Akkermansia muciniphila identified as key strain to alleviate gut barrier injury through Wnt signaling pathway
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eLife Assessment
The work by Han and collaborators describes valuable findings on the role of Akkermansia muciniphila during ETEC infection. If confirmed, these findings will add to a growing list of beneficial properties of this organism. The strength of the evidence used to justify the conclusions in the manuscript is solid, as the analyses broadly support the claims with only minor weaknesses.
This article has been Reviewed by the following groups
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Abstract
As the largest mucosal surface, the gut has built a physical, chemical, microbial, and immune barrier to protect the body against pathogen invasion. The disturbance of gut microbiota aggravates pathogenic bacteria invasion and gut barrier injury. Fecal microbiota transplantation (FMT) is a promising treatment for microbiome-related disorders, where beneficial strain engraftment is a significant factor influencing FMT outcomes. The aim of this research was to explore the effect of FMT on antibiotic-induced microbiome-disordered (AIMD) models infected with enterotoxigenic Escherichia coli (ETEC). We used piglet, mouse, and intestinal organoid models to explore the protective effects and mechanisms of FMT on ETEC infection. The results showed that FMT regulated gut microbiota and enhanced the protection of AIMD piglets against ETEC K88 challenge, as demonstrated by reduced intestinal pathogen colonization and alleviated gut barrier injury. Akkermansia muciniphila ( A. muciniphila ) and Bacteroides fragilis ( B. fragilis ) were identified as two strains that may play key roles in FMT. We further investigated the alleviatory effects of these two strains on ETEC infection in the AIMD mice model, which revealed that A. muciniphila and B. fragilis relieved ETEC-induced intestinal inflammation by maintaining the proportion of Treg/Th17 cells and epithelial damage by moderately activating the Wnt/β-catenin signaling pathway, while the effect of A. muciniphila was better than B. fragilis . We, therefore, identified whether A. muciniphila protected against ETEC infection using basal-out and apical-out intestinal organoid models. A. muciniphila did protect the intestinal stem cells and stimulate the proliferation and differentiation of intestinal epithelium, and the protective effects of A. muciniphila were reversed by Wnt inhibitor. FMT alleviated ETEC-induced gut barrier injury and intestinal inflammation in the AIMD model. A. muciniph ila was identified as a key strain in FMT to promote the proliferation and differentiation of intestinal stem cells by mediating the Wnt/β-catenin signaling pathway.
Article activity feed
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eLife Assessment
The work by Han and collaborators describes valuable findings on the role of Akkermansia muciniphila during ETEC infection. If confirmed, these findings will add to a growing list of beneficial properties of this organism. The strength of the evidence used to justify the conclusions in the manuscript is solid, as the analyses broadly support the claims with only minor weaknesses.
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Reviewer #3 (Public review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
As in previous revisions, there remains concerning ambiguity in the methodology used for microbiota sequence analysis and it would be difficult to replicate the analysis in any …
Reviewer #3 (Public review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
As in previous revisions, there remains concerning ambiguity in the methodology used for microbiota sequence analysis and it would be difficult to replicate the analysis in any meaningful way. In this revision, concerns about the rigor and reproducibility of this component of the manuscript have been increased. Readers should be cautious with interpretation of this data.
(1) In previous versions of the manuscript it would appear the correct bioproject accession was listed but, the actual link went to an unrelated project. The updated accession link appears to contain raw data; however, the authors state they used an Illumina HiSeq 2500. This would be an unusual choice for V3-V4 as it would not have read lengths long enough to overlap. Inspection of the first sample (SRR19164796) demonstrates that this is absolutely not the raw data, as there is a ~400 nt forward read, and a 0 length reverse read. All quality scores are set to 30. There is no logical way to go from HiSeq 2500 raw data and read lengths to what was uploaded to the SRA and it was certainly not described in the manuscript.
(2) No multiple testing correction was applied to the microbiome data.
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Author response:
The following is the authors’ response to the current reviews.
Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval.
Reviewer #3 (Public review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up …
Author response:
The following is the authors’ response to the current reviews.
Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval.
