Author Response
The following is the authors’ response to the original reviews.
Reviewer #1 (recommendations for the authors):
The following are comments that the authors may wish to address or clarify:
(1) The claim that respiration and fermentation occur concurrently in the agr mutant during aerobic growth is not strongly supported by the evidence presented…. However, since neither lactate production nor a difference in the NAD+/NADH ratio between the wild type and agr mutant was observed, it is challenging to assert that fermentation is occurring. Relying solely on a gene expression signature indicative of fermentation is, in my view, inadequate to conclusively establish that aerobic fermentation is taking place.
Lactate production. The data we provide in Figure 5-E of the original manuscript (Figure 5-C in the revised manuscript) indicates that lactate production is lower in the wild-type compared to the Δagr mutant.
The exact focus of Reviewer 1’s concern is not clearly specified, but may have been referring to how the result was described in the text:
“Although the stimulatory effect of the agr deletion on production of the fermentation product lactate was not observed in optimally aerated broth cultures after growth to late exponential growth phase, it was confirmed for organisms grown in broth under more metabolically demanding, suboptimal aeration conditions (Figure 5E). Overall, these results are consistent with transcription-level up-regulation of respiratory and fermentative pathways in agr-deficient strains.”
The greater sensitivity of suboptimal aeration conditions is unsurprising and relates to a low rate of fermentation during the vigorous aeration (shaking at 250 rpm) conditions commonly used to grow S. aureus. To clarify the point, we modified the text to provide additional context as follows:
Line 271: “Although the stimulatory effect of the agr deletion on production of the fermentation product lactate was not observed in optimally aerated broth cultures after growth to late exponential growth phase, it was confirmed for organisms grown in broth under more metabolically demanding, suboptimal aeration conditions (limitations in the rate of respiration when oxygen is limiting are expected to increase overall levels of fermentation) (Figure 5C). Overall, these results are consistent with transcription-level up-regulation of respiratory and fermentative pathways in agr-deficient strains.” NAD+/NADH ratio. Extended studies of the NAD+/NADH ratio, requested by Reviewer 1 under Comments 12 and 13, document an effect of the Δagr mutant not seen in Figure 5F in the original submission. Our responses to Comments 12 and 13 below address this issue.
(2) The mechanisms through which the ΔagrΔrot double mutant resists H2O2 are not clearly elucidated. While the authors suggest that the ΔagrΔrot double mutant expresses several genes involved in combating oxidative stress, essential genetic studies that would validate this hypothesis have not been conducted.
The data we provide indicate 1) that wild-type strains are tolerant to peroxide and 2) that wild-type strains are able to render inducible several known reactive oxygen species (ROS)-protective genes in the presence of peroxide in a rot-dependent manner. Δagr strains, which do not demonstrate this response, are more readily killed by peroxide. Additional data indicate that increased respiration caused by deletion of agr is associated with increased endogenous ROS. Higher levels of endogenous ROS can modulate tolerance to subsequent challenge by ROS (1). Collectively, these observations support a model of Δagr-induced hyper-susceptibility in which elevation of endogenous ROS results in a suboptimal ROS-defense response that plays a role in increased peroxide lethality.
We prefer to test this model in future studies directed at understanding the complexities of the interaction among agr-mediated tolerance, endogenous ROS levels, and induction of protective responses in S. aureus. Culprit protective genes, alone and in various combinations, will be inactivated in Δagr mutant and wild-type strains, tested in killing assays with and without agents that mitigate endogenous ROS, and subjected to RNAseq, proteomic, and metabolomic analyses, as part of a larger program to identify factors involved in S. aureus tolerance to lethal stress.
To clarify the issue raised by the reviewer we altered the wording in the following sentences as follows:
Line 335: “Elevated expression of protective genes suggests that the double mutant survives damage from H2O2 better because protective genes are rendered inducible (loss of Rot-mediated repression).”
Line 440: “Details of agr-mediated protection are sketched in Figure 10. At low levels of
ROS, agr is activated by a redox sensor in AgrA, RNAIII is expressed and represses the Rot repressor, thereby rendering protective genes (e.g., clpB/C, dps) inducible via an unknown mechanism (induction, candidate protective gene(s), and their connection to endogenous ROS levels are being pursued, independent of the current report).
(3) The reason behind the agr mutant's low metabolic efficiency, as evidenced by low levels (Fig 5A) despite enhanced respiration and acetate production, is not clearly explained. Could insights from the modeling shed light on why the ATP levels are low in the agr mutant?
