Fisetin inhibits Salmonella Typhimurium type III secretion system regulator HilD and reduces pathology in vivo

ABSTRACT Salmonella enterica is an important zoonotic intracellular bacterial pathogen that is capable of causing infections ranging from localized gastroenteritis to fatal systemic infection in humans and food-producing animals. The increasing antibiotic resistance in Salmonella isolates, especially the emergence of MDR and newer XDR strains, has compromised the efficacy of conventional antimicrobial therapy for Salmonella infections. Hence, it is desirable to develop alternative therapeutic means to tackle the antimicrobial resistance crisis. In this study, we screened plant-derived compounds to identify inhibitors of Salmonella invasion of host cells. These efforts identified fisetin as a possible protector against infection. Further mechanistic studies revealed that fisetin suppressed the function of type III secretion system 1 (T3SS-1), the virulence determinant critical for Salmonella invasion. Fisetin appears to interfere with the interaction between HilD and its relevant promoters, thereby decreasing the transcription of hilA, the central transcriptional regulator that functions to activate the expression of T3SS-1 effector proteins and structural elements. In addition, administration of fisetin in the Salmonella murine infection model resulted in reduced bacterial colonization, alleviation of histopathological destruction, and decreased proinflammatory cytokine levels. Taken together, our study establishes that the natural compound fisetin can be used as a lead compound for the development of anti-Salmonella drugs targeting T3SS-1. IMPORTANCE Salmonella spp. remains a major worldwide health concern that causes significant morbidity and mortality in both humans and animals. The spread of antimicrobial resistant strains has declined the efficacy of conventional chemotherapy. Thus, novel anti-infection drugs or strategies are needed. Anti-virulence strategy represents one of the promising means for the treatment of bacterial infections. In this study, we found that the natural compound fisetin could inhibit Salmonella invasion of host cells by targeting SPI-1 regulation. Fisetin treatment impaired the interaction of the regulatory protein HilD with the promoters of its target genes, thereby suppressing the expression of T3SS-1 effectors as well as structural proteins. Moreover, fisetin treatment could reduce pathology in the Salmonella murine infection model. Collectively, our results suggest that fisetin may serve as a promising lead compound for the development of anti-Salmonella drugs.


Introduction.
1. Lines 75-78.Information regarding illness and death in human -readers should be referred to the original publication.Citing (#5) is wrong and should be changed.2. Lines 78-79.The reference is from 2003 so I suggest looking for more recent works before saying "...gastroenteritis remains one of the primary causes...". 3. Lines 80-81.Please separate the information regarding humans and animals.
4. Lines 109-112.Previous works of the authors.The readers should be referred to published works or this information should be added to the current manuscript.
Results. 5.The title of this manuscript describes assays with S. Typhimurium.Nevertheless, some assays were performed with S. Enteritidis.I recommend including these results in the manuscript alongside S. Typhimurium and not as complementary information.6. Line 167 and Figure 2D.It is unclear how the secretion levels between treatments were compared for the authors to determine "... that 4 µg/mL fisetin resulted in significantly reduced secretion of SipA and SipB...".Please check.7. Line 220 and Figure 6.Please name the genes in both places in the same order.8. Line 237.The HilD promoter complex is not available in the manuscript and should be either described or referenced.9. Lines 246-251.This information should be transferred to Material and Methods.How did the authors decide about the dose of fisetin and the treatment procedures?10.Line 271 and Figure 8E.How did the authors compare pathological damage in tissues?It is not clear if they claim that "...fisetin remarkably alleviated the pathological damage...".Discussion 11.The discussion should discuss the results of this investigation.However, much of it should be transferred into the introduction.Please reorganize this whole section.12. Line 310.Please cite the statement "...antivirulence drugs are not supposed to ....". 13.Lines 336 -337.I do not agree with ".... administration of fisetin showed a significant therapeutic effect...".See other comments and please stick to the facts.14.Line 344.Please state that the animals infected with L. monocytogenes were mice.