Reviewer #3 (Public review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
As in previous revisions, there remains concerning ambiguity in the methodology used for microbiota sequence analysis and it would be difficult to replicate the analysis in any meaningful way. In this revision, concerns about the rigor and reproducibility of this component of the manuscript have been increased. Readers should be cautious with interpretation of this data.
(1) In previous versions of the manuscript it would appear the correct bioproject accession was listed but, the actual link went to an unrelated project. The updated accession link appears to contain raw data; however, the authors state they used an Illumina HiSeq 2500. This would be an unusual choice for V3-V4 as it would not have read lengths long enough to overlap. Inspection of the first sample (SRR19164796) demonstrates that this is absolutely not the raw data, as there is a ~400 nt forward read, and a 0 length reverse read. All quality scores are set to 30. There is no logical way to go from HiSeq 2500 raw data and read lengths to what was uploaded to the SRA and it was certainly not described in the manuscript.
What we uploaded to the SRA was Contigs files for sample, we have modified the description on line 694.
(2) No multiple testing correction was applied to the microbiome data.
The alpha diversity indexes were tested using T-test and wilcox test, and we showed the result of T-test in FigureS1B. The p-values were corrected for multiple testing using the Benjamini-Hochberg method, we have modified the description on line 322.
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The following is the authors’ response to the previous reviews.
Public Reviews:
Reviewer #2 (Public Review):
Ma X. et al proposed that A. muciniphila was a key strain that promotes the proliferation and differentiation of intestinal stem cells through acting on the Wnt/β-catenin signaling pathway. They used various models, such as piglet model, mouse model and intestinal organoids to address how A. muciniphila and B. fragilis offer the protection against ETEC infection. They showed that FMT with fecal samples, A. muciniphila or B. fragilis protected piglets and/or mice from ETEC infection, and this protection is manifested as reduced intestinal inflammation/bacterial colonization, increased tight junction/Muc2 proteins, as well as proper Treg/Th17 cells. Additionally, they demonstrated that A. muciniphila protected basal-out and/or apical-out intestinal organoids against ETEC infection via Wnt signaling.
Comments on revised version:
Please add proper references to indicate the invasion of ETEC into organoids after 1 h of infection.
We have added references on line 211.
References:
Xiao K, Yang Y, Zhang Y, Lv QQ, Huang FF, Wang D, Zhao JC, Liu YL. 2022. Long-chain PUFA ameliorate enterotoxigenic Escherichia coli-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in porcine intestinal epithelial cells. Br. J. Nutr. 128(5):835-850.
Qian MQ, Zhou XC, Xu TT, Li M, Yang ZR, Han XY. 2023. Evaluation of Potential Probiotic Properties of Limosilactobacillus fermentum Derived from Piglet Feces and Influence on the Healthy and E. coli-Challenged Porcine Intestine. Microorganisms. 11(4).
Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
After an additional revision, the bioinformatics section of the methods has changed significantly from previous versions and now indicates a third sequencer was used instead: Ion S5 XL. Important parameters required to replicate analysis have still not been provided. Inspection of the SRA data indicates a mix of Illumina MiSeq and Illumina HiSeq 2500. It is now unclear which sequencing technology was used as authors have variably reported 4 different sequencers for these samples. Appropriate metadata was not provided in the SRA, although some groups may be inferred from sample names. These changing descriptions of the methodologies and ambiguity in making the data available create concerns about rigor of study and results.
Due to confusing the sequencing method of this experiment with other experiment samples, we apologize for the multiple incorrect modifications of the method description. We have modified the method for microbiome sequencing technology on line 304. The sequencing technology is Illumina HiSeq 2500. The SRA metadata can be viewed at https://www.ncbi.nlm.nih.gov/sra/PRJNA837047. The sample names ep1-6 and ef1-6 were correspond to the EP and EF groups, respectively.
Recommendations For the Authors:
As in the previous revision:
-provide important parameters required to replicate analysis
-ensure that reporting of sequencing technology is correct as data listed on SRA appears to be derived from Illumina sequencers, and was deposited indicating as such.
