Clostridium perfringens Delta-Toxin Damages the Mouse Small Intestine
Round 1
Reviewer 1 Report
The manuscript is well-written and the experiments are properly designed. The new data further strengthen their earlier works that showed Clostridium perfringens delta-toxin causes intestinal damage through disrupting E-cad barrier. I therefore recommend immediate publication.
Author Response
Comments and Suggestions for Authors
The manuscript is well-written and the experiments are properly designed. The new data further strengthen their earlier works that showed Clostridium perfringens delta-toxin causes intestinal damage through disrupting E-cad barrier. I therefore recommend immediate publication.
Answer. Thank you for your recommendation.
Reviewer 2 Report
The manuscript describes the pathological damage in the ileal loop and the small intestine induced by delta toxin. My main criticism is that the authors use trypsin inhibitor in their experiments. Obviously, in the “real world” this toxin is secreted to the lumen where trypsin and maybe other proteases are present, therefore call into question the relevance of these findings. The authors cited the 2008 paper which have also used TI for their experiments, yet in the cited paper they have indicated that without the addition of TI the toxin was not active. Thus, the authors should revise the manuscript and include an explanation in the results section (lines 59-70) to their decision to include the TI in their experiments and also add data to the activity of this toxin in the absence of TI, using the same in vivo system. The authors should also add a paragraph in the discussion section and discuss the relevance of TI in this system and how it simulate the “real world”, compare it to other manuscripts that included TI in their experiments and more.
More major comments:
- Lines 128 – What was the author’s hypothesis that the pathological changes in the small intestinal villi will be different from the results obtained in the ileum sections?
- A paragraph should be added to the discussion section to compare the toxicity of beta-toxin to that of delta-toxin, in the same in vivo model. The authors should refer to the toxins concentration, severity of the pathological changes etc, in order to convince the readers that the delta-toxin is indeed as toxic as the beta-toxin.
Minor comments
- Lines 42-44 – add reverences to these two sentences.
- Line 69 – this is a anti-serum and not “antibody”. In addition, what is known about the neutralization activity and potency of this anti-serum?
- Line 107 – change “one for six” to “one of six”
- Lines 108 and 126 – it is not clear how many experiments were performed (6 or 10 and 3 or 6)?
- Line 122 – change red to green
- Line 128 – delete “in”
Author Response
Reviewer 2
Comments and Suggestions for Authors
The manuscript describes the pathological damage in the ileal loop and the small intestine induced by delta toxin. My main criticism is that the authors use trypsin inhibitor in their experiments. Obviously, in the “real world” this toxin is secreted to the lumen where trypsin and maybe other proteases are present, therefore call into question the relevance of these findings. The authors cited the 2008 paper which have also used TI for their experiments, yet in the cited paper they have indicated that without the addition of TI the toxin was not active. Thus, the authors should revise the manuscript and include an explanation in the results section (lines 59-70) to their decision to include the TI in their experiments and also add data to the activity of this toxin in the absence of TI, using the same in vivo system. The authors should also add a paragraph in the discussion section and discuss the relevance of TI in this system and how it simulate the “real world”, compare it to other manuscripts that included TI in their experiments and more.
Answer. We agree with you. We added the results in the absence of TI to pages 72-73 and 90-91. In addition, an explanation considering the role of TI in the real-world of type C infection was added to discussion in line 178-184.
More major comments:
- Lines 128 – What was the author’s hypothesis that the pathological changes in the small intestinal villi will be different from the results obtained in the ileum sections?
Answer: Observation of changes in small intestinal villi caused by delta-toxin was examined at 1 hour after administration. In contrast, pathological changes due to toxin in the ileal section were observed at 3 hours after administration. Thus, changes in villi induce by the toxin are an initial damaging effect of the toxin. Delta-toxin initially changes E-cadherin and induces shedding and caspase-3 activation, which are thought to cause histological damage of the ileum. From the above, changes in villi caused by delta-toxin are associated with intestinal injuries.
- A paragraph should be added to the discussion section to compare the toxicity of beta-toxin to that of delta-toxin, in the same in vivo model. The authors should refer to the toxins concentration, severity of the pathological changes etc, in order to convince the readers that the delta-toxin is indeed as toxic as the beta-toxin.
