX® has been shown to kill nosocomial and waterborne pathogens by production of reactive oxygen species (ROS), but the effect of AGXX® on the bacterial redox balance has not been demonstrated. Since treatment options for MRSA infections are limited, ROS-producing agents are attractive alternatives to combat multi-resistant strains. In this work, we used RNA-seq transcriptomics, redox biosensor measurements and phenotype analyses to study the mode of action of AGXX® microparticles in S. aureus USA300. Using growth and survival assays, the growth-inhibitory amount of AGXX® microparticles was determined as 5 μg/ml. In the RNA-seq transcriptome, AGXX® caused a strong thiol-specific oxidative stress response and protein damage as revealed by the induction of the PerR, HypR, QsrR, MhqR, CstR, CtsR, and HrcA regulons. The derepression of the Fur, Zur, and CsoR regulons indicates that AGXX® also interferes with the metal ion homeostasis inducing Fe2+- and Zn2+-starvation responses as well as export systems for toxic Ag+ ions. The induction of the SigB and GraRS regulons reveals also cell wall and general stress responses. AGXX® stress was further shown to cause protein S-bacillithiolation, protein aggregation and an oxidative shift in the bacillithiol (BSH) redox potential. In phenotype assays, BSH and the HypR-controlled disulfide reductase MerA were required for protection against ROS produced under AGXX® stress in S. aureus. Altogether, our study revealed a strong thiol-reactive mode of action of AGXX® in S. aureus USA300 resulting in an increased BSH redox potential and protein S-bacillithiolation.
Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.
Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its 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. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress.
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