Potential Molecular Targets of the Broad-Range Antimicrobial Peptide Tyrothricin in the Apicomplexan Parasite Toxoplasma gondii

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates the effects of the broad-spectrum antimicrobial peptide (AMP) tyrothricin (a mixture of tyrocidines and gramicidins) on the apicomplexan parasite Toxoplasma gondii. Through in vitro assays, the authors found that tyrothricin potently inhibits T. gondii proliferation (IC₅₀ < 100 nM) but also exhibits significant cytotoxicity towards human fibroblasts, murine T cells, and zebrafish embryos, indicating a narrow therapeutic window. The anti-parasitic activity was attributed primarily to its cyclic decapeptide component, tyrocidine A, while gramicidin A showed no effect. The study concludes that while tyrothricin (via tyrocidine A) shows strong anti-Toxoplasma activity, its host cell toxicity limits systemic therapeutic potential. The identified protein targets provide insights into its multimodal action and highlight structural differences compared to other AMPs like leucinostatin derivatives. Overall, the manuscript is scientifically sound.

 

1. The Results section presents extensive data (e.g., cytotoxicity assays, TEM images, proteomic lists) without clearly linking them to the mechanistic hypotheses discussed later. This creates a descriptive rather than narrative flow.
2. Some figures and tables are referenced in the text before they appear (e.g., Figure 1 is mentioned after Table 1 but appears earlier in the PDF), and captions lack sufficient methodological detail (e.g., statistical tests used in graphs).

Minor:

1. Ensure all figures and tables are cited in sequential order. Check that each is introduced before or at the point of discussion.
2. Expand figure captions to include key experimental conditions (e.g., drug concentrations, time points) and statistical methods (e.g., "Data are mean ± SD; *P < 0.05, Student’s t-test").
3. In Table 1, add a footnote defining how "viability" and "proliferation" percentages were calculated relative to controls for clarity.
4. In figure 1A-B, uM should be μM.

Author Response

Comments and Suggestions for Authors

This study investigates the effects of the broad-spectrum antimicrobial peptide (AMP) tyrothricin (a mixture of tyrocidines and gramicidins) on the apicomplexan parasite Toxoplasma gondii. Through in vitro assays, the authors found that tyrothricin potently inhibits T. gondii proliferation (IC₅₀ < 100 nM) but also exhibits significant cytotoxicity towards human fibroblasts, murine T cells, and zebrafish embryos, indicating a narrow therapeutic window. The anti-parasitic activity was attributed primarily to its cyclic decapeptide component, tyrocidine A, while gramicidin A showed no effect. The study concludes that while tyrothricin (via tyrocidine A) shows strong anti-Toxoplasma activity, its host cell toxicity limits systemic therapeutic potential. The identified protein targets provide insights into its multimodal action and highlight structural differences compared to other AMPs like leucinostatin derivatives. Overall, the manuscript is scientifically sound.

Response: thank you for this assessment.

1. The Results section presents extensive data (e.g., cytotoxicity assays, TEM images, proteomic lists) without clearly linking them to the mechanistic hypotheses discussed later. This creates a descriptive rather than narrative flow.

Response: The reviewer is right in stating that the data presented in this paper is descriptive. However, by demonstrating anti-Toxoplasma in vitro activity as well as host cell and immune cell / zebrafish embryo toxicity, we meant to justify the decision that further in vivo work was not really an option. These safety assessments are elements of a screening cascade we have used extensively in other works prior to embarking on studies in laboratory animals (e.g. Müller et al., 2022, 2023 and other papers cited in this manuscript). Instead of further focusing on the potential of tyrothricin, or its components tyrocidine and gramicidin, as treatment options, we decided to investigate which proteins could be targeted by these peptides. We therefore added the following sentence into the discussion (lane 543 ff):

The fact that tyrothricin not only displays considerable anti-T. gondii activity but also impairs the viability of HFF, immune cell subsets as well as zebrafish embryos renders this compound unsuitable for further in vivo evaluation, as the risk of adverse effects in animal studies was considered to be high. However, we decided to investigate which proteins could be actually targeted by this AMP.  

