Activation of the Spx redox sensor counters cysteine-driven Fe(II) depletion under disulfide stress
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eLife Assessment
This important study provides new insights into how Staphylococcus aureus adapts to disulfide stress through the redox-sensitive regulator Spx, which coordinates nutrient uptake, cysteine import, redox homeostasis, and bacterial growth. While the authors present compelling evidence supporting the central role of Spx in managing disulfide stress, several aspects require further clarification. In particular, the precise mechanisms regulating cysteine uptake and the proposed link between disulfide stress responses and iron limitation would benefit from additional explanation and experimental or conceptual justification.
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Abstract
In many low G+C Gram-positive bacteria, the global regulator Spx helps maintain thiol homeostasis during disulfide stress, when protein thiols form aberrant disulfide bonds that can lead to misfolding and oxidative damage. Spx-dependent gene expression is triggered when an intramolecular disulfide bond forms between two cysteines in its redox switch. Surprisingly, some Spx functions persist even in the absence of an active redox switch, highlighting the need to better understand the physiological significance of maintaining this regulatory feature. Here, we utilize a spx C10A mutant that encodes a redox-insensitive Spx variant to study the role of the Spx redox switch in Staphylococcus aureus . We show that the spx C10A mutant is hypersensitive to diamide-induced disulfide stress and exhibits widespread transcriptional dysregulation of genes that contribute to thiol maintenance and disulfide repair. Remarkably, the spx C10A mutant rapidly adapts to disulfide stress by increasing its intracellular pool of L-cysteine (L-Cys) through enhanced uptake, which helps restore a reduced intracellular environment. However, during this process increased L-Cys inadvertently depletes cytosolic Fe(II), leading to growth inhibition of the spx C10A mutant. Finally, we show that the Spx-dependent control of intracellular L-Cys is critical for S. aureus survival when it encounters human neutrophils. Overall, these findings suggest that staphylococcal adaptation to disulfide stress through intracellular L-Cys accumulation imposes significant fitness costs that S. aureus overcomes by rapid regulatory control of thiol homeostasis through a functional Spx redox switch.
Significance
All cells have a pool of low molecular weight thiols, such as cysteine, glutathione, bacillithiol, and coenzyme A, to maintain redox balance under oxidative and disulfide stress. Among these, cysteine is a very effective thiol but is highly reactive, and its intracellular concentration must be tightly regulated. In S. aureus , we found that cysteine accumulates intracellularly during disulfide stress and if left unchecked, can inadvertently deplete cytosolic Fe(II), leading to growth inhibition. To prevent cysteine toxicity, S. aureus activates the global regulator Spx, which rapidly induces genes that restore thiol homeostasis and limits cysteine accumulation.
Article activity feed
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eLife Assessment
This important study provides new insights into how Staphylococcus aureus adapts to disulfide stress through the redox-sensitive regulator Spx, which coordinates nutrient uptake, cysteine import, redox homeostasis, and bacterial growth. While the authors present compelling evidence supporting the central role of Spx in managing disulfide stress, several aspects require further clarification. In particular, the precise mechanisms regulating cysteine uptake and the proposed link between disulfide stress responses and iron limitation would benefit from additional explanation and experimental or conceptual justification.
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Reviewer #1 (Public review):
Summary and Strengths:
This manuscript presents a thoughtful and well-executed analysis of how S. aureus adapts to disulfide stress using a redox-sensitive regulator, Spx, as a lynchpin to coordinate nutrient uptake, redox balance, and growth. The work is strengthened by a systematic and complementary experimental approach that combines genetic, biochemical, and physiological measurements. The authors carefully test alternative explanations and build a coherent model linking stress sensing to downstream metabolic consequences. Several results, particularly those connecting cysteine uptake to growth defects, provide convincing support for the proposed trade-off. Overall, the authors largely achieve their aims, and the evidence generally supports the central conclusions. The conceptual framework and …
Reviewer #1 (Public review):
Summary and Strengths:
This manuscript presents a thoughtful and well-executed analysis of how S. aureus adapts to disulfide stress using a redox-sensitive regulator, Spx, as a lynchpin to coordinate nutrient uptake, redox balance, and growth. The work is strengthened by a systematic and complementary experimental approach that combines genetic, biochemical, and physiological measurements. The authors carefully test alternative explanations and build a coherent model linking stress sensing to downstream metabolic consequences. Several results, particularly those connecting cysteine uptake to growth defects, provide convincing support for the proposed trade-off. Overall, the authors largely achieve their aims, and the evidence generally supports the central conclusions. The conceptual framework and experimental approaches should be of broad interest to researchers studying S. aureus physiology and pathogenesis and to those studying bacterial stress responses and metabolic trade-offs.