Materials and Methods 15.Lines 352-353.Bacterial strains that come from previous works of the authors should be referred to published works or more information should be added to the current manuscript.16.Line 359.Please describe briefly the modification of LB medium.17.General information regarding routine methods like PCR, thermocycling conditions, western blot, etc should be cited and only modification should be stated.18. Please add information regarding the definition of the PCR primers that were used.Were they designed by the authors or other publications?19.The murine trial should be described with more information: a.The number of mice that were used and justified.b.Why were only female mice included?c.A fourth group is missing -treatment with fisetin as a control.The authors should explain why it was not included.d.More information is needed for the infective dose of S. Typhimurium -how was it fixed?In the results (lines 253-254), 100% mortality is described in the non-treated mice and only 20% survival for the treated ones.This is a very poor "improvement".Why was not the LD50 tested and used?e.The volume of inoculation should be stated.f.Why histological samples were taken from different mice inoculated with a lower dose of S. Typhimurium and not from already dead/euthanized mice of the other trial?Furthermore, it is not clear if the histological samples were taken on day 9 post-infection from dead or euthanized mice.g.Was the administration of fisetin done individually or in the drinking water?h.The method of euthanization should be described.i. Treated mice had significantly reduced bacterial colonization, alleviation of histopathological destruction, and decreased proinflammatory cytokine levels.Nevertheless, mice mortality was only slightly reduced and it was not statistically different so it is not correct to say that the "administration of fisetin can significantly protect Salmonella infection in a murine model" (line 37).No real protection was observed.Authors should stick to the facts.20.Title: The natural compound fise n protects against Salmonella Typhimurium infec on via inhibi on of the type III secre on system Li, et al Microbiology Spectrum Review Overall comments In general, the paper is well considered.The authors state that there is protec on without a significant change in mouse outcomes, which should be avoided.However, the reduc on in biomarkers and organ invasion is promising and supports their other conclusions, and their in vitro data is thorough.The sum of experimental data here is good and demonstrates a likely mechanism through gel-shi assay, protein abundance, and transcript abundance analysis.They then show ac vity in a murine model.While a change in death rates is not observed, they show a reduc on in important biomarkers, such as the level of organ infiltra on and cytokine prolifera on.
The paper would benefit from condensing of several figures in the results, though I leave it to the editor whether this is necessary given their journal format.The standard set by the journal is a review of the scien fic content and methodological rigor, which is sufficient except for some control data in Figures 8 and 9.
For Figure 8 and 9, there is no control showing the effects of fise n alone, and the non-infected state should be shown for comparison in both figures as a baseline.Given that fise n is bioac ve in several different contexts, including an -inflammatory, it is important to control for these effects.Figure 9 needs cytokine levels for the non-infected mice and fise n alone mice.
The manuscript should be reviewed for introductory and methods material in the results, as well as other issues highlighted in the comments below.Lastly, the discussion has too much introductory informa on, and should be significantly shortened.

Specific comments
Title: Needs revision.Protec on is against infec on.Authors demonstrate ac vity against HilD binding of promoter sequence in vitro, and consistent observa ons in other tests, so tle should be re-wri en to reflect this.Something like: Fise n inhibits Salmonella Typhimurium type III secre on system regulator HilD and reduces pathology in vivo.
While I agree that the molecule shows promise and a reduc on in disease biomarkers and pathological hallmarks, the lack of a significant difference in mouse outcomes means I advise against claiming protec on and encourage other descriptors.
Abstract: Sufficient, but needing some revision to be er reflect the results and for improved readability.
Line 25-27: Authors should state the an microbial resistance has been rising for Salmonella specifically, maybe even men on the emergence of more MDR and newer XDR strains.Line 26: remove "remarkably" Line 27: remove "highly", I would reconsider calling this "necessary".Consider replacing with "sought" or "desirable", as in "Hence, alterna ve therapeu c means to aid treatment of invasive Salmonella infec ons.Line 29: "plant-derived" is superior to "natural", remove "natural".Line 30: Sugges on: "These efforts iden fied fise n as…" Line 33: Replace "could" with "appears to" or something similarly stronger.Evidence in paper supports stronger language.Line 37: "significantly protect" should be skipped since there is no improvement in mouse survival.Authors can s ll reference the biomarkers a erwards as signs that fise n may have some value.