-update SRA metadata such that experimental groups are clear and match the nomenclature used in the manuscript (Particularly for samples which are labelled [A-Z][0-9]
- The multiple testing correction wasn’t applied.
-Due to confusing the sequencing method of this experiment with other experiment samples, we apologize for the multiple incorrect modifications of the method description. We have modified the method for microbiome sequencing technology on line 304. The sequencing technology is Illumina HiSeq 2500.
- The SRA metadata can be viewed at https://www.ncbi.nlm.nih.gov/sra/PRJNA837047. The sample names ep1-6 and ef1-6 were correspond to the EP and EF groups, respectively.
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eLife assessment
The work by Han and collaborators describes valuable findings on the role of Akkermansia muciniphila during ETEC infection. If confirmed, these findings will add to a growing list of beneficial properties of this organism. Although the strength of the evidence used to justify the conclusions in the manuscript is solid, the issues raised about the sequencing method used should be addressed.
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Reviewer #2 (Public Review):
Ma X. et al proposed that A. muciniphila was a key strain that promotes the proliferation and differentiation of intestinal stem cells through acting on the Wnt/b-catenin signaling pathway. They used various models, such as piglet model, mouse model and intestinal organoids to address how A. muciniphila and B. fragilis offer the protection against ETEC infection. They showed that FMT with fecal samples, A. muciniphila or B. fragilis protected piglets and/or mice from ETEC infection, and this protection is manifested as reduced intestinal inflammation/bacterial colonization, increased tight junction/Muc2 proteins, as well as proper Treg/Th17 cells. Additionally, they demonstrated that A. muciniphila protected basal-out and/or apical-out intestinal organoids against ETEC infection via Wnt signaling.
Comments …
Reviewer #2 (Public Review):
Ma X. et al proposed that A. muciniphila was a key strain that promotes the proliferation and differentiation of intestinal stem cells through acting on the Wnt/b-catenin signaling pathway. They used various models, such as piglet model, mouse model and intestinal organoids to address how A. muciniphila and B. fragilis offer the protection against ETEC infection. They showed that FMT with fecal samples, A. muciniphila or B. fragilis protected piglets and/or mice from ETEC infection, and this protection is manifested as reduced intestinal inflammation/bacterial colonization, increased tight junction/Muc2 proteins, as well as proper Treg/Th17 cells. Additionally, they demonstrated that A. muciniphila protected basal-out and/or apical-out intestinal organoids against ETEC infection via Wnt signaling.
Comments on revised version:
Please add proper references to indicate the invasion of ETEC into organoids after 1 h of infection.
-
Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
After an additional revision, the bioinformatics section of the methods has changed significantly from previous versions and now indicates a third sequencer was used instead: Ion S5 …
Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
After an additional revision, the bioinformatics section of the methods has changed significantly from previous versions and now indicates a third sequencer was used instead: Ion S5 XL. Important parameters required to replicate analysis have still not been provided. Inspection of the SRA data indicates a mix of Illumina MiSeq and Illumina HiSeq 2500. It is now unclear which sequencing technology was used as authors have variably reported 4 different sequencers for these samples. Appropriate metadata was not provided in the SRA, although some groups may be inferred from sample names. These changing descriptions of the methodologies and ambiguity in making the data available create concerns about rigor of study and results.
-
Author response:
The following is the authors’ response to the previous reviews.
Public Reviews:
Reviewer #2 (Public Review):
The authors indicated that the adherence of ETEC is to intestinal epithelial cells. However, it is also possible that the majority of ETEC may reside in the intestinal mucus, particularly under in vivo infection condition. The colonization of ETEC in the jejunum and colon of piglets (Fig 2C) and in the intestines of mice (Fig S2A) does not necessarily reflect the adherence of ETEC to epithelial cells. Please verify these observations with other methods, such as immunostaining. Also, while Salmonella enterica serovar Typhimurium or Listeria monocytogenes can invade organoids within 1 hour, it is unknown if ETEC invade into organoids in this study. Clarifying this will help resolve if A. muciniphila block the …
Author response:
The following is the authors’ response to the previous reviews.
