Surfactin-Induced β-(1,3)-Glucan Exposure in Aspergillus niger Cell Wall: A Molecular Perspective

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article presents an interesting analyses and conformed mode of action of surfactin against cell wall of fungal spores. Despite of a high scientific interest, the article needs improving, especially figures 1 and 3 (dynamic simulation system) need to be better explained for readers, what they present in particular.

Latin names are consequently printed without italics, they are marked in the text. 

Marked points which definetely need to be improved, except mentioned above:

line 133-134 - After 48h of incubation in a medium, there should be germinated spores and young mycelia, unless the temperature inhibited germination, but this should be clarified—the same in line 148.

Table 1 - all shorts should be explained under the table.

Figure 1 - Special colors should be explained for each part of the figure. 

Figure 2 - the same as above, because it is not possible to see the tested component. 

Figure 3 is not fulle described in the text and explained especially part A, B, C. In part D the blue line is not visible. 

Figure 5 comes before Fig. 4

I do not see Table 2. , Table 3 and 4, however 2 and 4 are mentioned in the text. 

 

 

Comments for author File: Comments.pdf

Author Response

Comments 1: The article presents an interesting analyses and conformed mode of action of surfactin against cell wall of fungal spores. Despite of a high scientific interest, the article needs improving, especially figures 1 and 3 (dynamic simulation system) need to be better explained for readers, what they present in particular.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have discussed this part deeply. Mention exactly where in the revised manuscript this change can be found in line221-227, 340-346.

Comments 2: Latin names are consequently printed without italics, they are marked in the text.

Response 2: Thank you for pointing this out. We have corrected them in this manuscript.

Comments 3: line 133-134 - After 48h of incubation in a medium, there should be germinated spores and young mycelia, unless the temperature inhibited germination, but this should be clarified—the same in line 148.

Response 3: Thank you for pointing this out. Mention exactly where in the revised manuscript this change can be found in line126-134.

Comments 4: Table 1 - all shorts should be explained under the table.

Response 4: Thank you for pointing this out. We agree with this comment. Mention exactly where in the revised manuscript this change can be found in line196-197.

Comments 5: Figure 1 - Special colors should be explained for each part of the figure.

Response 5: Thanks for your kind reminder. Mention exactly where in the revised manuscript this change can be found in line 200-202. The red is Surfactin C, the yellow is Iturin A, and the purple is the Fengycin A.

Comments 6: Figure 2 - the same as above, because it is not possible to see the tested component.

Response 6: Thanks for your kind reminder. Mention exactly where in the revised manuscript this change can be found in line 219-220. The red is Surfactin C, the yellow is Iturin A, and the purple is the Fengycin A.

Comments 7: Figure 3 is not fully described in the text and explained especially part A, B, C. In part D the blue line is not visible.

Response 7: Thank you for pointing this out. Mention exactly where in the revised manuscript this change can be found in line 246. The Figure A is a complete dynamic simulation system of Surfactin C and β-glucan, The Figure B showed the part of dynamic simulation system, and the C and D are enlarged images of Figure B.

Comments 8: Figure 5 comes before Fig. 4

Response 8: Thank you for pointing this out. We have modified them in manuscript.

Comments 9: I do not see Table 2, Table 3 and 4, however 2 and 4 are mentioned in the text.

Response 9: Thank you for kind reminder. We have supplemented Table 2 and Table 3. Mention exactly where in the revised manuscript this change can be found in line257, 312.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

the manuscript is focused on elucidating the molecular mechanism of surfactin using Aspergillus niger as target pathogen.

According to my expertise I can contribute especially regarding the biological part related to the "effect of Surfactin on Aspergillus niger". In my point of view the methods are not well described and the results not well presented. According tho the pictures provided it is not clear the difference with respect to the control. 

In addition, in vitro test related to the conidia germoination and colony growth should be perfomed. It would be better rather than working with liquid medium.

Finally, in vivo test by artifical inoculation of berries or other fruits should be performed.

For these reasons, I regret to say that in the current form, I can not support the publication on the manuscript.

 

Author Response

Comments 1: the manuscript is focused on elucidating the molecular mechanism of surfactin using Aspergillus niger as target pathogen. According to my expertise I can contribute especially regarding the biological part related to the "effect of Surfactin on Aspergillus niger". In my point of view the methods are not well described and the results not well presented. According to the pictures provided it is not clear the difference with respect to the control.