Importance: Needs revision for readability and English.Some minor revision to content.Line 49: "urgently demanded" -replace with "needed", "desired", or something similar.Line 52: "targe ng T3SS-1" -fise n appears to target SPI-1 regula on, not the T3SS itself.Line 56: Again, protec on is not warranted here, should men on reduc on of biomarkers.Line 57: Rewrite for clarity and grammar.
Introduc on: Generally well considered, but should be shortened to remove unnecessary details, such as the breadth of serovars and their cause as they are not needed to understand the paper, or the details of T3SS-2/SPI-2 func on.Line 83: "misuse" instead of "use" Line 85: Some more comment on the emergence of XDR Typhi may be warranted here.Line 105: "primarily present" -consider "naturally found" Line 116: Recurrent use of protec on oversells results.Line 117: Authors should consider that while they did not see a protec on from a death endpoint in the mouse model, fise n or a related agent may be used in combina on with other exis ng therapies.
Results: Some figures need to be combined to reduce the total number of figures, primarily figures 3 to 7, and Figure 8 and 9.The overall considera on of experiments is good, specific comments will be found line by line below.
Figure 1: Did authors have a posi ve control for the MIC assay where kanamycin/gentamicin prevented color change.May include with supplementary material, not cri cal for acceptance.Subfigure C: Use "ST + Fise n" as in Fig. 9. Subfigure E -include "µg/ml".Line 122: Introductory material, remove.Line 140: change to "non-an bacterial fise n inhibits S. Typhimurium invasion."Line 160: Methods material here isn't needed -curious readers can find this in the methods.Figure 8: 8B y-axis label can be simply "CFU/g ssue".Samples should be "ST + Fise n".Declare that ST is an abbrevia on of S. Typhimurium in the legend.NC stands for "Nega ve control" Please specify in legend.Also, include infec ng dose, which is different between A and B.
As men oned above, the experiments need an interven on-only control or the cita on or relevant literature.
Line 245: Again, the use of protec on here without a mouse endpoint is not warranted.
Line 249: It would help the reader to include a dosing method here, I assume oral gavage?Line 252: There is no sta s cal power here to claim a difference.
Line 252: Infec ous doses need to be listed to dis nguish bet0ween 8A and 8B.There is one for determining a survival phenotype, and one that is sub-lethal.Discussion: This sec on could use significant revision, and I recommend moving or removing the first two paragraphs.Of note, the XDR/MDR content here should be in the introduc on and helps inform the reader.
What is needed here is a broader view of other drugs or treatments that have shown promise or are in development, how the mouse model signs here show promise even though there was no significant survival phenotype.It may also be worth postula ng how a drug with ac vity like that demonstrated in the mouse model would intersect with modern therapy, and whether the authors see this as being developed further into a drug that alone combats disease, or is part of a regimen.
Methods: Appear appropriate and reproducible with the following excep on: Mul ple manufacturers for different components needed, including for fise n, and whether a different grade or manufacturer was used between cell culture and mouse experiments.

Supplemental material:
Table S4: The whole sequences probed for the EMSA should be declared along with the promoters used to amplify.

Responses to Reviewer #1
Reviewer #1 (Comments for the Author): Introduction.
1. Lines 75-78.Information regarding illness and death in human -readers should be referred to the original publication.Citing (#5) is wrong and should be changed.
Response: Thanks for your advice.We have replaced the reference #8.Line 82: "…million Salmonella infection-related annual deaths 8 " 2. Lines 78-79.The reference is from 2003 so I suggest looking for more recent works before saying "...gastroenteritis remains one of the primary causes...".
Response: Thanks for your comment.We have replaced the reference #9 with a recent publication.Please find this in lines 82-84 as well as the reference list in the revised manuscript.