Public Reviews:
Reviewer #2 (Public Review):
The authors indicated that the adherence of ETEC is to intestinal epithelial cells. However, it is also possible that the majority of ETEC may reside in the intestinal mucus, particularly under in vivo infection condition. The colonization of ETEC in the jejunum and colon of piglets (Fig 2C) and in the intestines of mice (Fig S2A) does not necessarily reflect the adherence of ETEC to epithelial cells. Please verify these observations with other methods, such as immunostaining. Also, while Salmonella enterica serovar Typhimurium or Listeria monocytogenes can invade organoids within 1 hour, it is unknown if ETEC invade into organoids in this study. Clarifying this will help resolve if A. muciniphila block the adherence and/or invasion of ETEC. Please also address if A. muciniphila metabolites could prevent ETEC infection in the organoid models.
In the original manuscript, the sentence “ETEC K88 adheres to intestinal epithelial cells and induces gut inflammation (Yu et al., 2018)” in line 447 is a reference cited for the purpose of connecting the previous and the following, and it is not our result. We have deleted this sentence on line 457. Previous studies have shown that ETEC enter into intestinal epithelial cells after only one hour of infection (Xiao et al., 2022; Qian et al., 2023). Whether A. muciniphila metabolites prevent ETEC infection in the organoid models is not the focus of this manuscript, it may be further explored by other members of the research group in the future.
References:
Xiao K, Yang Y, Zhang Y, Lv QQ, Huang FF, Wang D, Zhao JC, Liu YL. 2022. Long-chain PUFA ameliorate enterotoxigenic Escherichia coli-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in porcine intestinal epithelial cells. Br. J. Nutr. 128(5):835-850.
Qian MQ, Zhou XC, Xu TT, Li M, Yang ZR, Han XY. 2023. Evaluation of Potential Probiotic Properties of Limosilactobacillus fermentum Derived from Piglet Feces and Influence on the Healthy and E. coli-Challenged Porcine Intestine. Microorganisms. 11(4).
Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
After revision, the bioinformatics section of the methods is still jumbled and may indicate issues in the pipeline. Important parameters are not included to replicate analyses. Merging the forward and reverse reads may represent a problem for denoising. Chimera detection was performed prior to denoising.
Potential denoising issues for NovaSeq data was not addressed in the response. The authors did not clarify if multiple testing correction was applied; however, it may be assumed not as written. The raw sequencing data made available through the SRA accession (if for the correct project) indicates it was a MiSeq platform; however, the sample names do not appear to link up to this experimental design and metadata not sufficient to replicate analyses.
We have redescribed the method for microbiome sequencing analysis on lines 298-327.
Recommendations for the authors:
Reviewer #3 (Recommendations For The Authors):
SRA accession must be confirmed and metadata made available.
We updated the SRA data.
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eLife assessment
The work by Han and collaborators describes valuable findings on the role of Akkermansia muciniphila during ETEC infection. If confirmed, these findings will add to a growing list of beneficial properties of this organism. However, as it stands, the strength of the evidence used to justify the conclusions in the manuscript is incomplete.
-
Reviewer #2 (Public Review):
The authors indicated that the adherence of ETEC is to intestinal epithelial cells. However, it is also possible that the majority of ETEC may reside in the intestinal mucus, particularly under in vivo infection condition. The colonization of ETEC in the jejunum and colon of piglets (Fig 2C) and in the intestines of mice (Fig S2A) does not necessarily reflect the adherence of ETEC to epithelial cells. Please verify these observations with other methods, such as immunostaining. Also, while Salmonella enterica serovar Typhimurium or Listeria monocytogenes can invade organoids within 1 hour, it is unknown if ETEC invade into organoids in this study. Clarifying this will help resolve if A. muciniphila block the adherence and/or invasion of ETEC. Please also address if A. muciniphila metabolites could prevent …
Reviewer #2 (Public Review):
The authors indicated that the adherence of ETEC is to intestinal epithelial cells. However, it is also possible that the majority of ETEC may reside in the intestinal mucus, particularly under in vivo infection condition. The colonization of ETEC in the jejunum and colon of piglets (Fig 2C) and in the intestines of mice (Fig S2A) does not necessarily reflect the adherence of ETEC to epithelial cells. Please verify these observations with other methods, such as immunostaining. Also, while Salmonella enterica serovar Typhimurium or Listeria monocytogenes can invade organoids within 1 hour, it is unknown if ETEC invade into organoids in this study. Clarifying this will help resolve if A. muciniphila block the adherence and/or invasion of ETEC. Please also address if A. muciniphila metabolites could prevent ETEC infection in the organoid models.