Comparative modeling of central metabolic pathways, in combination with in vitro metabolic analyses of Δagr and wild-type strains, revealed the metabolic inefficiency but cannot explain it. The basis for the metabolic inefficiency conferred by agr inactivation is unknown. The possibility that aberrant sorting of cell wall surface proteins could lead to metabolic inefficiency was raised in the Discussion where we wrote:
“Our work supports this idea by showing that increased respiration caused by deletion of agr is associated with increased ROS-mediated lethality. The basis for the metabolic inefficiency conferred by agr inactivation is unknown. Given that Δagr mutants are unable to downregulate surface proteins during stationary phase (2, 3), it is possible that deletion of agr perturbs the cytoplasmic membrane or the machinery that sorts proteins across the cell wall. In support of this notion, jamming SecY translocation machinery of E. coli results in downstream events shared with antibiotic lethality, including accelerated respiration and accumulation of ROS (4). In this scenario, the formation of a futile macromolecular cycle may accelerate cellular respiration to meet the metabolic demand of unresolvable problems caused by elevated surface sorting.”
For clarification, we modified the text as follows:
Line 461: “Our work supports this idea by showing that increased respiration caused by deletion of agr is associated with increased ROS-mediated lethality. How agr deficiency is connected to the corruption of downstream processes that result in metabolic inefficiency and increased endogenous ROS levels is unknown. Given that Δagr mutants are unable to downregulate surface proteins during stationary phase (2, 3), it is possible that deletion of agr perturbs the cytoplasmic membrane or the machinery that sorts proteins across the cell wall.”
agr has been linked to defects in peptidoglycan autolysis (5). Cho et al. (2019) found that β-lactam treatment can induce a futile cycle of peptidoglycan synthesis and degradation that has been linked to increased production of endogenous ROS (6). Thus, an alternative, nonmutually exclusive route to a futile cycle and elevated endogenous ROS levels in agr-deficient cells other than surface protein dysregulation may be via decreased cell wall cross-linking. We prefer not to include this and other speculations, because they are not necessary or revealing and because they would detract from the manuscript by disrupting its sense of narrative and brevity.
(4) The observation that menadione can protect the agr mutant from H2O2 is perplexing. The authors propose that even though menadione generates superoxide through redox cycling, this superoxide might inhibit the TCA cycle, thereby restricting respiration, which could be advantageous for the agr mutant. To substantiate this hypothesis, it would be imperative to demonstrate that a double mutant ΔagrΔacnA exhibits long-lived protection against H2O2.
Rowe et al. (2020) definitively showed that a burst of menadioneassociated ROS inactivates the TCA cycle in S. aureus, leading to reduced respiration and ATP production (7). Both aconitase activity and ATP levels in menadione-treated cultures were complemented by the antioxidant N-acetyl cysteine. In the present work we demonstrate, using the same experimental conditions as Rowe et al., that menadione protected the Δagr mutant from peroxide killing but had little effect on the wild-type strain. Addition of N-acetyl cysteine in the presence of menadione restored H2O2 susceptibility to the Δagr mutant and had no effect on the wild-type. Collectively, these observations support the idea that menadione inactivates the TCA cycle, leading to reduced respiration, and increased protection of the Δagr mutant from peroxide killing.
As requested, we tested whether the ΔagrΔacnA double mutant exhibits protection against H2O2. The new data we now provide (Figure 8—figure supplement 2A) show that a ΔacnA mutation completely protected the Δagr mutant from peroxide killing after growth to late exponential growth phase, but it had little if any effect on the wild-type strain. To evaluate long-lived protection, we compared survival rates of ΔagrΔacnA mutant and Δagr cells following dilution of overnight cultures and regrowth prior to challenge with H2O2, which revealed partial protection of the Δagr mutant (Figure 8— figure supplement 2B).
We explained these results with the following:
Line 351: “Rowe et al. (2020) showed that menadione exerts its effects on endogenous ROS by inactivating the TCA cycle in S. aureus. To determine whether this mechanism can also induce protection in the Δagr mutant, we inactivated the TCA cycle gene acnA in wild-type and Δagr strains (Figure 8—figure supplement 2). We found that ΔacnA mutation completely protected the Δagr mutant from peroxide killing after growth to late exponential growth phase but had little effect on the wild-type strain. This finding supports the idea that TCA cycle activity contributes to an imbalance in endogenous ROS homeostasis in the Δagr mutant, and that this shift is a critical factor for Δagr hyperlethality. When we evaluated long-lived protection by comparing survival rates of ΔagrΔacnA mutant and Δagr cells following dilution of overnight cultures and regrowth prior to challenge with H2O2, ΔacnA remained protective, but less so (Figure 8—figure supplement 2). These partial effects of an ΔacnA deficiency suggest that Δagr stimulates long-lived lethality for peroxide through both TCA-dependent and TCA-independent pathways.”
