Redox regulation by reversible protein S-thiolation in bacteria

Abstract

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.

Keywords: bacillithiol; glutathione; mycothiol; oxidative stress; protein S-thiolation; thiol-redox buffer.

Figure 1

Generation of Reactive Oxygen Species (ROS) during respiration and HOCl production by activated neutrophils during infections. ROS are generated in bacteria during respiration by stepwise one-electron transfer to O₂ producing superoxide anion, hydrogen peroxide and hydroxyl radical. The highly reactive hydroxyl radical is also produced from H₂O₂ and Fe²⁺ in the Fenton reaction. During infections, activated neutrophils generate superoxide anion by the NADPH oxidase (NOX) that is converted to H₂O₂ by the superoxide dismutase (SOD). Myeloperoxidase (MPO) is released upon degranulocytosis producing the highly reactive hypochlorous acids (HOCl) from H₂O₂ and Cl⁻ as potent killing agent for pathogenic bacteria.

Figure 2

Reactive Electrophilic Species (RES) with partial positive charges (δ+) (A) and the reaction of quinones with thiols via the S-alkylation and oxidation chemistry (B). (A) In quinones and aldehydes the electrons are drawn to carbonyl oxygen leaving the partial positive charges at neighboring carbon atoms that become electrophilic. Diamide is an electrophilic azocompound that causes disulfide stress. (B) Quinones can react as electrophiles with the nucleophilic thiol group of Cys residues via thiol-S-alkylation leading to irreversible thiol-S-adduct formation. In the oxidative mode, quinones are incompletely reduced to semiquinone radicals that generate superoxide anions and can oxidize protein thiols to disulfides.

Figure 3

Thiol-chemistry of ROS and HOCl with thiol-containing proteins. The Cys thiol group is oxidized by ROS to an unstable Cys sulfenic acid intermediate (Cys-SOH) that reacts further with proximal thiols to form intramolecular and intermolecular disulfides or mixed disulfides with LMW thiols (RSH), such as glutathione, bacillithiol or cysteine, termed as S-thiolations. HOCl leads to chlorination of protein thiols to sulfenylchloride intermediates (Cys-SCl) that react further to form disulfides. In the absence of proximal thiols, the chlorinated Cys is overoxidized to Cys sulfinic and sulfonic acids. Disulfides function as redox switches to control protein activity and protect thiol groups against overoxidation to Cys sulfinic and sulfonic acids.

Figure 4

Structures of major bacterial low molecular weight (LMW) thiols. Major LMW thiols are glutathione (GSH) in eukaryotes and Gram-negative bacteria, mycothiol (MSH) in Actinomycetes and bacillithiol (BSH) in Firmicutes. Coenzyme A (CoASH) also serves as a LMW thiol-redox buffer in some bacteria, like in S. aureus and B. anthracis.

Figure 5

Redox proteomics methods to study protein S-glutathionylation at a global scale. Mass spectrometry-based methods for identification of S-glutathionylations include the glutaredoxin-coupled NEM-biotin switch assay (A), the biotin-Gsp assay, (B) or the N,N-biotinyl glutathione disulfide (BioGSSG) assay, (C) (Lind et al., ; Brennan et al., ; Kehr et al., ; Zaffagnini et al., 2012a). In the biotin-Gsp assay, E. coli GspS is expressed in mammalian cells and converts GSH and biotinyl-spermine (biotine-spm) to biotin-glutathionylspermidine (biotin-Gsp). Proteins in ROS-treated cells are modified by biotin-Gsp-S-thiolation (Chiang et al., ; Lin et al., 2015). The biotin-spm is removed from the enriched biotin-Gsp-S-thiolated peptides by GspA and the GSS-peptides are identified by mass spectrometry.

Figure 6

The functions of bacillithiol (BSH) in B. subtilis and S. aureus. Bacillithiol functions in detoxification of ROS, RES, HOCl, and antibiotics (fosfomycin, rifampicin) in B. subtilis and S. aureus. BSH is oxidized by ROS to bacillithiol disulfide (BSSB). Electrophiles (RX) are conjugated to BSH by the BSH S-transferase BstA to form BS-electrophiles (BSR) which are cleaved by the BSH S-conjugate amidase Bca to CysSR and mercapturic acids (AcCySR) that are exported from the cell. BSH serves as a cofactor for the epoxide hydrolase FosB which adds BSH to fosfomycin to open the ring structure for its detoxification. BSH functions in methylglyoxal detoxification as a cofactor for the glyoxalases I/II (GlxA and GlxB) in B. subtilis. GlxA converts BSH-hemithioacetal to S-lactoyl-BSH that is further converted by GlxB to D-lactate. BSH serves as Zn buffer under conditions of Zn excess in B. subtilis. In S. aureus, BSH is important under infection-related conditions and increased the survival of S. aureus in phagocytosis assays using murine macrophages. Under conditions of NaOCl stress, proteins are oxidized to mixed disulfides with BSH, termed as S-bacillithiolations which is reversed by bacilliredoxins.