2. Some figures and tables are referenced in the text before they appear (e.g., Figure 1 is mentioned after Table 1 but appears earlier in the PDF), and captions lack sufficient methodological detail (e.g., statistical tests used in graphs).

Response: We have checked chronological appearance of figures and Tables in the text. In the case described by reviewer 1, Figure 1 appears after Table 1, as in the text. Some details were added in the figure captions as requested.

Minor:

1. Ensure all figures and tables are cited in sequential order. Check that each is introduced before or at the point of discussion.

Response: thank your for pointing this out. Figures and Tables are cited in sequential order.

2. Expand figure captions to include key experimental conditions (e.g., drug concentrations, time points) and statistical methods (e.g., "Data are mean ± SD; *P < 0.05, Student’s t-test").

Response: done Figure 1, 2, 7, Table 1

3. In Table 1, add a footnote defining how "viability" and "proliferation" percentages were calculated relative to controls for clarity.

Response: thank you for this comment, this was done

4. In figure 1A-B, uM should be μM.

Thank you for this, we changed to µM.

Reviewer 2 Report

Comments and Suggestions for Authors

Summary: This study investigates the in vitro efficacy and mechanism of action of tyrothricin, a polypeptide complex derived from Brevibacillus parabrevis, against the intracellular parasite Toxoplasma gondii. The authors screened seven antimicrobial peptides (AMPs), identifying tyrothricin as the only effective candidate with an IC50​<100 nM. The study dissects the complex, demonstrating that the cyclic peptide Tyrocidine A is the active anti-parasitic component, whereas the linear Gramicidin A is inactive. The authors utilized a multi-disciplinary approach including cytotoxicity assays on human foreskin fibroblasts and murine splenocytes, in vivo toxicity assays using zebrafish embryos, transmission electron microscopy, and TMRE assays for mitochondrial potential. Finally, they employed differential affinity chromatography coupled with mass spectrometry to identify potential protein targets of Tyrocidine A. Key findings indicate that Tyrocidine A induces mitochondrial matrix dissolution and cristae loss, reduces mitochondrial membrane potential, and causes vacuolization. However, the study notes significant cytotoxicity to host cells and zebrafish embryos at concentrations close to the effective dose, and observes that the parasitic inhibition is transient, with parasites recovering after 96 hours. Proteomics identified 521 specific binding partners for Tyrocidine A, including GRA1, mitochondrial electron transport proteins, and surface antigens (SRS).

General Comments: The manuscript presents a logically structured and technically robust investigation into the anti-parasitic potential of AMPs. The authors are to be commended for the comprehensive nature of the study, moving from basic screening to ultrastructural analysis and target identification via proteomics. The inclusion of the "inactive" component (Gramicidin A) as a negative control in the DAC-MS experiments is a strong experimental design choice that adds confidence to the proteomics data.

Specific Comments:

1. Peptide Stability and Transient Effects (Section 3.3) The authors note that ultrastructural damage is transient, and parasites recover after 96 hours. Did the authors refresh the medium containing the peptide daily? AMPs are often susceptible to proteolytic degradation in serum-containing media over 4 days. If the drug was added only once at t=0, the recovery is likely due to peptide degradation rather than parasite adaptation. This distinction is vital for interpreting the "transient" nature of the effect. Please clarify the dosing schedule in the methods for the long-term assays.
2. Specificity of Proteomics Hits (Section 3.6) The DAC-MS identified 521 proteins binding to Tyrocidine A. Tyrocidines are cationic, amphipathic peptides known to act as detergents. A hit list of 521 proteins represents a significant portion of the proteome. There is a high risk that many of these are non-specific electrostatic interactions between the cationic peptide and anionic parasite proteins. While the Gramicidin A control helps, Gramicidin is neutral/linear and may not control for charge-based "stickiness." The authors should discuss this limitation in the discussion.
3. The abstract mentions the identification of 522 proteins, but the results section mentions 526 total and 521 specific to Tyrocidine. Please ensure consistency in the numbers throughout the text.
4. The methods state embryos were exposed at different concentrations. Please confirm if dechorionation was performed. The chorion can act as a barrier to some peptides. If not dechorionated, the toxicity might be underestimated.