(5) Figure 10 presents a model suggesting that Rot-mediated repression of respiration is essential for long-lasting resistance to H2O2 lethality. However, the connection between decreased respiration and long-lived resistance to ROS is not evident, especially considering that the respiration rate varies over the growth phase and does not seem to align with the long-lived and steady protection provided by agr. However, the authors could investigate this by examining whether inactivating qox in the agr mutant restores its resistance to H2O2. The experiments with menadione are not particularly persuasive, as menadione could have additional effects on the cells that are not accounted for.
As requested, we tested whether the ΔagrΔqoxC double mutant exhibits protection against H2O2. qox deficiency was hyperlethal in wild-type and Δagr strains, even with the lowest concentration of H2O2 used in our assay. Indeed, surviving cells were undetectable, precluding comparison of survival differences between wild-type and Δagr mutant strains. This striking finding can be explained by prior work highlighting the profound and pleotropic effects of qox deficiency on metabolism that involve not only control of respiration but also participation in other physiological processes such as cell growth and morphological differences. For example, in Bacillus, qox deficiency decreases TCA cycle flux and increases overflow metabolism (8). Additionally, we confirmed prior work in S. aureus showing that qox deficiency decreases growth rate and yield (9, 10), dramatically increases production of pigment that functions as an oxidation shield, and decreases hemolytic activity (11). Moreover, we found that that qox deficiency results in a striking increase (~150%) in endogenous ROS in both wild-type and agr mutant cells, likely explaining the hyperlethality phenotype. Thus, interpretation of killing assay results must account for the complex and likely reciprocal interactions among Δqox-mediated metabolic changes, agrA-mediated redox sensing, and Δagrmediated changes in metabolism. Since killing data are not necessary or revealing without this information, we prefer to address the role of qox in future studies directed at understanding the complexities of the interaction among agr-mediated tolerance, endogenous ROS levels, and induction of protective responses in S. aureus.
(6) The repeated use of the term 'agr wild type' throughout the text is somewhat distracting. It might be clearer to simply use 'wild type,' as it is implied that this refers to the agr+ genotype.
We modified the text by replacing 'agr wild-type' with “wild-type” as suggested by the Reviewer.
(7) In the text, the authors imply that the extended lag phase of the agr mutant is observed solely in nutrient-limited CDM. However, Figure 1 and Figure Supplement 3A reveal that the strains were actually cultivated in CDM supplemented with glucose and Casamino acids, which makes the medium rich in both carbon and nitrogen, in addition to other nutrients present in CDM. The authors should clarify the composition of the media used and assess whether the term 'nutrient-limited CDM' is accurate in this context.
The extended lag phase of the Δagr mutant is observable in TSB but it is more easily appreciated in CDM, perhaps owing to a larger range of carbohydrates and other nutrient types (TSB a rich and complex medium for which the composition is unknown) and a higher concentration of glucose (2.5 mM versus 2.2 mM).
For clarification, we modified line 135 as follows:
Line 184: “Lag-time differences between strains were more obvious in experiments using less complex, chemically defined medium (CDM)…”
(8) Figure 1 - Figure Supplement 3C represents the growth rate in terms of [OD/min]. However, it would be more accurate to calculate the growth rate (μ) based on the change in the natural logarithm of optical density (OD) relative to the corresponding change in time, using appropriate units (preferably, h⁻¹). Additionally, the method employed for measuring growth rates should be detailed in the Materials and Methods section.
Our responses to Reviewer 2 Minor Comment 1 below address this issue.
(9) The resolution of the inset charts in Figure 4B is poor, and the Y-axis lacks labels. The figure legend should also specify whether the flux distribution (represented by thick black arrows in Fig 4B) is predicted for the wild type or the mutant.
We modified Figure 4B and the legend accordingly.
(10) On Page 9, the term "RT-PCR" should be corrected to "RT-qPCR."
We thank the Reviewer for their attention to detail in picking up our error. We modified text accordingly.