Lines 82-84 in the revised manuscript: "In particular, gastroenteritis remains one of the primary causes of morbidity and mortality in children younger than 5 years 9 ."3. Lines 80-81.Please separate the information regarding humans and animals.
Response: Thanks for your comment.We have revised this sentence.
Lines 87 in the revised manuscript: "…for diseases caused by Salmonella in humans and animals 11 ."4. Lines 109-112.Previous works of the authors.The readers should be referred to published works or this information should be added to the current manuscript.
Response: Thanks for your comment.The screening of invasion inhibitors was conducted in the present study but not from our previous work, and the screening method was shown in the Materials and Methods (Please see the lines 349) and Figure 1A.
Revised Figure 1 Results.
5. The title of this manuscript describes assays with S. Typhimurium.Nevertheless, some assays were performed with S. Enteritidis.I recommend including these results in the manuscript alongside S. Typhimurium and not as complementary information.
Response: Thanks for your comment.We tried to follow your recommendation and combine the results of S. Enteritidis and S. Typhimurium (Figure 1 plus Supplementary Figure 1).We feel that the combined figure is too repetitious.
As we performed the same experiments for S. Enteritidis, the data presentation is quite similar as for the S. Typhimurium.Therefore, we leave the S. Enteritidis in the supplementary figure 1.However, if the reviewer hopes to move this data to the main manuscript, we can present the data as follows.Response: Thanks for your comment.We have used SipA-and Flag-specific antibodies to detect the secretion levels of SipA and SipB.FliC, the flagellin of bacteria, whose secretion is SPI-1 independent, was used as a loading control.Obviously, compared to the fisetin-free culture, the secretion of SipA and SipB in S. Typhimurium cultured with 4 µg/mL fisetin was reduced.To further quantify the alteration between different treatments, we calculated the density ratios of SipA/SipB against FliC using imageJ.Please find these data in revised Figure 2.
Revised Figure 2 7. Line 220 and Figure 6.Please name the genes in both places in the same order.
Response: Thanks for your advice.We have adjusted the order of genes in the manuscript to match those in the Figure 6.Please see the figure 6 and lines 220-222 in the revised manuscript.
Response: Thanks for your comment.We have revised the labeling of Figure 7. HilD-DNA was changed into HilD-P hilA and HilD-P hilD complex.
Revised Figure 7 9. Lines 246-251.This information should be transferred to Material and Methods.How did the authors decide about the dose of fisetin and the treatment procedures?
Response: Thanks for your suggestion.We have deleted this information as your suggestion.
We have deleted Lines 247-251 in the original submission: "Three days before S. Typhimurium infection, mice were administered streptomycin in their drinking water.Mice in the treatment group were given 100 mg/kg fisetin at 12 h intervals, while the control mice were administered an equal volume of solvent on the same schedule." In our preliminary experiments, orally administration of mice with 100 mg/kg of fisetin at 12 h-interval for 4 days did not cause toxicity to mice.Due to the long survival time of mice in the Salmonella gastroenteritis model, we chose to administer gavage every 12 hours for 4 days in an attempt to observe better treatment outcomes.However, this regimen was still not ideal as no significant improvement of the survival rate was observed.We have discussed the potential reasons in the discussion section.10.Line 271 and Figure 8E.How did the authors compare pathological damage in tissues?It is not clear if they claim that "...fisetin remarkably alleviated the pathological damage...".
Response: Thanks for your comment.The pathological damages in the tissues were described in Lines 262-268: "The pathological tissue section showed that in the infection control mice, hepatocytes were lysed and dissipated, which was accompanied by fatty degeneration and a congested central vein; the spleen was soaked by inflammatory cells, and capillaries showed extensive hyperemia and congestion; the cecum exhibited submucosal edema, and the lumen accumulated a large number of exfoliative necrotic epithelial cells and intestinal villi (Fig. 8D)."In S. Typhimurium-infected mice that receiving fisetin treatment, the abovementioned histopathological changes were alleviated.However, we realized that use "remarkably" is not appropriate here since no quantification was applied.We have revised the description as follows.