Author Response

General Comments: The manuscript presents a logically structured and technically robust investigation into the anti-parasitic potential of AMPs. The authors are to be commended for the comprehensive nature of the study, moving from basic screening to ultrastructural analysis and target identification via proteomics. The inclusion of the "inactive" component (Gramicidin A) as a negative control in the DAC-MS experiments is a strong experimental design choice that adds confidence to the proteomics data

Response: thank you for this assessment

Comment 1: Peptide Stability and Transient Effects (Section 3.3) The authors note that ultrastructural damage is transient, and parasites recover after 96 hours. Did the authors refresh the medium containing the peptide daily? AMPs are often susceptible to proteolytic degradation in serum-containing media over 4 days. If the drug was added only once at t=0, the recovery is likely due to peptide degradation rather than parasite adaptation. This distinction is vital for interpreting the "transient" nature of the effect. Please clarify the dosing schedule in the methods for the long-term assays.

Response: Thank you for this highly valuable comment. For the pre-screening and IC50 determinations, which typically run over a period of 72-96 h, the medium plus/minus AMPs was added only once at the beginning. For TEM studies, we have actually replenished the cultures with fresh medium containing tyrothricin after 48 h. We have added this information into the M&M section (lane 176).

Comment 2: Specificity of Proteomics Hits (Section 3.6) The DAC-MS identified 521 proteins binding to Tyrocidine A. Tyrocidines are cationic, amphipathic peptides known to act as detergents. A hit list of 521 proteins represents a significant portion of the proteome. There is a high risk that many of these are non-specific electrostatic interactions between the cationic peptide and anionic parasite proteins. While the Gramicidin A control helps, Gramicidin is neutral/linear and may not control for charge-based "stickiness." The authors should discuss this limitation in the discussion.

Response: Correct point of criticism and limitation of our approach. Nevertheless, we have found earlier that the linear AMP leucinostatin also interacted with a large number (269) of T. gondii proteins and over 500 immune cell proteins (with a structurally related non-active peptide included in the analysis, see discussion, lane 563 and corresponding reference). The differences in the structures of tyrocidine A and gramicidin A, however, a limitation of our approach. To clarify these obvious limitations, we added the following sentence into the discussion (lane 567-572): No protein was identified to bind only to gramicidin A, which coincides with the absence of anti-parasitic activity. On the other hand, it appears conceivable that an AMP capable of interacting with multiple proteins would exert higher anti-parasitic activity, as well as cytotoxicity. Most likely not all tyrocidine A-binding proteins identified here are actual drug targets, since the circular cationic peptide is prone to bind to anionic parasite proteins. Whether these are relevant in terms of targets interactions is not clear, and remains to be investigated.

Comment 3: The abstract mentions the identification of 522 proteins, but the results section mentions 526 total and 521 specific to Tyrocidine. Please ensure consistency in the numbers throughout the text.

Response: Thank you for this remark. In fact, overall 525 proteins were identified, 521 binding only to tyrocidine A and 4 binding both peptides. This was now corrected in the abstract, results and discussion sections.

Comment 4: The methods state embryos were exposed at different concentrations. Please confirm if dechorionation was performed. The chorion can act as a barrier to some peptides. If not dechorionated, the toxicity might be underestimated.

Response: The zebrafish experiments were performed as described in Anghel et al (2020), and no dechorionation was performed. Therefore it is possible that toxicity is underestimated, but still high enough to not go for in vivo experiments

 

Reviewer 3 Report

Comments and Suggestions for Authors

1.The introduction correctly notes that the systemic toxicity of many antimicrobial peptides limits their therapeutic use. However, the rationale for studying tyrothricin specifically, with its known toxic profile, remains unclear. Could you clarify the fundamental novelty or hypothesis surrounding the mechanism of action of tyrothricins against T. gondii, which justifies its detailed study despite known selectivity issues? Was it expected that its components might exhibit unexpected specificity against apicomplexan parasites?

2.The DAC-MS protocol uses gramicidin A immobilized on sepharose as a control for target protein identification. Since tyrocidin A and gramicidin A have significant differences in chemical structure, charge, and hydrophobicity, their non-specific binding to cell lysate components may be incomparable. Can you please comment on this?

