Staphylococcal Enterotoxins Modulate Platelet Response During Storage of Platelet Concentrates and Impair Silkworm Survival
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe paper is well-written, scientifically relevant manuscript that investigates the role of Staphylococcal enterotoxins SEG and SEH in platelet activation, cytokine secretion, miRNA modulation, and virulence by using the silkworm model. However i do have some concerns here:
-Although the authors demonstrate associations, the mechanisms whereby SEG/SEH modulate platelet activation and miRNA expression are not explored.
-No experiments focus on the interactions between toxins and platelet receptors, or internalization and signaling such as via TLRs, MAPK, NF-κB, etc.
-The cytokine kit used is a semi-quantitative array. Authors consider pixel intensity as quantitative fold differences, which might not be accurate.
-Some of the results appear to be inconsistent; for instance, both SE mutants show increases as compared to the WT, which is counterintuitive.
-Important sequencing parameters missing: read depth per sample, normalization method, number of identified miRNAs, thresholds for inclusion
-Some parts had unclear statistical analysis such as multiple comparison corrections for cytokine arrays are not described.
-Interpretation of borderline p-values, such as p=0.06, should be more cautious.
-Limitations of the silkworm model are not fully discussed. This model is convenient; however, it differs from mammalian immune systems. How well does this model translate to humans? Discussion does mention limitations but needs more depth.
-No direct measurement of the production of enterotoxin in PCs. This study assumes that SEs are expressed during storage, yet it does not quantify the protein levels of SEG/SEH in PCs. This is a major gap.
-The figures and supplementary data require improvement. Some figures lack error bars, sample size notes or statistical annotations. Cytokine heatmaps and miRNA volcano plots would be more visually clear.
-Extend mechanistic discussion, Include possible pathways for toxin-induced platelet activation: TLR2/TLR4 involvement, MHC or superantigen-like signaling, cAMP or MAPK pathways, Even if speculative, this strengthens the interpretation.
-Add: summary table of sequencing depth & mapping percentages, List of significantly expressed miRNAs brief GO or pathway enrichment analysis
-Expand Discussion of silkworm relevance, Explain: evolutionary conservation of innate immunity, limitations in comparison with mammalian models, Justification of the model for translational conclusions
-Provide clinical implications Discussion should more directly elaborate on: How biomarkers could be incorporated into PC screening - miRNAs/cytokines How SE inhibition can help improve pathogen reduction strategies.
Author Response
Reviewer 1
The paper is well-written, scientifically relevant manuscript that investigates the role of Staphylococcal enterotoxins SEG and SEH in platelet activation, cytokine secretion, miRNA modulation, and virulence by using the silkworm model.
However, i do have some concerns here:
-Although the authors demonstrate associations, the mechanisms whereby SEG/SEH modulate platelet activation and miRNA expression are not explored.
RESPONSE: We thank the reviewer for this insightful comment. SEG and SEH are superantigens that potentially activate platelets indirectly. Through a classical superantigen pathway, SEG and SEH plausibly bind platelet receptors nonspecifically causing massive activation of platelet and triggering miRNA expression and cytokine release. This has been added to the Introduction and Discussion sections of the manuscript.
-No experiments focus on the interactions between toxins and platelet receptors, or internalization and signaling such as via TLRs, MAPK, NF-κB, etc.
RESPONSE: We appreciate the reviewer for this comment. We assessed SEG/SEH interaction with platelet via a binding assay using flow cytometry to measure expression of P-selectin independently for platelets spiked with wildtype S. aureus in comparison with platelet inoculated with derivate SE-mutant strains (See Figure 1a). Also, there are reports of platelet aggregometry functional assay showing SEB’s involvement in the activation of specific intracellular signaling modules like PKC and eicosanoid metabolism in platelet. However, we did not test this for SEG/SEH as it was outside the scope of this work.
-The cytokine kit used is a semi-quantitative array. Authors consider pixel intensity as quantitative fold differences, which might not be accurate.
RESPONSE: We thank the reviewer for this important comment. We agree that the cytokine array is inherently semi-quantitative. Pixel intensity values reflect relative signal strength rather than absolute protein concentration. In our analysis, we used pixel densities only to compare relative changes between unspiked platelet concentrates and those spiked with S. aureus after subtracting background signal and normalizing with control on each membrane, not to derive absolute fold-differences. We have now clarified this in Figure 2 (y-axis) as well as in the manuscript and reframed our conclusions to emphasize relative trends.