Response 1: Thank you for pointing this out. we have modified the methods and results. Additionally, more data of figure 1 and 2 are shown in table 1, with significant difference. Mention exactly where in the revised manuscript this change can be found line 196.

Comments 2: In addition, in vitro test related to the conidia germination and colony growth should be performed. It would be better rather than working with liquid medium.

Response 2: Thanks for your kind suggestions. We have done the experiment in vitro, and the result in Table 2 showed that the minimum inhibitory concentration (MIC) of Surfactin against Aspergillus niger was 40 µg/mL. Additionally, the related inhibition using oxford cup method has been supplemented as follow (Figure S1).

Comments 3: Finally, in vivo test by artificial inoculation of berries or other fruits should be performed.

Response 3: Thank you for pointing this out. We conducted relevant experiments on table grapes and blueberries, with supporting references listed below. We also performed the same analyses on strawberries, these results will be presented in a separate publication (Figure S2). Thank you very much.

References:

Zhang B, Li Y, Zhang Y, Qiao H, He J, Chen X, Fan J. High-cell-density culture enhances the antimicrobial and freshness effects of Bacillus subtilis S1702 on table grapes (Vitis vinifera cv. Kyoho). (2019). Food Chemistry.

Xu L, Zhang B, Qin Y, Li F, Yang S, Lu P, Fan J. Preparation and characterization of antifungal coating films composed of sodium alginate and cyclolipopeptides produced by Bacillus subtilis. (2019). International Journal of Biological Macromolecules.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article can be regarded as an innovative approach to get a better insight into the molecular mechanisms of the inhibitory activity of cycòopeptides (CLPs) against the fungal pathogen Aspergillus niger. Using molecular dynamics simulation, it was demonstrated that CLPs, mainly surfactin,  compromise cell wall integrity binding to β-glucan, a major component of fungus cell wall.  The study was complemented  by morphological observations at both scannong and transmission electron microscopy of alterations of A. niger conidia treated with surfactin.

The subject is original. The experimental design is appropriate. The results are clearly presented and the English style is fine. I noticed two formal flaws that can be easily addressed:

• Latin names of genera and species must be always italicized (Aspergillus niger) and abbreviated (A. niger) after the first citation. See the notes in the text (attached PDF file).
• Surfactin may be written surfactin or Surfactin but use alwys the same form throughout the whole text.

Moreover I have two major doubts:

• Fig. 4. Conidia of A. niger are typically sphaerical; in the picture they appear flattened (also in the non treated control), I suspect a preparation artifact (may be during dehydration). Caption: provide a more detailed description of TEM images.
• I am wondering if the subject fits the scope of a journal dealing with food. 

Comments for author File: Comments.pdf

Author Response

Comments 1: The article can be regarded as an innovative approach to get a better insight into the molecular mechanisms of the inhibitory activity of Cyclic lipopeptides (CLPs) against the fungal pathogen Aspergillus niger. Using molecular dynamics simulation, it was demonstrated that CLPs, mainly surfactin, compromise cell wall integrity binding to β-glucan, a major component of fungus cell wall. The study was complemented by morphological observations at both scannong and transmission electron microscopy of alterations of A. niger conidia treated with surfactin. The subject is original. The experimental design is appropriate. The results are clearly presented and the English style is fine. I noticed two formal flaws that can be easily addressed: Latin names of genera and species must be always italicized (Aspergillus niger) and abbreviated (A. niger) after the first citation. See the notes in the text (attached PDF file).

Response 1: Thank you a lot. Latin names have been italicized in this manuscript.

Comments 2: Surfactin may be written surfactin or Surfactin but use always the same form throughout the whole text.

Response 2: Thank you for pointing this out. We have unified them in this manuscript.

Comments 3: Fig. 4. Conidia of A. niger are typically sphaerical; in the picture, they appear flattened (also in the non-treated control), I suspect a preparation artifact (may be during dehydration).

Response 3: Thanks for your kind suggestion. We have taken pictures according to the references.

Comments 4: Caption: provide a more detailed description of TEM images.

Response 4: Thank you for pointing this out. We have supplemented the detailed description of TEM images in line 145.

Comments 5: I am wondering if the subject fits the scope of a journal dealing with food.

Response 5: Thanks a lot.

Author Response File: Author Response.pdf