3.To evaluate the cytotoxicity of tyrothricin on HFF cells, only two concentrations (0.1 and 1 µM) were used. However, to calculate the selectivity index (SI), the concentration that causes 50% cell death (CC50) is required. Have full dose-response curves been conducted to determine the CC50 of tyrothricin and its components on HFF cells? If so, please provide these data and the calculated SI values.

4.In the tetramethylrhodamine ethyl ether experiment, total fluorescence is measured in a culture containing both infected and uninfected HFF cells to assess changes in mitochondrial membrane potential. How do you differentiate between the contribution of parasite mitochondria and host cell mitochondria to the fluorescence change? Is it possible that the observed 40% decrease primarily reflects an effect on HFF cells?

5.Transmission electron microscopy data indicate that ultrastructural damage to parasite mitochondria caused by tyrothricin is reversible within 72-96 hours, despite the persistent presence of the peptide. How should we interpret this observation in the context of our search for lethal molecular targets?

6. Based on the data on decreased metabolic activity (Alamar Blue) of ConA-stimulated splenocytes, it was concluded that tyrothricin has an immunosuppressive effect on T cells. How can you rule out that the observed signal reduction is a consequence of general cytotoxicity against actively proliferating cells rather than specific suppression of the immune response?
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7.In the discussion section, the tone should be consistent with the data. Since the results demonstrate high toxicity and transient effects, the main message should shift from a description of "therapeutic potential" and "multiple targets" to an analysis of non-selective cytotoxicity and challenges in identifying specific targets for cationic peptides. This will create a more honest and compelling narrative.

8. The conclusions are misleading, as they are based on an incorrect interpretation of invalid proteomic data (specifically, the DAC-MS experiment which used the chemically distinct gramicidin A as a negative control) and ignore the key results of the original study: high overall toxicity and the transient nature of the antiparasitic effect.

 

Author Response

Thank you for your valuable comments on our manuscript.

Comment 1: The introduction correctly notes that the systemic toxicity of many antimicrobial peptides limits their therapeutic use. However, the rationale for studying tyrothricin specifically, with its known toxic profile, remains unclear. Could you clarify the fundamental novelty or hypothesis surrounding the mechanism of action of tyrothricins against T. gondii, which justifies its detailed study despite known selectivity issues? Was it expected that its components might exhibit unexpected specificity against apicomplexan parasites?

Response: The rational for studying tyrothricin and its components in more detail is based on the fact that tyrothricin is composed of several different AMPs, two of which are tyrocidine A and gramicidin A. The activities of these two components against T. gondii have not been known before, and unexpectedly, gramicidin A did not at all contribute to the anti-T. gondii activity, while tyrocidine exhibited activities very similar to the original thyrocidine preparation, including cytotoxic effects. Thus, while not useful for potential in vivo evaluation, it is of interest to identify proteins that physically bind to these two AMPs.

Comment 2: The DAC-MS protocol uses gramicidin A immobilized on sepharose as a control for target protein identification. Since tyrocidine A and gramicidin A have significant differences in chemical structure, charge, and hydrophobicity, their non-specific binding to cell lysate components may be incomparable. Can you please comment on this?

Response: The reviewer is right in pointing out that tyrocidine and gramicidin A are clearly different in structure and physicochemical properties. However, of the overall 525 proteins that were identified, 521 were exclusively bound to tyrocidine A, and only 4 proteins bound to both peptides tyrocidine A and gramicidin A. Not a single protein was bound specifically to gramicidin A. Thus, unspecific binding does not appear to be a problem, at least for gramicidin A. This virtual non-binding to gramicidin coincides clearly with the absence of anti-parasitic activity, and it make sense that an AMP that is capable of interacting specifically with multiple proteins (521 in our study) would exert high anti-parasitic activity. We do not claim that all tyrocidine A-binding proteins identified here are actual drug targets (electrostatic interactions can cause unspecific binding), this would require many more confirmatory experimentation, but this study can give an idea which processes could be impaired upon exposure to tyrocidine.