(11) It is ambiguous whether the agr mutant is producing more acetate, based on the information provided in Figure 5B. Since the cells might have entered the post-exponential phase at 5 hours, they could start consuming acetate. Consequently, the elevated acetate concentration in the agr mutant might result from a delay in acetate consumption rather than increased production. To discern between the production and consumption of acetate, it is essential to measure acetate concentrations at earlier time points as well as the corresponding glucose concentrations in the media. This will help ascertain when the agr mutant enters the post-exponential phase. A similar concern also exists in the case of lactate (Fig 5E) since it is not clear when lactate was measured.
As requested, we measured acetate levels at earlier time points (1, 2, 3, 4, h of growth). New Figure 5B shows that the Δagr mutant accumulated more acetate than the wild-type strain during exponential growth at 3 h, well before entry into postexponential phase (see growth curves in Figure 1—figure supplement 1).
In the original report, lactate levels were measured at 4 h for organisms grown under suboptimal aeration conditions (see Reviewer 1, Comment 1). When we measured lactate accumulation at 3 h it remained higher in the Δagr mutant compared to the wildtype. Likewise, acetate levels at 3 h under suboptimal aeration conditions remained elevated in the Δagr mutant compared to the wild-type. These results support the idea that inactivation of agr promotes production rather than decreased consumption of acetate and lactate in the culture medium.
(12) In Figure 5G-H, presenting the actual NAD+ and NADH values side-by-side would facilitate a more straightforward interpretation of the data by the readers.
(13) On Page 9, the text states that respiration and fermentation lower the NAD+/NADH ratio. However, this seems contradictory as these processes would typically increase the NAD+/NADH ratio. Furthermore, it would be beneficial for the authors to provide supporting evidence for the statement made at the beginning of Page 10, which claims that there is greater consumption of NADH in the agr mutant.
Responses to Comments 12 and 13 were grouped together.
We thank the Reviewer for their attention to detail in picking up our error about the NAD+/NADH ratio. The ratio is expected to be elevated by increases in respiration and fermentation, not lowered, owing to increased consumption of NADH.
Figure 5I in the submitted manuscript indicated a small but insignificant decrease in the NAD+/NADH ratio of the Δagr mutant. Thus, the NAD+/NADH ratio remained tightly bounded, but if anything was decreased, not increased.
We explained this finding as follows:
Line 284: “Collectively, these observations suggest that a surge in NADH production and reductive stress in the Δagr strain induces a burst in respiration and fermentation.”
The NAD+/NADH ratio in Figure 5F of the submitted manuscript was calculated from NADH and total (NAD+/NADH) levels. As requested, we measured individual NAD+ and NADH concentrations. We found that the decrease in the NAD+/NADH ratio of the Δagr mutant was now large, significant, and largely due to a relative increase in NADH.
We have included these new data in a revised Figure 5 in the revised version of the manuscript and clarify the relationship among the NAD+/NADH ratio, respiration, and fermentation in the Δagr mutant by modifying the wording of the text as follows:
Line 280: “Since respiration and fermentation generally increase NAD+/NADH ratios and since these activities are increased in Δagr strains (Figure 5C and 5E-F), we expected a higher NAD+/NADH ratio relative to wild-type cells. However, we observed an increase decrease in the NAD+/NADH ratio due to a large surge in NADH accompanied by a modest drop in NAD+ compared to wild-type. Collectively, these observations suggest that a surge in NADH production and reductive stress in the Δagr strain induces a burst in respiration, but levels of NADH are saturating, thereby driving fermentation in the presence of oxygen.
Reviewer #2 (Recommendations For The Authors):
(1) The RNA-seq analysis revealed that the Δagr strain exhibited increased expression of genes involved in respiration and fermentation, suggesting enhanced energy generation. However, metabolic modeling based on transcriptomic data indicated a decrease in tricarboxylic acid (TCA) cycle and lactate flux per unit of glucose uptake in the Δagr mutant. Additionally, intracellular ATP levels were significantly lower in the Δagr mutant compared to the wild-type strain, despite the carbon being directed into an acetate-generating, ATP-yielding carbon "overflow" pathway. Furthermore, growth analysis in nutrient-constrained medium demonstrated a decrease in the growth rate and yield of the Δagr mutant. Given that S. aureus actively utilizes the electron transport chain (ETC) to replenish NAD pools during aerobic growth on glucose, supporting glycolytic flux and pyruvate dehydrogenase complex (PDHC) activity while restricting TCA cycle activity through carbon catabolite repression (CCR), it is suggested that the authors analyze glucose consumption rates in conjunction with the determination of intracellular levels of pyruvate, AcCoA, and TCA cycle intermediates such as citrate and fumarate. These additional experiments will provide valuable insights into the metabolic fate of glucose and pyruvate and their subsequent impact on cellular respiration and fermentation in the Δagr mutant.