-
Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
After revision, the bioinformatics section of the methods is still jumbled and may indicate issues in the pipeline. Important parameters are not included to replicate analyses. Merging …
Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
After revision, the bioinformatics section of the methods is still jumbled and may indicate issues in the pipeline. Important parameters are not included to replicate analyses. Merging the forward and reverse reads may represent a problem for denoising. Chimera detection was performed prior to denoising.
Potential denoising issues for NovaSeq data was not addressed in the response. The authors did not clarify if multiple testing correction was applied; however, it may be assumed not as written. The raw sequencing data made available through the SRA accession (if for the correct project) indicates it was a MiSeq platform; however, the sample names do not appear to link up to this experimental design and metadata not sufficient to replicate analyses.
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
In this paper, the authors investigate the impact of fecal microbiota transfer (FMT) on intestinal recovery from enterotoxigenic E. coli infection following antibiotic treatment. Using a piglet model of intestinal infection, the authors demonstrate that FMT reduces weight loss and diarrhea and enhances the expression of tight junction proteins. Sequencing analysis of the intestinal microbiota following FMT showed significant increases in Akkermansia muciniphila and Bacteroides fragilis. Using additional mouse and organoid models, the authors examine the impact of these microbes on intestinal recovery and modulation of the Wnt signaling pathway. Overall, the data support the notion that FMT following ETEC infection is …
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
In this paper, the authors investigate the impact of fecal microbiota transfer (FMT) on intestinal recovery from enterotoxigenic E. coli infection following antibiotic treatment. Using a piglet model of intestinal infection, the authors demonstrate that FMT reduces weight loss and diarrhea and enhances the expression of tight junction proteins. Sequencing analysis of the intestinal microbiota following FMT showed significant increases in Akkermansia muciniphila and Bacteroides fragilis. Using additional mouse and organoid models, the authors examine the impact of these microbes on intestinal recovery and modulation of the Wnt signaling pathway. Overall, the data support the notion that FMT following ETEC infection is beneficial, however, additional investigation is required to fully elucidate the mechanisms involved.
Strengths:
Initial experiments used a piglet model of infection to test the value of FMT on recovery from E. coli. The FMT treatment was beneficial and the authors provide solid evidence that the treatment increased the diversity of the microbiota and enhanced the recovery of the intestinal epithelium. Sequencing data highlighted an increase in Akkermansia muciniphila and Bacteroides fragilis after FMT.
The mouse data are consistent with the observations in pigs, and reveal that daily gavage with A. muciniphila or B. fragilis enhances intestinal recovery based on histological analysis, expression of tight junction proteins, and analysis of intestinal barrier function.
The authors demonstrate the benefit of probiotic treatment following infection using a range of model systems.
Weaknesses:
Without sequencing the pre-infection pig microbiota or the FMT input material itself, it's challenging to firmly say that the observed bloom in Akkermansia muciniphila and Bacteroides fragilis stemmed from the FMT.
Response: We have determined the relative abundance of each bacterium in fecal bacterial suspension, referring to Hu et al. (2018). The absolute abundances of Akkermansia muciniphila and Bacteroides fragilis in the FMT were 1.3 × 103 ± 2.6 × 103 and 4.5 × 103 ± 6.1 × 103 respectively.
Reference:
Hu LS, Geng SJ, Li Y, et al. Exogenous Fecal Microbiota Transplantation from Local Adult Pigs to Crossbred Newborn Piglets. Front. Microbiol. 2018, 8.
