Figures and data
Redox activation of Spx is critical for diamide-induced disulfide stress resistance.
A. Characteristic features of the S. aureus Spx protein B. Growth curve of S. aureus (WT) and spx redox switch mutants. C. and E. Dose-dependent impact of diamide on growth of S. aureus. The relative growth (y-axis) represents the ratio of the area under the growth curve (AUC) for cultures treated with increasing diamide concentrations to that of the untreated control ((−) diamide). D. Spx turnover was assessed by western blot analysis using anti-Spx antibodies. Protein translation was inhibited by 100 µg/mL chloramphenicol prior to determining protein turnover. For all experiments, n= 3. Data represents, mean ± SD. Error bars for some data points may not be visible due to small error values.
Complementation of spxC10A.
Dose-dependent impact of diamide on growth of various S. aureus strains. CspxC10A, spxC10A mutant complemented with pJC1111: spx at SaPI site, VspxC10A, spxC10A mutant with pJC1111 (vector control) at SaPI site; n= 3, Data represent mean ± SD.
Half-life of Spx.
The half-life of different Spx variants were determined from Spx decay intensities observed over 0-15 min for WT+D and spxC10A+D, and 0-4.5 min for WT and spxC10A. The Spx intensities derived from western blot analysis was normalized to the loading control (enolase). The half-life (t1/2) was calculated as the ratio of Log10(0.5) to the slope of the log-transformed protein decay intensity. +D, diamide.
The spxC10A mutant efficiently adapts to disulfide stress.
A. Volcano plot of differentially expressed genes (DEGs) in the spxC10A mutant relative to WT strain. B. DEGs in the spxC10A mutant relative to WT strain after 15 mins of diamide treatment (0.5 mM). +D, diamide treatment. C. Expression of thiol-disulfide exchange pathway genes in the spxC10A mutant following diamide treatment. D. Degree of oxidation (OxD) of bacillithiol over time following 1mM diamide challenge. OxD was assessed using Brx-roGFP2 biosensor as described in material and methods. n= 4, mean ± SD. For all experiments unless otherwise noted, n= 3.
KEGG pathway analysis of RNAseq dataset.
Differentially expressed pathways in the spxC10A mutant relative to the WT strain after 15 mins of 0.5 mM diamide treatment. Analysis was carried out using KOBAS 3.0.
L-Cys accumulation in S. aureus reduces disulfide stress but impairs growth.
A. Bubble plot of enriched regulons in the spxC10A mutant relative to WT strain after 15 mins of diamide treatment (0.5 mM). DEGsR, total number of differentially expressed genes in the regulon; TGDR, total number of genes designated as part of the regulon; +D, diamide treatment. B. Low molecular weight thiol content in cells was determined using the DTNB assay as described in Materials and Methods. gDCW, gram dry cell weight. n= 6, mean ± SD, Two-way ANOVA, Tukey’s post-test. C. Dose-dependent impact of selenocysteine on growth of different S. aureus strains challenged with 1 mM diamide. D. Degree of oxidation (OxD) of bacillithiol as a function of time following diamide treatment (1mM). Diamide (D) was treated at time 0. n= 4. E. Dose-dependent impact of diamide on growth of S. aureus. F., G. and H. Growth of S. aureus strains in chemically defined media (CDM) with different combinations of sulfur-containing amino acids. For all experiments unless otherwise noted, n= 3. Data represents, mean ± SD, ***P ≤ 0.001, ****P ≤ 0.0001.
Disulfide stress mediates Fe(II) starvation in S. aureus.
A. Volcano plot of DEGs following diamide-induced disulfide stress in WT strain. +D, diamide treatment. B. Bubble plot of enriched regulons in the WT strain following diamide treatment. DEGsR, total number of differentially expressed genes in the regulon; TGDR, total number of genes designated as part of the regulon. C. ICP-MS analysis of iron pools in S. aureus. DCW, dry cell weight. n= 9, mean ± SD, Tukey’s post-test. C. Dose-dependent impact of diamide on growth of S. aureus strains under D. anaerobic E. dipyridyl (0.31 mM) F. Fe(II) and Fe(III) and G. aerobic conditions. For all experiments unless otherwise noted, n= 3, Data represents, mean ± SD. **P ≤ 0.01, ****P ≤ 0.0001.
Volcano plot.
Differentially expressed genes in spxC10A mutant strain following diamide-induced disulfide stress. +D, diamide treatment.
L-Cys uptake in response to disulfide stress mediates Fe(II) starvation.
A. Schematic depicting timeline of streptonigrin treatment and growth assessment of S. aureus cultures after diamide exposure. Diamide was removed prior to streptonigrin treatment. The specific growth rates (µ.h-1) of B. WT, spxC10A and C. tcyA/P, tcyA/PspxC10A were determined from growth curves using the equation (lnN2 − lnN1)/(t2 − t1), where N2 and N1 are OD600 at times t2 and t1. Aconitase activity was measured from either, D. crude cell extracts of cultures grown with or without 1 mM diamide or, E. from samples grown in media supplemented with 4 mM FeSO4. F. Neutrophil killing assay. Opsonized S. aureus was incubated with neutrophils at an MOI of 1 for 4 h, and bacterial viability was determined. Percent growth in the presence of neutrophils is quantified relative to the strain cultured in the absence of neutrophils (100% = no neutrophil killing, <100% = neutrophil killing, >100% = growth enhancement in the presence of neutrophils). n= 7, mean ± SEM, One-way ANOVA, Tukey’s post-test. For all other experiments, n= 3, mean ± SD, Two-way ANOVA, Tukey’s post-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
L-Cys-mediated Fe(II) starvation in the spxC10A mutant under disulfide stress occurs regardless of glucose in the medium.
Cultures were grown in TSB (-) glucose with increasing concentrations of diamide. The relative growth was determined from the fractional area under the growth curve of diamide treated cultures to that of untreated culture.
ROS levels in neutrophils.
Neutrophils were cultured with S. aureus (MOI = 10) for two hours. Neutrophils were stained with DHR123, approximately 15 minutes prior to fixation in 4% PFA. Neutrophils were blocked and stained with anti-CD15 and anti-CD16 antibodies and the mean fluorescence intensity (MFI) for DHR123 in the neutrophil population was quantified by flow cytometry. MFI was normalized to unstimulated neutrophils. n= 5, mean ± SEM, One-way ANOVA, Tukey’s post-test, ***P ≤ 0.001.
Model depicting regulation of L-Cys uptake by Spx.
Top panel: L-CySS uptake and biosynthesis are tightly regulated in S. aureus by the CymR regulator. Under diamide-induced disulfide stress, Spx is activated through its redox switch and turns on pathways that counter oxidative damage including genes involved in the thiol disulfide exchange system and bacillithiol biosynthesis. A basal level of L-CySS uptake also occurs depending on the level of CymR oxidation which is rapidly converted to L-Cys in the cytosol and contributes to the LMW thiols. Bottom panel: Inactivation of the Spx redox switch decreases the ability of S. aureus to counter disulfide stress through induction of the Spx regulon. However, under these conditions CymR repression is more strongly relieved due to excess oxidation resulting in increased L-CySS uptake and L-Cys biosynthesis. L-Cys accumulation helps normalize the intracellular redox state. However, excess L-Cys is predicted to chelate Fe(II) resulting in growth inhibition. Thus, S. aureus must finely balance Spx activity during disulfide stress to maintain both growth and redox homeostasis.
Strains and plasmids used in this study.