(2) The authors highlighted the importance of redox balance in Δagr cells by emphasizing the tendency of these cells to prioritize NAD+-generating lactate production over generating additional ATP from acetate. However, the results regarding acetate and lactate production in Δagr cells during aerobic growth suggest that carbon is directed towards acetate generation rather than lactate.
Responses to Comments 1 and 2 have been combined.
As requested, we measured glucose consumption and intracellular levels of several different metabolites in the wild-type and Δagr mutant strain. The results are consistent with the idea that increased acetogenesis and fermentation in Δagr mutant cells contribute to increased ATP production and NAD+ recycling, respectively. These two processes appear to be relatively favored over the flux of pyruvate carbon into the TCA cycle of the Δagr mutant.
We explained our finding as follows:
Line 288: “To help determine the metabolic fate of glucose, we measured glucose consumption and intracellular levels of pyruvate and TCA-cycle metabolites fumarate and citrate in the wild-type and Δagr mutant strains. At 4 h of growth to late-exponential phase, intracellular pyruvate and acetyl-CoA levels were increased in the Δagr mutant compared to wild-type strain, but levels of fumarate and citrate were similar (Figure 5— figure supplement 1D-E). Glucose was depleted after 4 h of growth, but glucose consumption after 3 h of growth (exponential phase) was increased in the Δagr mutant compared to the wild-type strain (Figure 5—figure supplement 1A). These observations, together with the decrease in the NAD+/NADH ratio and increase in acetate and lactate production described above, are consistent with a model in which respiration in Δagr mutants is inadequate for 1) energy production, resulting in an increase in acetogenesis, and 2) maintenance of redox balance, resulting in an increase in fermentative metabolism, lactate production, and conversion of NADH to NAD+. Increased levels of acetate compared to lactate under optimal aeration conditions suggests that demand for ATP is in excess of demand for NAD+.”
Future work will compare additional extracellular and intracellular (e.g., formate, ethanol, acetoin) metabolites to test these and other models using a combination of approaches (e.g., mass spectrometry, nuclear magnetic resonance, genetic deletion studies, transcriptomics) and will determine the mechanisms underlying metabolic differences in wild-type and Δagr mutant strains.
To maintain a sense of narrative we added a new subheading after the explanation of our findings:
Line 311: “Transcriptional changes due to Δagr mutation are long-lived and result in down-regulation of H2O2-stimulated genes relative to those in an agr wild-type.”
(3) The authors mentioned that respiration and fermentation typically reduce the NAD+/NADH ratios, and since these activities are elevated in Δagr strains (Figure 5F-G), they initially anticipated a lower NAD+/NADH ratio compared to wild-type agr cells. However, the increase in respiration and activation of fermentative pathways leads to a decrease in NADH levels, therefore resulting in an increase in the NAD+/NADH ratio.
We have clarified the issue with new experiments and by modifying the wording as shown in the response to Reviewer 1 Comment 13.
(4) To improve the clarity and completeness of this work, it would be advantageous for the authors to provide specific details regarding the glucose concentration in the TSB media and the aeration conditions during growth, including the flask-tomedium ratio. These additional experimental parameters are essential for ensuring the reproducibility and comprehensiveness of the study, allowing for a more precise understanding and interpretation of the observed metabolic changes in the Δagr strain.
We modified the Methods as suggested.
Minor comments:
(1) The growth rate in Figure 1-figure supplement 3 should not be presented as a simple calculation of OD/min and needs to be recalculated.
We recalculated the growth rate and modified Figure 1 as suggested. The exponential phase was used to determine growth rate (µ) from two datapoints, OD1 and OD2 flanking the linear portion of the curve, following the equation lnOD2-lnOD1/t2-t1, as described (12).
(2) Δrot (BS1301) should be removed from Figure 2 (A) legend as it is not presented in the panel A.
We modified Figure 2 as suggested.
(3) The authors should specify in the Figure 3 (D) legend that the kinetics of killing by H2O2 was performed for ΔrnaIII and ΔagrBD mixtures.
We modified Figure 3 as suggested.
(4) In the Figure 4 legend for (C), the statement "See Supplementary file 2 for supporting information" should be changed to "See Supplementary file 3 for supporting information."
We modified Supplementary file name as suggested.
References cited in responses
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(8) Zamboni N, Sauer U. 2003. Knockout of the high-coupling cytochrome aa3 oxidase reduces TCA cycle fluxes in Bacillus subtilis. FEMS Microbiol Lett 226:121-126.
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