Lines 268-270 in the revised manuscript: "Compared to the infection control mice, treatment of S. Typhimurium-infected mice with fisetin could alleviate the pathological damages of the spleen, liver, and cecum (Fig. 8D)." Discussion 11.The discussion should discuss the results of this investigation.However, much of it should be transferred into the introduction.Please reorganize this whole section.
Response: Thanks for your comment.We have reorganized this section.The first paragraph was transferred into the introduction section.In addition, we also discussed the poor survival outcomes of fisetin treatment.
Response: Thanks for your suggestion.We have added a reference for this statement.Please find the citation in the reference list (#28) Line 298: "…supposed to impose evolutional pressure as antibiotic treatment 28 " 13.Lines 336 -337.I do not agree with ".... administration of fisetin showed a significant therapeutic effect...".See other comments and please stick to the facts.
Response: Thanks for your comment.We have revised the sentence as your suggestion.
Line 324 in the revised manuscript:".... administration of fisetin showed certain therapeutic effect..." the.14.Line 344.Please state that the animals infected with L. monocytogenes were mice.
Response: Thanks for your advice.We have revised it accordingly.Response: Thanks for your comment.In order to stimulate T3SS-1 secretion, Salmonella strains were cultured at 37 °C with high-salt LB medium containing 0.3 M NaCl.
Line 360 in the revised manuscript: "Salmonella strains were cultured at 37 °C with high-salt LB medium containing 0.3 M NaCl" 17.General information regarding routine methods like PCR, thermocycling conditions, western blot, etc should be cited and only modification should be stated.
Response: Thanks for your advice.We have deleted the description of routine methods accordingly.These procedures were performed according to previous publications.
We have deleted Lines 445-449 in the original submission: "After separation by SDS-PAGE, proteins on the gel were transferred onto PVDF membranes (Pall Life Sciences) by a wet transfer system.Following a blocking step with 5% (w/v) skim milk for 1 h at room temperature, the membranes were probed with appropriate primary antibodies for 1 h."Lines 449 in the revised manuscript: "The Western blot procedure was performed as previously described 42 ."18. Please add information regarding the definition of the PCR primers that were used.Were they designed by the authors or other publications?
Response: Thanks for your comment.The qRT-PCR primers that were described in previous studies were deleted and replaced with a reference.
Please find the revision in revised Table S2.Other primers listed in Table S2, S3 and S4 were designed in this study.
Lines 472-474 in the revised manuscript: "The qRT-PCR primers specific for sipA, sipB, sipC, hilA, hilC, hilD, rtsA and gyrB were described earlier 43 ; while primers specific for prgH, prgI, prgK and invG were listed in Table S3." 19.The murine trial should be described with more information: a.The number of mice that were used and justified.
Response: Thanks for your comment.In the mice survival rates, 10 mice were used in each group.In the experiments involved in inoculating mice with sublethal doses, such as bacterial colonization in the organs, histopathology observations, and cytokine measurement, there were five mice each group, where the experiment to detect proinflammatory cytokines used data from only three mice.We have revised in the manuscript.
Nanocapsules loaded with iron-saturated bovine lactoferrin have antimicrobial therapeutic potential and maintain calcium, zinc and iron metabolism.Nanomedicine (London, England), 10( 8 Revised Figure 8 d.More information is needed for the infective dose of S. Typhimurium -how was it fixed?In the results (lines 253-254), 100% mortality is described in the non-treated mice and only 20% survival for the treated ones.This is a very poor "improvement".Why was not the LD50 tested and used?
Response: Thanks for your comment.The infective doses of S. Typhimurium were fixed based on our extensive preliminary experiments.1x10 7 CFUs is the minimal infection dose that can cause death of all infected mice within 9 days.While 5x10 6 CFUs is the optimal dose that causes significant pathological changes and organ bacterial colonization without leading to any death.20% survival is indeed poor "improvement".No toxicity was reported up to 2000 mg/kg b. wt. of fisetin when administered orally as a single oral dose to mice (Currais et al., 2014).Therefore, the LD 50 should be very high.We actually tried higher doses which did not improve the survival rate either.We have analyzed the potential reasons in the discussion section.In general, the paper is well considered.The authors state that there is protection without a significant change in mouse outcomes, which should be avoided.However, the reduction in biomarkers and organ invasion is promising and supports their other conclusions, and their in vitro data is thorough.