Answer. We agree with you. We added the sentences in discussion line 184-187.
Minor comments
- Lines 42-44 – add reverences to these two sentences.
Answer. Reviewer 2 states "reverences", but we think "references". We added the references in line 43 and 44.
Line 69 – this is an anti-serum and not “antibody”. In addition, what is known about the neutralization activity and potency of this anti-serum?
Answer. OK. We mistook. We changed the word in line 70. We reported this antibody in a PLOS one (Seike et al. PlosOne 11, e0147957 (2016)). We have confirmed that this antibody neutralizes the cytotoxicity of the toxin.
- Line 107 – change “one for six” to “one of six”
Answer. OK. We corrected the word, as suggested in line 112.
- Lines 108 and 126 – it is not clear how many experiments were performed (6 or 10 and 3 or 6)?
Answer. OK. We corrected the sentences, as suggested in line 112-113 and 131-132.
- Line 122 – change red to green
Answer. OK. We mistook. We changed the word in line 127 and 149.
- Line 128 – delete “in”
Answer. OK. We mistook. We deleted the word in line 133.
Reviewer 3 Report
In this study, using purified delta toxin from C. perfringnes, the authors measured the extent of fluid accumulation, paracellular permeabiliity, villus shortening, E-cadherin degradation, and lastly, shedding of intestinal epithelial cells. Overall, the experiments proposed here were carried out clearly and the results were explained clearly as well. However, this is a observational driven study, and it is this reviewer's view that more insights could be gain by performing additional experiments or include more discussion. Specific comments are listed below:
1. Earlier in the introduction, the authors mentioned that the role of delta-toxin in pathogenesis is unknown since C. perfringens strain B and C contains other toxins. The authors' expertise are not in making mutants in C. perfringens, however, it's still unclear after reading this manuscript that during infection, delta toxin plays a significant role. Are the concentration of toxins used in this study relevant to biological condition?
2. Delta-toxin has been thought to act in the same manner as alpha toxin. Can the authors either perform similar test along with alpha toxin, or provide more discussion on how the results generated from this manuscript can help answer that questions?
3. The authors’ previous study using Caco-2 cells and purified delta-toxin has established many similar observations gained from this study. Can the authors explain what is the novelty behind this study?
Author Response
Comments and Suggestions for Authors
In this study, using purified delta toxin from C. perfringnes, the authors measured the extent of fluid accumulation, paracellular permeabiliity, villus shortening, E-cadherin degradation, and lastly, shedding of intestinal epithelial cells. Overall, the experiments proposed here were carried out clearly and the results were explained clearly as well. However, this is a observational driven study, and it is this reviewer's view that more insights could be gain by performing additional experiments or include more discussion. Specific comments are listed below:
1. Earlier in the introduction, the authors mentioned that the role of delta-toxin in pathogenesis is unknown since C. perfringens strain B and C contains other toxins. The authors' expertise are not in making mutants in C. perfringens, however, it's still unclear after reading this manuscript that during infection, delta toxin plays a significant role. Are the concentration of toxins used in this study relevant to biological condition?
Answer. We agree with you. It is unclear whether the toxin concentration used is related to the biological condition, as pointed out. To date, there have been no reports of delta-toxin showing damage to the intestine, and for the first time, it has been clarified that delta-toxin causes the intestinal injury. In other words, this study focuses on the damage to the small intestine of delta-toxin. Since C. perfringens produce various toxins, it is difficult to find out the conditions, but we will study further in the future.
2. Delta-toxin has been thought to act in the same manner as alpha toxin. Can the authors either perform similar test along with alpha toxin, or provide more discussion on how the results generated from this manuscript can help answer that questions?
Answer OK. I added the explanation in line 208-212.
3. The authors’ previous study using Caco-2 cells and purified delta-toxin has established many similar observations gained from this study. Can the authors explain what is the novelty behind this study?
Answer. OK. We have previously reported that delta-toxin activates ADAM10 in caco-2 cells to cleave E-cadherin and damages the barrier. In this report, from the previous report, it has been clarified that delta-toxin activates ADAM10 to cause intestinal injury even in the mouse small intestine. That is, it is a new finding that ADAM10 is activated to exhibit intestinal tract injury action.
Round 2
Reviewer 2 Report
Accept in present form