Comment 3: To evaluate the cytotoxicity of tyrothricin on HFF cells, only two concentrations (0.1 and 1 µM) were used. However, to calculate the selectivity index (SI), the concentration that causes 50% cell death (CC50) is required. Have full dose-response curves been conducted to determine the CC50 of tyrothricin and its components on HFF cells? If so, please provide these data and the calculated SI values.

Response: We have not aimed to calculate a selectivity index, since cytotoxicity was obvious, and the aim of this study was not to propose tyrothricin and its components as novel anti-Toxoplasma compounds that would be suitable for treatment, but rather to investigate the proteins and associated processes potentially targeted by these AMPs.

Comment 4: In the tetramethylrhodamine ethyl ether experiment, total fluorescence is measured in a culture containing both infected and uninfected HFF cells to assess changes in mitochondrial membrane potential. How do you differentiate between the contribution of parasite mitochondria and host cell mitochondria to the fluorescence change? Is it possible that the observed 40% decrease primarily reflects an effect on HFF cells?

Response: Yes, since the reduction is very similar in both uninfected and infected HFF, this is possible. The corresponding statement is on lane 346 ff: Tyrothricin treatment in uninfected as well as in T. gondii infected HFF resulted in a decrease of TMRE uptake of 40% in both cases, indicating that tyrothricin affects the MMP in both T. gondii tachyzoites as well as in HFF.

Comment 5: Transmission electron microscopy data indicate that ultrastructural damage to parasite mitochondria caused by tyrothricin is reversible within 72-96 hours, despite the persistent presence of the peptide. How should we interpret this observation in the context of our search for lethal molecular targets?

Response: The observation that T. gondii is capable of adapting to drug induced adverse conditions has been made for other compounds, as indicated in the discussion (Refs 54, 59,). True lethal molecular targets are very difficult to find, also in the context of the fact that a drug will not only act on one single target. We have not embarked on this discussion in this manuscript, but could, if necessary

Comment 6: Based on the data on decreased metabolic activity (Alamar Blue) of ConA-stimulated splenocytes, it was concluded that tyrothricin has an immunosuppressive effect on T cells. How can you rule out that the observed signal reduction is a consequence of general cytotoxicity against actively proliferating cells rather than specific suppression of the immune response?

Response: this is a good point, and we cannot rule this out. But first, the viability impairment in LPS-stimulated B cells was lower. Secondly, we have not mentioned that there is an immunosuppressive effect, but just that there is a viability impairment of T cells. This could, of course, impact on immunological competence.

Comment 7: In the discussion section, the tone should be consistent with the data. Since the results demonstrate high toxicity and transient effects, the main message should shift from a description of "therapeutic potential" and "multiple targets" to an analysis of non-selective cytotoxicity and challenges in identifying specific targets for cationic peptides. This will create a more honest and compelling narrative.

Response: The reviewer is right in his statement, and, in accordance with reviewer 1, we have added a paragraph in the discussion that points out that point (see lanes 541-545)

Comment 8: The conclusions are misleading, as they are based on an incorrect interpretation of invalid proteomic data (specifically, the DAC-MS experiment which used the chemically distinct gramicidin A as a negative control) and ignore the key results of the original study: high overall toxicity and the transient nature of the antiparasitic effect.

Response: We cannot agree with this statement. The proteomic data is not invalid, but provides a basis for formulating hypotheses with respect to potential targets. Admittedly gramicidin A is not the perfect control for a non-active peptide. We have added a sentence on this aspect into the discussion (lane 567-572).

Whether these hypotheses will be confirmed or not, will be seen in future studies. We have surely not ignored the aspects of high overall toxicity (shared by many other AMPs, including leucinostatin) and the transient nature of the anti-parasitic effects, which are well documented in this paper. It is of course possible (also mentioned in this manuscript), that a range of effects exerted by thyrothricin are host cell mediated. However, corresponding studies are not within the framework of this manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

The study identifies tyrothricin as a novel anti-Toxoplasma gondii agent, emerging as the most potent candidate among the screened peptides. The authors further extend their investigation by systematically characterizing its cytotoxicity profile and employing affinity-based proteomic approaches to elucidate its potential molecular targets within T. gondii. Overall, the manuscript is technically sound and scientifically well-conceived, and it addresses an important gap in the development of alternative therapeutics against toxoplasmosis.