The sum of experimental data here is good and demonstrates a likely mecha-nism through gel-shift assay, protein abundance, and transcript abundance analysis.They then show activity in a murine model.While a change in death rates is not observed, they show a reduction in important biomarkers, such as the level of organ infiltration and cytokine proliferation.
Response: Thanks for your comment.
The paper would benefit from condensing of several figures in the results, though I leave it to the editor whether this is necessary given their journal format.The standard set by the journal is a review of the scientific content and methodological rigor, which is sufficient except for some control data in Fig- For Figure 8 and 9, there is no control showing the effects of fisetin alone, and the non-infected state should be shown for comparison in both figures as a baseline.Given that fisetin is bioactive in several different contexts, including anti-inflammatory, it is important to control for these effects.Figure 9 needs cytokine levels for the non-infected mice and fisetin alone mice.
Response: Thanks for your comment.In our experiments, we actually contained fisetin alone group.However, since treatment with fisetin could not count the bacterial load of organs, these data were not included in our original submission in order to maintain the consistency of Figure 8B with other experiments.In the revised Figure 8 and 9, we have added these data.
The manuscript should be reviewed for introductory and methods material in the results, as well as other issues highlighted in the comments below.Lastly, the discussion has too much introductory information, and should be significantly shortened.
Response: Thanks for your comment.We have carefully reviewed the results section and removed some introductory and method descriptions.Please find these in our detailed responses to specific comments.In addition, we also reorganized the discussion section as your suggestions.Specific comments 1.Title: Needs revision.Protection is against infection.Authors demonstrate activity against HilD binding of promoter sequence in vitro, and consistent observations in other tests, so title should be re-writien to reflect this.Something like: Fisetin inhibits Salmonella Typhimurium type III secretion system regulator HilD and reduces pathology in vivo.
While I agree that the molecule shows promise and a reduction in disease biomarkers and pathological hallmarks, the lack of a significant difference in mouse outcomes means I advise against claiming protection and encourage other descriptors.
Response: Thanks for the constructive comments.We have realized that using "protection" is not appropriate according to the poor mouse outcomes in the survival rates experiment.Therefore, we have revised the title as your advice.
The revised title: "Fisetin inhibits Salmonella Typhimurium type III secretion system regulator HilD and reduces pathology in vivo" 2 Abstract: Sufficient, but needing some revision to better reflect the results and for improved readability.
Response: Thanks for your comment.We have made changes for the "abstract" section according to your detailed suggestions.
Line 25-27: Authors should state the antimicrobial resistance has been rising for Salmonella specifically, maybe even mention the emergence of more MDR and newer XDR strains.
Response: Lines 26-27 in the revised manuscript: "…especially the emergence of more MDR and newer XDR strains…" Line 26: remove "remarkably" Response: Line 27 in the revised manuscript, "remarkably" was removed.
We have deleted Line 26 in the original submission: "…has remarkably compromised the efficacy of conventional…" Line 27 in the revised manuscript: "…has compromised the efficacy of conventional antimicrobial therapy for Salmonella infections…" Line 27: remove "highly", I would reconsider calling this "necessary".Consider replacing with "sought" or "desirable", as in "Hence, alternative therapeutic means to aid treatment of invasive Salmonella infections.
Response: Line 29 in the revised manuscript: "highly" was removed and "necessary" was substituted with "desirable".
Line 29 in the revised manuscript: "Hence, it is desirable to develop alternative therapeutic means…" Line 29: "plant-derived" is superior to "natural", remove "natural".
Response: Line 30 in the revised manuscript, we have used "plant-derived" instead of "natural".