-Some of the results appear to be inconsistent; for instance, both SE mutants show increases as compared to the WT, which is counterintuitive.
RESPONSE: We appreciate the reviewer for pointing out this out. We noticed that the description of Figure 2 was inconsistent in the text. Figure 2 shows specific cytokines with increased expression in SE-mutants vs wildtype-spiked PCs. The issue has been properly addressed in the Results and Discussion sections.
-Important sequencing parameters missing: read depth per sample, normalization method, number of identified miRNAs, thresholds for inclusion
RESPONSE: We thank the reviewer for pointing this out. We appreciate the opportunity to clarify the sequencing and data-processing parameters. We have now added to the normalization approach used in the Methods section and prepared a new table (Supplementary Table 1) showing total number of miRNAs detected across samples.
-Some parts had unclear statistical analysis such as multiple comparison corrections for cytokine arrays are not described.
RESPONSE: We appreciate this comment. Statistical analyses including the baseline for each comparison pairs have now been added. See Methods section.
-Interpretation of borderline p-values, such as p=0.06, should be more cautious.
RESPONSE: We appreciate the reviewer’s comment regarding borderline p-values. We acknowledge that P=0.06 does not reach statistical significance under the standard p=0.05 threshold. In the revision, we have reframed the statement in the Result section, as not statistically significant.
-Limitations of the silkworm model are not fully discussed. This model is convenient; however, it differs from mammalian immune systems. How well does this model translate to humans? Discussion does mention limitations but needs more depth.
RESPONSE: We thank the reviewer for this valuable comment. We agree that the limitations of the silkworm model warrant deeper discussion. We have expanded the Discussion section to more explicitly acknowledge these limitations and to emphasize that translation to human biology requires validation in mammalian models or human cell-based systems, which was beyond the scope of this study.
-No direct measurement of the production of enterotoxin in PCs. This study assumes that SEs are expressed during storage, yet it does not quantify the protein levels of SEG/SEH in PCs. This is a major gap.
RESPONSE: We thank the reviewer for this important comment. In our previous publications, reference 13 “Staphylococcal Enterotoxins Enhance Biofilm Formation by Staphylococcus aureus in Platelet Concentrates’’ and reference 40 “Proof of concept for detection of staphylococcal enterotoxins in platelet concentrates as a novel safety mitigation strategy,” we demonstrated that SEs were expressed in PCs contaminated with the same S. aureus strains used in the present study. Specifically, SEG/SEH were detected in PCs during storage. This prior work informed the biological premise of the current study. The focus of this present study was to investigate platelet immunological responses following exposure to S. aureus wild-type and SE-deficient mutant strains. We have clarified in the Method section that SEG and SEH protein levels in PCs were previously detected.
-The figures and supplementary data require improvement. Some figures lack error bars, sample size notes or statistical annotations. Cytokine heatmaps and miRNA volcano plots would be more visually clear.
RESPONSE: Thank you for your constructive feedback regarding the figures and supplementary data. We have carefully revised the visual components of the manuscript to address these concerns. All figures presenting quantitative data have been updated to include appropriate error bars and explicit sample size (n) in the figure legends. We have also added significance indicators (p values) to clarify the statistical differences between groups.
-Extend mechanistic discussion, Include possible pathways for toxin-induced platelet activation: TLR2/TLR4 involvement, MHC or superantigen-like signaling, cAMP or MAPK pathways, Even if speculative, this strengthens the interpretation.
RESPONSE: Thank you for this insightful suggestion. We agree that extending the mechanistic discussion would strengthen the interpretation of our findings. We have substantially expanded the Discussion section to include potential pathways through which staphylococcal enterotoxins may modulate platelet activation, even though mechanistic confirmation lies beyond the scope of the present study.
-Add: summary table of sequencing depth & mapping percentages, List of significantly expressed miRNAs brief GO or pathway enrichment analysis
RESPONSE: Thank you for this helpful suggestion. We have revised the manuscript to incorporate your recommendations. We have added a new table (Table S1) summarizing sequencing depth and listing some significantly differentially expressed miRNAs, and average total reads across the samples.
-Expand Discussion of silkworm relevance, Explain: evolutionary conservation of innate immunity, limitations in comparison with mammalian models, Justification of the model for translational conclusions
RESPONSE: We appreciate the reviewer’s suggestion and have expanded the Introduction and Discussion sections to fully address the relevance and limitations of the silkworm infection model. Specifically, we now highlight that Bombyx mori possesses evolutionarily conserved innate immune pathways including Toll-like signaling, antimicrobial peptide production, and cellular immunity which show meaningful parallels to mammalian innate responses and support its use for studying early host–pathogen interactions.