Weaknesses:
Clarifying several interpretive points would further strengthen confidence in the proposed model. Some conclusions rely on data presentations or experimental designs that are not immediately clear to the reader. In particular, aspects of the protein stability analysis, global regulatory comparisons, and assays linking cysteine uptake to iron limitation would benefit from clearer justification and more precise interpretation. In addition, certain conclusions could be more carefully framed to reflect partial rather than complete rescue effects.
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Reviewer #2 (Public review):
Summary:
The manuscript titled "Activation of the Spx redox sensor counters cysteine-driven Fe(II) depletion under disulfide stress" by Hall and colleagues describes that an active redox switch is required for surviving under the diamide-induced disulfide stress. Furthermore, the SpxC10A mutant exhibits transcriptional dysregulation of genes involved in thiol maintenance and disulfide repair. The authors further demonstrate a role for Spx in regulating the uptake of L-cysteine, which otherwise leads to the chelation of intracellular iron and thus the repression of growth.
Strengths:
The authors demonstrate that the SpxC10A mutant accumulates high levels of thiols, leading to the chelation of intracellular iron and subsequent repression of the SpxC10A mutant's growth.
Weaknesses:
The authors did not show a …
Reviewer #2 (Public review):
Summary:
The manuscript titled "Activation of the Spx redox sensor counters cysteine-driven Fe(II) depletion under disulfide stress" by Hall and colleagues describes that an active redox switch is required for surviving under the diamide-induced disulfide stress. Furthermore, the SpxC10A mutant exhibits transcriptional dysregulation of genes involved in thiol maintenance and disulfide repair. The authors further demonstrate a role for Spx in regulating the uptake of L-cysteine, which otherwise leads to the chelation of intracellular iron and thus the repression of growth.
Strengths:
The authors demonstrate that the SpxC10A mutant accumulates high levels of thiols, leading to the chelation of intracellular iron and subsequent repression of the SpxC10A mutant's growth.
Weaknesses:
The authors did not show a direct regulation of L-cysteine uptake through CymR.
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Reviewer #3 (Public review):
Summary:
The paper from Hall et al. reports the effects of an altered function spx allele on the physiology of S. aureus. Since Spx is essential in this organism, the authors compare WT with a spx C10A allele that retains Spx functions that are independent of the formation of a C10-C13 disulfide. However, the major role of Spx in maintaining disulfide homeostasis in this organism appears to be reduced by this mutation, including a reduction (relative to WT) in the DIA-induction of thioredoxin, thioredoxin reductase, and BSH biosynthesis and reduction enzymes.
Strengths:
Based on a wide range of studies, the authors develop a model in which Spx is required for adaptation to disulfide stress, and this adaptation involves (in part) induction of both cystine/Cys uptake and the Fur regulon. Overall, the results …
Reviewer #3 (Public review):
Summary:
The paper from Hall et al. reports the effects of an altered function spx allele on the physiology of S. aureus. Since Spx is essential in this organism, the authors compare WT with a spx C10A allele that retains Spx functions that are independent of the formation of a C10-C13 disulfide. However, the major role of Spx in maintaining disulfide homeostasis in this organism appears to be reduced by this mutation, including a reduction (relative to WT) in the DIA-induction of thioredoxin, thioredoxin reductase, and BSH biosynthesis and reduction enzymes.
Strengths:
Based on a wide range of studies, the authors develop a model in which Spx is required for adaptation to disulfide stress, and this adaptation involves (in part) induction of both cystine/Cys uptake and the Fur regulon. Overall, the results are compelling, but further efforts to clarify the presentation will aid readers in being able to follow this very complicated story.
Weaknesses:
(1) More details are needed on how relative growth is defined and calculated (e.g., line 145 and Figure 1C). The raw data (growth curves) should be included when reporting relative growth so that readers can see what changed (lag, growth rate, final OD?). Later in the paper, the authors refer to "the diamide-induced growth delay of the spxC10A mutant" (line 379), but this is not apparent from the presented data.