"…we screened plant-derived compounds to identify inhibitors of Salmonella invasion…" in line 30.Line 30: Suggestion: "These efforts identified fisetin as…" Response: Line 32 in the revised manuscript: "These efforts identified fisetin as…" Line 33: Replace "could" with "appears to" or something similarly stronger.Evidence in paper supports stronger language.
Response: Line 35 in the revised manuscript: "Fisetin appears to interfere with the interaction between HilD and its..." Line 37: "significantly protect" should be skipped since there is no improvement in mouse survival.Authors can still reference the biomarkers afterwards as signs that fisetin may have some value.
Response: Thanks for your advice.We have deleted "administration of fisetin can significantly protect Salmonella infection in a murine model" and rephrased this sentence.
Lines 38-41 in the revised manuscript: "In addition, administration of fisetin in Figure 1A.There is no mention of the 29 compounds in the text.Please change the Figure or add more information to the manuscript.21. Figure 1E.Please define if this figure describes concentrations or OD and use the correct terms in the description.22. Figure 2. Please add "****" to p<0.0001.23.Figures 4A and 6C.Please add significance level and description to only one *.24. Figure 9.Only one level of significance is shown.Please omit the others.25. Figure 1S.Please add the description of levels of significance Staff Comments:

Figure 2 :
Figure 2: Consider labeling C and D as "supernatant" to dis nguish visually from Figure 3. NOTE: Please include expected molecular weights in figures (MULTIPLE FIGURES) or text.

Figure 9 :
Figure 9: Label y-axes with the cytokine measured.Note use of ST + Fise n here should be used for Figure 7 and elsewhere.Consider combining with Figure 8. Please include cytokine levels for the control mice as well.
6. Line 167 and Figure 2D.It is unclear how the secretion levels between treatments were compared for the authors to determine "... that 4 µg/mL fisetin resulted in significantly reduced secretion of SipA and SipB...".Please check.
),1289-1314.doi:10.2217/nnm.14.209Kong, W., Wanda, S.-Y., Zhang, X., Bollen, W., Tinge, S. A., Roland, K. L., &   Curtiss, R. (2008).Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment.Proceedings of the National Academy of Sciences of the United States of America, 105(27), 9361-9366.doi:10.1073/pnas.0803801105c.A fourth group is missing -treatment with fisetin as a control.The authors should explain why it was not included.Responses: Thanks for the comment.In our experiments, we actually contained the drug treatment control.Since treatment with fisetin could not count the bacterial load of organs, these data were deleted in order to maintain the consistency of Figure 8B with other panels.According to your suggestion, we have added the group of only treatment with fisetin to Figure 8. Please find our revision in revised Figure 8A, Figure 8C and Figure 8D.
for your comment."*, P< 0.05." have been added in the manuscript.Please see the lines 739 and 761 of the revised manuscript.Lines 739 in the revised manuscript: "…**, P< 0.01; *, P< 0.05."Lines 761 in the revised manuscript: "…P< 0.001; **, P< 0.01; *, P< 0.05."24. Figure 9.Only one level of significance is shown.Please omit the others.Response: Thanks for your comment.Figure 9 added data.The level of significance was added to its legend."…****, P< 0.0001; ***, P< 0.001; **, P< 0.01; *, P< 0.05."Please see the line 794 of the revised manuscript.25. Figure 1S.Please add the description of levels of significance.Response: Thanks for your comment."****, P< 0.0001; **, P< 0.01" have been added in the manuscript.Please see the line 807 of the revised manuscript.Line 807 in the revised manuscript: "… independent experiments.****, P< 0.0001; **, P< 0.01."Responses to Reviewer #2 Reviewer #2 (Comments for the Author): Title: The natural compound fisetin protects against Salmonella Typhimurium infection via inhibition of the type III secretion system Li, et al Microbiology Spectrum Review Overall comments ures 8 and 9. Response: Thanks for your comment.We actually have tried to combine Figure 8 and 9 into one figure.However, it looks too crowd.We went through recent issues of Microbiology spectrum, and 9 main figures is allowed for the journal.