-Provide clinical implications Discussion should more directly elaborate on: How biomarkers could be incorporated into PC screening - miRNAs/cytokines How SE inhibition can help improve pathogen reduction strategies.
RESPONSE: We thank the reviewer for this suggestion. We have expanded the Discussion to highlight the clinical implications of our findings. Specifically, how miRNAs and cytokines could serve as early indicators of S. aureus contamination or toxin activity, potentially complementing existing pathogen detection methods. Also on how targeting SE activity, either pharmacologically or via modified pathogen reduction strategies, could further improve PC safety by mitigating toxin-mediated platelet activation and inflammatory responses. These additions clarify translational relevance and potential applications of our work in improving PC transfusion safety.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript presents high scientific value and a strong level of novelty. Only a few minor revisions are needed to improve clarity and methodological precision.
- The Introduction requires a clearer justification for selecting the strain producing enterotoxins H and G (SEH and SEG). In the context of the classical staphylococcal enterotoxins (SEA–SEE), which are widely recognized as major virulence factors, the authors should explain why the focus was placed on these less commonly studied variants. This rationale should be supported by appropriate references regarding their potential clinical relevance or specificity in the experimental model.
- A gene knockout–based approach alone is insufficient to conclusively demonstrate the absence of SEH and SEG protein production. To confirm that the enterotoxins are not functionally produced, protein-level validation is essential.
• Western Blot or ELISA results should be provided to confirm the absence of the toxins in the culture supernatants of the deletion mutants.
• A positive control, such as recombinant enterotoxin protein, is recommended to validate the sensitivity of the detection method.
• The manuscript lacks information on the potential production of enterotoxins in the presence of platelets. This is a critical issue that may influence the experimental outcomes. It is necessary to assess whether PRP components modulate toxin production or whether the toxins retain activity in this medium.
If protein-level data are not available, the subsection titles and the Results section should be revised to reflect the actual scope of the analyses performed (e.g., focusing on the effects of the deletion mutants rather than the toxins themselves). Although the Discussion addresses these concerns, greater precision is required in the Methods and Results sections. - The term “Day 0” in the Materials and Methods section must be clearly defined. It must be explicitly stated whether “Day 0” refers to the moment immediately before strain administration or to a specific number of hours afterwards. This clarification is crucial for accurate interpretation of the results, particularly in the context of microRNA (miRNA) expression, which exhibits dynamic temporal changes.
- While the novelty of the study limits the availability of comparable work for discussion, one relevant publication examines the effect of TSST-1 on platelet activation and could be worth including:
Guo M, Yi T, Wang Q, Wang D, Feng P, Kesheng D, Chunyan H. TSST-1 protein exerts indirect effect on platelet activation and apoptosis. Platelets. 2022;33(7):998–1008. - In the Supplementary Data, Figure S1 lacks a scale on the x-axis. This should be added to ensure clarity and proper interpretation of the data.
Author Response
Reviewer 2
ï‚· The Introduction requires a clearer justification for selecting the strain producing enterotoxins H and G (SEH and SEG). In the context of the classical staphylococcal enterotoxins (SEA–SEE), which are widely recognized as major virulence factors, the authors should explain why the focus was placed on these less commonly studied variants. This rationale should be supported by appropriate references regarding their potential clinical relevance or specificity in the experimental model.
RESPONSE: We thank the reviewer for raising this important point. We agree that the Introduction required clearer justification for the selection of an SEG/SEH-producing strain. This study is based on two of our previous publications (references 17 and 43), where genomic analyses revealed the absence of SEA-SEE in all studied transfusion relevant S. aureus strains, and that most of the strains encode SEG and SEH. We also demonstrated by transcriptome and protein analyses that SEG and SEH are transcribed and translated and could be detected in PCs during storage, supporting their relevance in the transfusion setting. These findings provided the foundational rationale for selecting a strain producing SEH and SEG in the present study. In response, we have substantially revised the Introduction, Methods and Discussion sections to articulate the biological and clinical relevance of SEG and SEH and to clarify why these enterotoxins were the appropriate focus for this study.
ï‚· A gene knockout–based approach alone is insufficient to conclusively demonstrate the absence of SEH and SEG protein production. To confirm that the enterotoxins are not functionally produced, protein-level validation is essential.