(2) Are the spx C10A, spx C13A, and spx C10A,C13A all really equivalent? In all cases, the Spx protein is presumably made (as confirmed for C10A in panel 1D). However, the only evidence to suggest that they are equivalent is the similar growth effects in panel 1C, and (as noted above), this data presentation can mask differences in how the mutations affect protein activity.
(3) Figure 1D and Figure 1 Supplement 2 report results related to the effect of diamide treatment on protein half-life (t1/2). Only single results are shown for both panels, and the conclusions do not seem to be statistically robust. For example, in Figure 1, Supplement 2 concludes that Spx C10A has a t1/2 is 3.38 min (this should be labeled correctly in the Figure legend as the red line). and WT Spx is 8.69 min. However, Figure 1D suggests that the protein levels at time 0 may not be equivalent, and this is lost in the data processing. Indeed, there are significant differences in Spx levels between time 0 - and + DIA, which is curious. Further, the authors' conclusion relies very heavily on line-fitting that includes a final point that has very low signal intensity (as judged from Figure 1D) and therefore is likely the least reliable of all the data. It might be worth showing curve fitting for multiple gels. Regardless of the overfitting of the data, the general conclusion that Spx is partially stabilized against proteolysis by ClpXP, and that the C10A mutant is reduced in stabilization, is probably correct.
(4) Figure 2 concludes that despite differences in the mRNA profiles between WT and spx C10A after 15 min. of DIA treatment, the overall level of responsiveness of the bacillithiol pool is unchanged. The authors find it "surprising" that the BSH pool responds normally despite some differences in gene expression. This is not surprising. The major events visualized in panel 2D are the chemical oxidation of BSH to BSSB and, presumably, the re-reduction by Bdr(YpdA). While it is seen that BSH synthesis (bshC) and ypdA expression may be less induced by DIA in the C10A mutant (2C), there is no evidence that the basal levels are different prior to stress. Therefore, the chemical oxidation and enzymatic re-reduction might be expected to occur at similar rates, as observed.
(5) Line 215. For the reason stated above, there is no reason to invoke Cys uptake as needed for the reduction of BSSB. Further, since CySS (presumably an abbreviation for cystine) is imported, this itself can contribute to disulfide stress.
(6) Line 235. Following on the above point, "diamide-induced disulfide stress increased L-CySS uptake in the spxC10A mutant to re-establish the BSH redox equilibrium." This is counterintuitive since LCySS is itself a disulfide and is thought to be reduced to 2 L-Cys in cells by BSH (leading to an increase in BSSB, not a reduction). Is there a known cystine reductase? Could cystine or L-cys be affecting gene regulation? (e.g., through CymR or Spx or ?). Cystine can also lead to mixed disulfide formation (e.g., could it modify Spx on C13?).
(7) l. 247 "a functional Spx redox switch allows S. aureus to avoid this trade-off and maintain thiol homeostasis without excessive L-CySS uptake." Can the authors expand on how this is thought to work? Does Spx normally affect cystine uptake? I thought this was CymR? I am not following the logic here.
(8) Line 258. "The fur mutant, which is known to accumulate iron...". My understanding is that fur mutant strains typically have higher bioavailable (free) Fe pools. This is seen in E. coli, for example, using EPR methods. However, they also often have lower total Fe due to the iron-sparing response, which represses the expression of abundant, Fe-rich proteins. Please provide a reference that supports this statement that in S. aureus fur mutants have higher total iron per cell.
(9) Figure 4. For the reasons stated above (point 1), it is hard to interpret data presented only as "Rel. Growth". Perhaps growth curve data could be included in a supplement.
(10) The interpretation of Figure 4 is complicated. It is not clear that there is necessarily a change in bioavailable Fe pools, although it does seem clear that Fe homeostasis is perturbed. It has been shown that one strong effect of DIA on B. subtilis physiology is to oxidize the BSH pool to BSSB (as shown also here), and this leads to a mobilization of Zn (buffered by BSH). Elevated Zn pools can inactivate some Fe(II)-dependent enzymes, which could account for the rescue by Fe(II) supplementation. Zn(II) can also dysregulate PerR and likely Fur regulons.
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