Figure 7

Physiological roles of S-bacillithiolations in B. subtilis and other Firmicutes. NaOCl leads to S-bacillithiolation of OhrR, MetE, YxjG, PpaC, SerA, AroA, GuaB, YumC, TufA, and YphP in B. subtilis (Chi et al., 2011). S-bacillithiolation of OhrR inactivates the repressor and causes induction of the OhrA peroxiredoxin that confers NaOCl resistance. S-bacillithiolation of the methionine synthase MetE at its active site Cys730 and other enzymes of the Cys and Met biosynthesis pathway (YxjG, PpaC, SerA, MetI) leads to methionine auxotrophy (Chi et al., 2011, 2013). In addition, other amino acids biosynthesis enzymes, translation factors and ribosomal proteins are S-bacillithiolated in Firmicutes bacteria. Thus, we hypothesize that S-bacillithiolation leads to a transient translation stop during the time of NaOCl detoxification to prevent further protein damage. NaOCl stress causes oxidation of BSH to BSSB and a two-fold decreased BSH/BSSB redox ratio that possibly contributes to S-bacillithiolation. The reduction of MetE-SSB and OhrR-SSB is catalyzed by bacilliredoxins (BrxA/B) in B. subtilis.

Figure 8

Reduction of protein S-glutathionylations, S-bacillithiolations and S-mycothiolations by glutaredoxin, bacilliredoxin and mycoredoxin pathways. The S-glutathionylated proteins are reduced by glutaredoxins (Grx) leading to a Grx-SSG intermediate that is reduced by GSH leading to GSSG which is recycled back to GSH by the NADPH-dependent GSSG reductase (Gor). Analogous bacilliredoxin and mycoredoxin pathways have been characterized in BSH- and MSH-utilizing Gram-positive bacteria. The S-bacillithiolated proteins are reduced by bacilliredoxins (Brx) leading to Brx-SSB formation. Brx-SSB is reduced by BSH with the generation of BSSB that likely requires the NADPH-dependent BSSB reductase YpdA for regeneration of BSH. In Actinomycetes, mycoredoxin1 catalyzes reduction of S-mycothiolated proteins leading to Mrx1-SSM generation that is recycled by MSH and the NADPH-dependent MSSM reductase Mtr.

Figure 9

The functions of mycothiol (MSH) in Mycobacteria and Corynebacteria. Mycothiol (MSH) is oxidized by ROS to mycothiol disulfide (MSSM). MSSM is reduced back to MSH by the mycothiol disulfide reductase Mtr on expense of NADPH. MSH-dependent peroxidases, such as Mpx, Tpx, and AhpE function in peroxide detoxification. Electrophiles (RX) are conjugated to MSH by the MSH S-transferase Mst to form MS-electrophiles (MSR) which are cleaved by the MSH S-conjugate amidase Mca to mercapturic acids (AcCySR) that are exported from the cell. The Mca-homologs LmbT, LmbV, and LmbE function also in the assembly and biosynthesis of the sulfur-containing lincosamide antibiotic lincomycin in Streptomyces lincolnensis (Zhao et al., 2015). MSH serves as a cofactor for the alcohol dehydrogenase AdhE/MscR in Mycobacteria and Corynebacteria for detoxification of formaldehyde to formate and MSNO to MSO2H. MSH functions in detoxification of maleylpyruvate as a cofactor for maleylpyruvate isomerase in C. glutamicum. Arsenate reductases CgArsC1 and CgArsC2 conjugate MSH and arsenate As(V) to form As(V)-SM that is reduced to As(III) by Mrx1. In M. tuberculosis, MSH is important under infection conditions and for growth and survival. Under conditions of NaOCl stress, proteins are oxidized to mixed disulfides with MSH, termed as S-mycothiolations which is reversed by mycoredoxins.

Figure 10

Physiological roles of S-mycothiolations in Corynebacterium glutamicum. The metabolic pathways for glycolysis, biosynthesis of methionine, thiamine, GMP, MSH, and glycogen metabolism are shown including identified S-mycothiolated proteins. The identified S-mycothiolated or oxidized proteins are labeled with colors (S-mycothiolated proteins are red; reversibly oxidized proteins are magenta; both reversibly oxidized and S-mycothiolated are blue). 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, Bcp, MsrA) that are not shown here. The figure is adapted from (Chi et al., 2014).