• Western Blot or ELISA results should be provided to confirm the absence of the toxins in the culture supernatants of the deletion mutants.
• A positive control, such as recombinant enterotoxin protein, is recommended to validate the sensitivity of the detection method.
RESPONSE: We thank the reviewer for this comment. Both SE-mutants were generated by CRISPR-Cas9 technique. Functional analyses in one of our previous studies (reference 17) confirmed that seG and seH deletional mutants lack enterotoxin-dependent biofilm enhancement, supporting the functional absence of SEH and SEG. Moreover, direct protein-level validation by Western Blot alongside purified positive controls was performed indicating the absence of both proteins in the mutants. Reference 17 describes how the SE-deficient strains were constructed and is referred in the Methods section.
The manuscript lacks information on the potential production of enterotoxins in the presence of platelets. This is a critical issue that may influence the experimental outcomes. It is necessary to assess whether PRP components modulate toxin production or whether the toxins retain activity in this medium.
If protein-level data are not available, the subsection titles and the Results section should be revised to reflect the actual scope of the analyses performed (e.g., focusing on the effects of the deletion mutants rather than the toxins themselves). Although the Discussion addresses these concerns, greater precision is required in the Methods and Results sections.
RESPONSE: We thank the reviewer for raising this important point. We agree that the manuscript requires clear evidence of the production of staphylococcal enterotoxins in PCs. This study is based on two of our previous publications (references 17 and 43), where genomic analyses revealed the presence of genes encoding for enterotoxins including seG and seH in the transfusion relevant S. aureus CBS2016-05 strain. We also demonstrated by comparative RNAseq analyses that these genes are upregulated in PCs and have shown by immunoblotting assays that both SEG and SEH are detectable in PCs during storage. These findings provided the foundational rationale of the present study. In response, we have substantially revised the Introduction, Methods and Discussion sections, stating clearly that the current investigation was premised on our previous findings using the same S. aureus strain.
ï‚· The term “Day 0” in the Materials and Methods section must be clearly defined. It must be explicitly stated whether “Day 0” refers to the moment immediately before strain administration or to a specific number of hours afterwards. This clarification is crucial for accurate interpretation of the results, particularly in the context of microRNA (miRNA) expression, which exhibits dynamic temporal changes.
RESPONSE: We thank the reviewer for highlighting this ambiguity. We agree that precise definition of “Day 0” is essential. In the revised manuscript, we now clearly specify that Day 0 refers to the timepoint when PCs were inoculated with S. aureus and is the baseline sampling time immediately after bacterial contamination. This clarification has been added to the Methods section.
ï‚· While the novelty of the study limits the availability of comparable work for discussion, one relevant publication examines the effect of TSST-1 on platelet activation and could be worth including:
Guo M, Yi T, Wang Q, Wang D, Feng P, Kesheng D, Chunyan H. TSST-1 protein exerts indirect effect on platelet activation and apoptosis. Platelets. 2022;33(7):998–1008.
RESPONSE: We thank the reviewer for highlighting this relevant study. We have now incorporated the suggested reference, which demonstrates that the staphylococcal superantigen TSST-1 can induce indirect platelet activation and apoptosis. This work supports the broader concept that S. aureus superantigens modulate platelet behavior and strengthens the context for our investigation of SEG and SEH. We have added relevant sentences in both the Introduction and Discussion sections to acknowledge this study and highlight how our work builds upon and extends these findings.
ï‚· In the Supplementary Data, Figure S1 lacks a scale on the x-axis. This should be added to ensure clarity and proper interpretation of the data.
RESPONSE: We thank the reviewer for pointing out this omission. We have now revised Supplementary Figure S1 to include a clearly labeled X-axis scale, ensuring that the figure is fully interpretable.
Reviewer 3 Report
Comments and Suggestions for AuthorsThis is an interesting study to show the effect of SEG and SEH to platelet activation and other biological features. The experiments seem to be scientifically well performed. However, there seems to be critical points to the study design and interpretation of results. These should be clarified and descriptions of manuscript should be largely revised.
- In this study, authors tried to analyze the effect of SEs according to the study design. However, authors used S. aureus isolate CBS2016-05 and SE deficient mutant isolates. But authors did not use a purified SEs. If authors wanted to know the biological effect of SEs, purified SEs with different concentrations should be used. Why were these not used? In the present study design, bacterial cells and their components, and bacterial proteins and toxins produced by the strain might have influenced in the experiments. Such possibility are not able to be avoided.
