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. 2018 Feb 20;28(6):410-430.
doi: 10.1089/ars.2016.6897. Epub 2017 Jan 18.

Protein S-Bacillithiolation Functions in Thiol Protection and Redox Regulation of the Glyceraldehyde-3-Phosphate Dehydrogenase Gap in Staphylococcus aureus Under Hypochlorite Stress

Marcel Imber  1 , Nguyen Thi Thu Huyen  1 , Agnieszka J Pietrzyk-Brzezinska  2 , Vu Van Loi  1 , Melanie Hillion  1 , Jörg Bernhardt  3 , Lena Thärichen  4   5 , Katra Kolšek  5 , Malek Saleh  1 , Chris J Hamilton  6 , Lorenz Adrian  7 , Frauke Gräter  4   5 , Markus C Wahl  2 , Haike Antelmann  1
Affiliations

Protein S-Bacillithiolation Functions in Thiol Protection and Redox Regulation of the Glyceraldehyde-3-Phosphate Dehydrogenase Gap in Staphylococcus aureus Under Hypochlorite Stress

Marcel Imber et al. Antioxid Redox Signal. .

Abstract

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.

Keywords: Gap; S-bacillithiolation; Staphylococcus aureus; bacilliredoxin; thiol-redox proteomics.

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Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
Percentages of thiol oxidation for 228 Cys peptides that are identified in Staphylococcus aureus USA300 and visualized using Voronoi redox treemaps. The percentages of thiol oxidation of 228 Cys residues that are identified using OxICAT in S. aureus USA300 in the control (A) and 30 min after exposure to 150 μM NaOCl stress (B) are visualized using Voronoi redox treemaps. The gray–red color gradient denotes 0–100% oxidation. The Voronoi redox treemap in (C) shows the percentages of oxidation changes under NaOCl stress using a blue–red color gradient ranging from −75% to +75% oxidation. The treemap in (D) serves as the legend showing the functional classifications of proteins. The treemaps are generated using the Paver software (Decodon) based on the OxICAT data presented in Supplementary Tables S1 and proteins were classified according to the S. aureus USA300 TIGRfam annotation. NaOCl, sodium hypochlorite.
<b>FIG. 2.</b>
FIG. 2.
Close-ups of the redox treemaps of S. aureus USA300 showing S-bacillithiolated enzymes and redox regulators (SarZ, MgrA, and Fur). Enlarged sections of the redox treemaps are shown that include the identified S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ) and NaOCl-sensitive redox-sensing regulators (MgrA, SarZ, and Fur). The close-ups show the percentages of thiol oxidation under control, NaOCl stress, and the percentage of oxidation change under NaOCl stress versus control as revealed in Figure 1 using the same color gradient. The symbol * denotes conserved Cys.
<b>FIG. 3.</b>
FIG. 3.
Northern blot analysis showing transcriptional induction of the SarZ-regulated ohrA gene (USA300HOU_0835) under NaOCl stress. RNA was isolated from S. aureus USA300 grown in Belitsky minimal medium under control and NaOCl stress conditions and subjected to Northern blot analysis for ohrA (USA300HOU_0835) transcription. Transcription of ohrA is upregulated due to SarZ thiol oxidation and inactivation under NaOCl stress as revealed by OxICAT analysis in vivo.
<b>FIG. 4.</b>
FIG. 4.
OxICAT analysis revealed a 29% increased oxidation of the Gap Cys151 peptide (A) and Gap was identified as most abundant S-bacillithiolated protein in S. aureus under NaOCl stress as shown by BSH-specific Western blot analysis (B). (A) The OxICAT mass spectrometry results are shown for the Gap Cys151 peptide in S. aureus USA300 under control and 30 min after NaOCl stress. The reduced Gap Cys151 peptides in the cell extract are labeled with light 12C-ICAT, followed by reduction of all reversible thiol oxidation, including the S-bacillithiolated Cys151 peptides and subsequent labeling of previously oxidized Cys151 peptide by heavy 13C-ICAT reagent. According to the quantification by the MaxQuant software, the Cys151 peptide was 8.3% oxidized in the control and its oxidation level increased to 37.7% under NaOCl stress. (B) Nonreducing BSH-specific Western blot analysis identified Gap as most abundant S-bacillithiolated protein in S. aureus USA300 and COL strains under NaOCl stress. Two independent biological replicates are shown for S. aureus COL denoted as COL-1 and COL-2. Gap is S-bacillithiolated at the active site Cys151 under NaOCl stress as revealed by subsequent LC-MS/MS analysis (Supplementary Fig. S1A). BSH, bacillithiol; LC-MS/MS, liquid chromatography tandem mass spectry.
<b>FIG. 5.</b>
FIG. 5.
Voronoi treemaps visualize Gap as the most abundant Cys protein in the total Cys proteome of S. aureus USA300. The treemap legend (left) indicates the classification of the S. aureus USA300 proteome into functional categories according to TIGRfam annotations. The cell size corresponds to the spectral counts of each protein identified in the proteome of S. aureus USA300 and classified according to TIGRfam. The Cys-containing proteins are color coded using a yellow–red color gradient based on their numbers of Cys residues (Supplementary Table S2). Proteins without Cys residues are displayed in gray.
