The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus

Abstract

How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR [Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor] represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high-resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60' interprotomer cross-links, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR.

Figure 1. CstR forms a modest affinity complex with Cu(I) and metal binding does not negatively regulate cst DNA binding

(A) Representative Cu(I)-bicinchoninic acid competition assays with apo CstR. Binding curves were obtained under anaerobic conditions. Open symbols represent the A₅₆₂ of the Cu(I):BCA₂ complex and the solid lines represent the global fitting of three individual experiments to a single-site binding, direct competition model. The global Cu(I):CstR binding constant was calculated to 1.0±0.4×10¹⁴ M⁻¹. Green: 29.6 μM Cu(I), 70 μM BCA. Red: 18.9 μM Cu(I), 50 μM BCA. Blue: 10 μM Cu(I), 30 μM BCA. (B) Representative fluorescence anisotropy titration of Cu(I)-bound CstR to a fluorescently labeled cst OP1 DNA fragment. The macroscopic binding constant, K_tet, was determined to be 4.3±1.7×10⁷ M⁻¹. The red line is a simulated curve defined by the binding parameters for apo-reduced CstR (see Fig. 6) under the same solution conditions (K_tet,= 6.3±0.5×10⁷ M⁻¹).

Figure 2. CstR is required for S. aureus defense against sulfide stress

(A) Schematic representation of the cst operon, NWMN_0026-NWMN_0029, and operator binding sites OP1 and OP2 (Grossoehme et al., 2011). Arrows indicate promoter regions. (B–E) Growth curves of wild-type (WT) and ΔcstR S. aureus mutant strains transformed with the indicated CstR allele carried on pOS1 complementation vector under aerobic conditions at 37 °C with shaking in the absence (open symbols) or presence (filled symbols) of 0.2 mM NaHS. (B) WT (circles), ΔcstR:pOS1 (triangles), ΔcstR:CstR (diamonds), and ΔcstR:CstR^C31A/C60A (squares) S. aureus grown in HHWm minimal media supplemented with 0.5 mM thiosulfate (HHWm+TS). (C) WT and ΔcstR:CstR^C31A/C60A S. aureus strains grown in HHWm supplemented with 0.25 mM cystine. (D) WT and ΔcstR:CstR^C31A/C60A grown on rich TSB media. (E) Growth of ΔcstR:CstR^C31A (circles) and ΔcstR:CstR^C60A (squares) in HHWm+TS. (F) WT and ΔcstR:CstR^C31A/C60A S. aureus grown in the absence (open symbols) or presence (filled symbols) of 25 μM sodium tetrasulfide (Na₂S₄). (G) WT and ΔcstR:CstR^C31A/C60A S. aureus grown in the absence (open symbols) or presence (filled symbols) of 0.2 mM sodium sulfide (Na₂S). The data points represent a single representative growth curve.

Figure 3. cst genes are required for cellular sulfide resistance

Representative growth curves of individual cst operon gene deletion strains (circles) and corresponding complementation strain (squares) grown in the absence (open symbols) or presence of 0.2 mM NaHS (closed symbols) in HHWm+TS. (A) ΔtauE (no complementation strain shown) (B) Δ3cstA and ΔcstA:CstA. (C) ΔcstB and ΔcstB:CstB. (D) Δsqr and Δsqr:SQR.

Figure 4. The cst operon is regulated by hydrogen sulfide in vivo

Quantitative RT-PCR experiments for WT and mutant S. aureus cultures grown to an OD₆₀₀ of 0.2 and challenged with 0.2 mM NaHS added to the growth medium at t=0. Aliquots for analysis were collected at 10 and 30 min post addition. All cultures were grown in HHWm+TS unless otherwise noted. (A) Relative expression levels for individual cst operon genes post addition of NaHS. (B) Levels of tauE and cstA expression in TSB (left) or HHWm+Cys (right). (C) ΔcstR (left), ΔcstR:CstR (middle), and ΔcstR:CstR^C31A/C60A (right). (D) ΔcstR:CstR^C31A (left) and ΔcstR:CstR^C60A (right) individual cysteine mutants of CstR. (E) RT-PCR analysis for WT S. aureus exposed to acute toxicity of 2.4 mM hypochlorite (ClO⁻), 10 mM sulfite (SO₃²⁻), 0.2 mM selenite (SeO₃²⁻), 0.5 mM nitric oxide (NO) as MAHMA NONOate, 5 mM nitrite (NO₂⁻), 25 nM paraquat, 10 mM hydrogen peroxide (H₂O₂), or 1 mM diamide. (F) ΔcstR:CstR^C31A/C60A S. aureus exposed to 1 mM diamide stress. N = 3 error bars represent one s.d. from the mean, with fold-expression relative to wild-type, unstressed cells. Two-way ANOVA analysis was performed relative to 16S RNA at the indicated time point (*** = p < 0.001, ** = p < 0.005, * = p < 0.050, and n.s. = not statistically significant).