Nevertheless, several minor issues should be addressed to further strengthen the rigor and clarity of the study:

A careful re-examination of Table 1 reveals that several other peptides also exhibit measurable anti-Toxoplasma activity, albeit at lower potency than tyrothricin. To enable a more robust comparison and to better substantiate the selection of tyrothricin as the lead candidate, it would be beneficial to include dose–response curves (e.g., IC₅₀ determination) for these active peptides. This would enhance quantitative interpretation and provide deeper insight into their relative efficacies.

The assessment of mitochondrial dysfunction using the Tetramethylrhodamine ethyl ester (TMRE) uptake assay is a key component of the mechanistic investigation. However, the inclusion of representative fluorescence microscopy images and/or flow cytometry plots would significantly strengthen the evidence for alterations in mitochondrial membrane potential (ΔΨm). Visual and quantitative data would improve reproducibility, clarity, and confidence in the reported mitochondrial effects.

 In Figure 8, it would be informative to also include the proteins associated with gramicidin, allowing for a direct comparative analysis between tyrothricin and a structurally and functionally related peptide. A concise paragraph discussing shared and distinct protein targets, as well as potential overlaps or divergences in their modes of action, would greatly enhance the mechanistic interpretation and contextual relevance of the affinity proteomics results.

Author Response

The study identifies tyrothricin as a novel anti-Toxoplasma gondiiagent, emerging as the most potent candidate among the screened peptides. The authors further extend their investigation by systematically characterizing its cytotoxicity profile and employing affinity-based proteomic approaches to elucidate its potential molecular targets within T. gondii. Overall, the manuscript is technically sound and scientifically well-conceived, and it addresses an important gap in the development of alternative therapeutics against toxoplasmosis

Response: thank your for your assessment

Comment 1: A careful re-examination of Table 1 reveals that several other peptides also exhibit measurable anti-Toxoplasma activity, albeit at lower potency than tyrothricin. To enable a more robust comparison and to better substantiate the selection of tyrothricin as the lead candidate, it would be beneficial to include dose–response curves (e.g., IC₅₀ determination) for these active peptides. This would enhance quantitative interpretation and provide deeper insight into their relative efficacies.

Response: Looking at Table 1, we cannot see any of the other peptides with measurable activity against T. gondii, and dose response experiment would require extensively high AMP concentrations and possibly also host cell toxicity could become a problem. Tyrothricin is the only AMP that impaired T. gondii proliferation at both concentrations. The preliminary screening was done at two concentrations and we did more detailed dose-response studies only with the compound that exhibited interesting activities.

Comment 2: The assessment of mitochondrial dysfunction using the Tetramethylrhodamine ethyl ester (TMRE) uptake assay is a key component of the mechanistic investigation. However, the inclusion of representative fluorescence microscopy images and/or flow cytometry plots would significantly strengthen the evidence for alterations in mitochondrial membrane potential (ΔΨm). Visual and quantitative data would improve reproducibility, clarity, and confidence in the reported mitochondrial effects.

Response: The reviewer is right with this statement. However, we have not included any visual TMRE data, and will not be able to do so. We suggest that the current data on TMRE is not stand-alone, and the obvious alterations in the mitochondrion upon treatment provide also rather convincing evidence for mitochondrial dysfunction.

Comment 3: In Figure 8, it would be informative to also include the proteins associated with gramicidin, allowing for a direct comparative analysis between tyrothricin and a structurally and functionally related peptide. A concise paragraph discussing shared and distinct protein targets, as well as potential overlaps or divergences in their modes of action, would greatly enhance the mechanistic interpretation and contextual relevance of the affinity proteomics results.

Response: There are no proteins that bind exclusively to gramicidin A, and only 4 that bind to both peptides. These are listed in Table 6.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No further comments

Reviewer 3 Report

Comments and Suggestions for Authors

I thank the authors for their work on revising and clarifying my comments. I do not have any new comments.