<b>FIG. 6.</b>
FIG. 6.
Inactivation of Gap of S. aureus in response to H2O2 in vitro. (A, B) Reduced Gap (40 μM) was oxidized with 100 μM, 1, and 10 mM H2O2 for 5 min in the absence (A) or presence of 10-molar excess of BSH (400 μM) (B) in reaction buffer (100 mM Tris HCl, 1.35 mM EDTA, pH 8.0). The remaining Gap activity was measured in the presence of G3P and NAD+ spectrophotometrically, following NADH production at 340 nm. The Gap activity was calculated as absorbance change from the slope of the reaction in the first 80 s, as described in the Materials and Methods section. (C) To assess the reversibility of Gap inactivation by H2O2, Gap was treated with 1 and 10 mM H2O2 alone or with H2O2 and BSH, followed by reduction with 10 mM DTT. (D) Schematic showing the irreversible inhibition of Gap activity due to overoxidation of the active site Cys with H2O2 alone, while Gap activity was reversibly inhibited with H2O2 and BSH due to S-bacillithiolation. (E) S-bacillithiolation of Gap in the presence of 10 mM H2O2 and BSH was confirmed using a BSH-specific Western blot analysis before and after subsequent DTT reduction. DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; G3P, glyceraldehyde-3-phosphate; H2O2, hydrogen peroxide.
<b>FIG. 7.</b>
FIG. 7.
Inactivation of Gap of S. aureus in response to NaOCl in vitro. (A, B) Reduced Gap was treated with 0.1–1 mM NaOCl for 5 min without (A) or with 10-molar excess of BSH (B) in reaction buffer (100 mM Tris HCl, 1.35 mM EDTA, pH 8.0). The remaining Gap activity was measured spectrophotometrically, following NADH production at 340 nm. The Gap activity was calculated as absorbance change from the slope of the reaction in the first 80 s, as described in the Materials and Methods section. (C) To analyze the reversibility of Gap inactivation by NaOCl, Gap was inactivated with 1 mM NaOCl in the absence or presence of BSH, followed by DTT reduction. Gap activity was irreversibly inhibited after treatment with NaOCl due to overoxidation since Gap activity could be not restored by DTT. In the presence of NaOCl and BSH, Gap was reversibly inactivated due to S-bacillithiolation since DTT reduction resulted in 85% recovery of Gap activity. (D) Schematic showing that NaOCl leads to the transient sulfenylchloride formation as unstable intermediate that reacts further with BSH to form S-bacillithiolated Gap. In the absence of BSH, Gap-SCl is quickly overoxidized resulting in irreversible inhibition of Gap activity in vitro.
<b>FIG. 8.</b>
FIG. 8.
Recycling of S-bacillithiolated Gap requires the bacilliredoxin Brx in vitro. (A) Gap activity is reversibly inhibited by S-bacillithiolation in vitro and can be restored by reduction using the bacilliredoxin Brx (SAUSA300_1321). Debacillithiolation required the Brx active site Cys. The BrxAGC mutant showed weak activity to reduce Gap-SSB, while the Brx resolving Cys mutant (BrxCGA) could restore Gap activity similar to the wild-type Brx protein. S-bacillithiolated Gap was generated in vitro by treatment of 25 μM Gap with 2.5 mM H2O2 in the presence of 250 μM BSH. For debacillithiolation, 2.5 μM Gap-SSB was incubated with 12.5 μM Brx, BrxAGC, and BrxAGC proteins for 30 min. Gap activity was measured after addition of G3P and NAD+ by spectrophotometric monitoring of NADH generation at 340 nm. (B) The level of debacillithiolation of Gap-SSB in vitro by Brx and BrxCys mutant proteins was monitored using nonreducing BSH-specific Western blot analysis. The SDS-PAGE is shown as loading control (right). The numbers 1–5 shown in the BSH Western blot and in the SDS-PAGE refer to the legend shown in (A). (C) Schematic for the reduction of S-bacillithiolated Gap using the bacilliredoxin Brx. SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
<b>FIG. 9.</b>
FIG. 9.
Structural insights into the Gap active site after overoxidation and S-bacillithiolation. (A) Crystal structure of the overoxidized active site Cys151 (Cys-SO3H, oC151) of Gap. (B, C) Computational model of BSH docked into the active site of the Gap apoenzyme (B) and holoenzyme with the NAD+ coenzyme (C) using a covalent docking algorithm that takes into account the possibility of bond formation between ligand and receptor. Shown is the best pose of 10 best poses of the S-bacillithiolated active site. (D) Superposition of Gap-SO3H with the S-bacillithiolated apo- and holoenzyme active sites. (E, F) The S-bacillithiolated active site pocket of the apoenzyme (E) and holoenzyme (F) structures rotated by 25° over y axis in respect to (B, C). (G, H) Surface representation of apoenzyme (G) and holoenzyme (H) with docked BSH.
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