Figure 5. Extracellular sodium hydrogen sulfide (NaHS) enters the cytoplasm of S. aureus resulting in a concomitant increase in thiosulfate (TS) and decrease in cysteine

Cellular LMW sulfur metabolites were derivatized with mBBr were detected using a fluorescence-detected profiling method (Fahey & Newton, 1987) before (t=0) and after (t=10 min and t=30 min) the addition of 0.2 mM NaHS to the culture medium (see Fig. S6 for representative liquid chromatograms). (A) Sulfide; (B) thiosulfate; and (C) cysteine, each expressed in nmol mg⁻¹ protein.

Figure 6. Reaction of CstR with polysulfide negatively regulates DNA operator binding

Fluorescence anisotropy titrations of fully reduced CstR (open circles) and NaHS- (closed squares) or Na₂S₄-reacted CstR (closed diamonds) with fluorescently-labeled cst OP1-containing DNA. Data were fit to a sequential tetramer binding model where two non-dissociable tetramers bind stepwise to one operator DNA binding site. Stepwise binding constants, K₁ and K₂, were used to determine the average macroscopic binding constant, K_tet (K_tet = (K₁•K₂)^½). WT CstR-OP1 affinity is 7.4 (±1.8)×10⁷ M⁻¹ (see Table S1 for all previously determined K_tet values) (Luebke et al., 2013, Grossoehme et al., 2011), while K_tet for NaHS- and Na₂S₄-reacted CstRs have upper limits of 0.06±0.05×10⁷ M⁻¹ and 0.03±0.03×10⁷ M⁻¹, respectively. Binding curves represent a single representative titration. Conditions: 10 nM cst OP1 DNA, 10 mM HEPES, 0.2 M NaCl, pH 7.5, 25 °C.

Figure 7. CstR reacts with NaHS, Na₂S₄, and GSSH to form a series of mixed di-, tri-, and tetrasulfide crosslinks

LC-ESI-MS spectra of intact reduced CstR (A), CstR following reaction with a 5-fold S:Cys molar excess of (B) Na₂S, (C) NaHS* (D) Na₂S₄, (E) NaHS, (F) GSH, (G) GSSG, and (H) GSSH. Black traces represent the ratio of reduced (left) or cross-linked (right) CstR in the deconvoluted mass spectra. Red traces represent m/z ratios of the +8 or +16 charge states of reduced or cross-linked dimeric CstR species, respectively, with corresponding post-translational modification assignments shown. RS-H indicates reduced CstR and RS-SR’ represents an interprotomer disulfide bond between Cys31 and Cys60’. Each ‘S’ represents a mass shift of +32 Da relative to the RS-SR’ disulfide in the deconvoluted spectra. For a sample like GSSG (panel G), the +8 and +16 m/z overlap but can be deconvoluted based on the m/z distribution of the reduced vs. oxidized forms (Luebke et al., 2013). For a summary of the observed masses, refer to Supplemental Table S2. (*) indicates a reaction performed with NaHS in 10 mM HEPES, 200 mM NaCl, pH 7.0. All other reactions were performed in 10 mM PO₄³⁻, 200 mM NaCl, 1 mM EDTA, pH 7.0.

Figure 8. High-resolution tandem mass spectrometry confirms di-, tri-, and tetrasulfide mass shift assignments

High-resolution LTQ-orbitrap tandem mass spectra of NaHS-reacted CstR tryptic peptides in the +4 charge state (left) and corresponding fragmentation patterns (right). Peptide A (red), ²⁴MMEEGKDCKVITQISASK⁴², includes Cys31 and Peptide B (blue), ^48′LMGIIISENLIECVK^62′, includes Cys60’. Peptide fragments were assigned relative to either peptide “A” or “B” where Bb₃ corresponds to the peptide fragment b ion ^48′LMG^50′ with a mass of 302 Da. Cross-linked peptides are denoted as “AB_yn” where peptide “A” remained intact and fragmentation occurred on peptide “B”. Inset: map of fragmentation pattern. (A) Disulfide. (B) Trisulfide. (C) Tetrasulfide.

Figure 9. An abbreviated rendering of sulfur assimilation and hydrogen sulfide metabolism in S. aureus strain Newman

The 4-number (wxyx) S. aureus strain Newman gene locus tags (NWMN_wxyz) are given for each enzyme that is annotated with a standard abbreviation based on recent work (Soutourina et al., 2009). OASS: O-acetyl-L-serine sulfhydrylase; SAT + OASS: cysteine synthase complex. The TS quinone-dependent dehydrogenase marked with an asterisk and shaded grey (NWMN_0676) is likely not functional given the absence of a gene encoding the small subunit. §, highlighted to implicate a reductive path to the generation of H₂S as a substrate for OASS from cellular protein-bound persulfides (Ida et al., 2014). The large yellow and red arrows illustrate the concept of sulfide homeostasis, which is dictated by the coordinate action of H₂S oxidation (by proteins encoded by the cst operon) and assimilatory pathways.