- Authors used S, aureus isolate CBS2016-05, and deficient mutants of SEH and SEG. Does this wildtype strain possess and produce only SEG and SEH? Dees it have any other SE genes? Because many SE genes, i.e., SEA-SEE, SEG-SEZ, SEl26, SEl27, SE01, SE02, have been identified in S. aureus and the presence of SE genes are different depending on strain. If CBS2016-05 has any other SE genes other than SEG and SEH genes, there is a possibility that other SE might have influenced the results in this study.
- Did authors evaluated the actual production amount of SEG and SEH of S. aureus strain CBS2016-05? Even if the SE genes are present in bacterial isolates, actual production (expression level) may be different depending on strain. Such factors should be taken into account in this study and interpretation of results.
Author Response
Reviewer 3
This is an interesting study to show the effect of SEG and SEH to platelet activation and other biological features. The experiments seem to be scientifically well performed. However, there seems to be critical points to the study design and interpretation of results. These should be clarified and descriptions of manuscript should be largely revised.
- In this study, authors tried to analyze the effect of SEs according to the study design. However, authors used S. aureus isolate CBS2016-05 and SE deficient mutant isolates. But authors did not use a purified SEs. If authors wanted to know the biological effect of SEs, purified SEs with different concentrations should be used. Why were these not used? In the present study design, bacterial cells and their components, and bacterial proteins and toxins produced by the strain might have influenced in the experiments. Such possibility are not able to be avoided.
RESPONSE: We thank the reviewer for this thoughtful comment. We agree that purified enterotoxins can be useful for isolating toxin-specific effects; however, the objectives of the present study were directed toward understanding how SEG and SEH contribute to platelet responses within the physiological context of bacterial contamination of platelet concentrates, rather than evaluating their effects in isolation. Our study design was informed by two previous publications (references 17 and 43), where we demonstrated that PC–associated isolates exhibit transcriptomic adaptations that may alter virulence and secretory profiles. We provided direct evidence that SEG and SEH are secreted and detectable in PCs during storage. Therefore, assessing SEG/SEH activity in the context of whole bacteria is highly relevant to understanding how transfusion-derived strains behave within PCs. This established the biological premise that SEs act within the complex milieu of PCs, where they coexist with other bacterial products. We fully acknowledge that other bacterial factors may contribute to the observed effects, and we have now added text in the Discussion to clarify this limitation. We also stated that future studies could incorporate purified SEG and SEH to complement the current whole-bacterium approach and to delineate direct versus synergistic toxin effects.
- Authors used S, aureus isolate CBS2016-05, and deficient mutants of SEH and SEG. Does this wildtype strain possess and produce only SEG and SEH? Dees it have any other SE genes? Because many SE genes, i.e., SEA-SEE, SEG-SEZ, SEl26, SEl27, SE01, SE02, have been identified in S. aureus and the presence of SE genes are different depending on strain. If CBS2016-05 has any other SE genes other than SEG and SEH genes, there is a possibility that other SE might have influenced the results in this study.
RESPONSE: We appreciate the reviewer’s comment. The enterotoxin gene profile of S. aureus CBS2016-05 was fully defined in our previous genomic study (reference 17). This strain encoded enterotoxins, including seG and seH, but lacks the classical SE genes (SEA–SEE). In our separate study (reference 43), we demonstrated that SEG and SEH are secreted during storage of PCs contaminated with S. aureus CBS2016-05. Thus, the SE-dependent effects observed in this work can be attributed primarily to SEG and SEH, with minimal influence from other enterotoxins. The Introduction and Discussion sections have been modified to clarify that the present work is based on our previous findings of SEs identification in transfusion relevant S. aureus strains and their detection in stored PCs.
- Did authors evaluated the actual production amount of SEG and SEH of S. aureus strain CBS2016-05? Even if the SE genes are present in bacterial isolates, actual production (expression level) may be different depending on strain. Such factors should be taken into account in this study and interpretation of results
RESPONSE: We thank the reviewer for this comment. While we did not quantify absolute SEG and SEH production levels in the current study, in our previous publication (reference 43), we demonstrated by immunoblot assays that CBS2016-05 secretes both SEG and SEH during PC storage, confirming active expression under relevant conditions.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for answering my questions. good luck
Reviewer 3 Report
Comments and Suggestions for AuthorsThe revised version has been well written with necessary informaion. Authors responded to the reviewers questions appropriately, and this reviewer understand the scientific soundness of this study.