The lack of details for the murine infection model, such as weight loss and quantification of bacterial loads over time, make it challenging for a reader to fully appreciate how treatment with Akkermansia muciniphila and Bacteroides fragilis is altering the course of infection. Bacterial loads of E. coli were only quantified at one time point, and the mice that received A. muciniphila and B. fragilis had very low levels of E. coli. Therefore, it is not clear if all mice were subjected to the same level of infection in the first place. The reduced translocation of E. coli to the organs and enhanced barrier function may just reflect the low level of infection in these mice. Further, the authors' conclusion that the effect is specific to A. muciniphila or B. fragilis would be more convincing if the experiments included an inert control bacterium, to demonstrate that gavage with any commensal microbe would not elicit a similar effect.
The weight loss was added in Figure S2A. All mice were subjected to the same level of infection in the first place.
Many of the conclusions in the study are drawn from the microscopy results. However, the methods describing both light microscopy and electron microscopy lack sufficient detail. For example, it is not clear how many sections and fields of view were imaged or how the SEM samples were prepared and dehydrated. The mucus layer does not appear to be well preserved, which would make it challenging to accurately measure the thickness of the mucus layer.
For light microscopy, 3-4 fields were selected from each mouse to count about 30 crypts. The method of electron microscopy was complemented on line 263-270. We have removed data of the mucus layer.
Gene expression data appears to vary across the different models, for example, Wnt3 expression in mice versus organoids. Additional experiments may be required to clarify the mechanisms involved. Considering that both of the bacteria tested elicited similar changes in Wnt signaling, this pathway might be broadly modulated by the microbiota.
The reason why the Wnt3 expression pattern is different in mice and in porcine intestinal organoids may be caused by the different infection periods of ETEC in vivo and in vitro. Furthermore, in vivo, the stem cell niche of intestinal stem cells is not only regulated by intestinal epithelial cells, but also affected by mesenchymal cells in connective tissues (Luo et al., 2022). However, in vitro models, stem cell niche is only regulated by epithelial secretory factors, which may also account for the differences in in vitro and in vivo results.
It has been reported that B. fragilis pretreatment significantly increased the relative abundance of A. muciniphila in the intestine of CDI mice, and the growth and maintenance of A. muciniphila were involved in the restoration of intestinal barrier integrity after CDI infection, indicating that there might exist a bacterial metabolic symbiosis between A. muciniphila and B. fragilis (Deng et al., 2018).
References:
Luo HM, Li MX, Wang F, et al. The role of intestinal stem cell within gut homeostasis: Focusing on its interplay with gut microbiota and the regulating pathways. Int. J. Biol. Sci. 2022, 18(13): 5185-5206.
Deng HM, Yang SQ, Zhang YC, et al. Bacteroides fragilis Prevents Clostridium difficile Infection in a Mouse Model by Restoring Gut Barrier and Microbiome Regulation. Front. Microbiol. 2018, 9.
The unconventional choice to not include references in the results section makes it challenging for the reader to put the results in context with what is known in the field. Similarly, there is a lack of discussion acknowledging that B. fragilis is a potential pathogen, associated with intestinal inflammation and cancer (Haghi et al. BMC Cancer 19, 879 (2019) ), and how this would impact its utility as a potential probiotic.
Bacteroides fragilis is one of the symbiotic anaerobes within the mammalian gut and is also an opportunistic pathogen which often isolated from clinical specimens. Bacteroides fragilis was first isolated from the pathogenic site and considered to be pathogenic bacteria. However, with the deepening of research, it is gradually realized that in the long-term evolution process, Bacteroides fragilis colonized in the gut has established a friendly relationship with the host, which is an essential component for maintaining the health of the host, especially for obesity, diabetes and immune deficiency diseases. We have supplemented the discussion on line 598-603.
Reviewer #2 (Public Review):
Ma X. et al proposed that A. muciniphila was a key strain that promotes the proliferation and differentiation of intestinal stem cells by acting on the Wnt/β-catenin signaling pathway. They used various models, such as the piglet model, mouse model, and intestinal organoids to address how A. muciniphila and B. fragilis offer protection against ETEC infection. They showed that FMT with fecal samples, A. muciniphila or B. fragilis protected piglets and/or mice from ETEC infection, and this protection is manifested as reduced intestinal inflammation/bacterial colonization, increased tight junction/Muc2 proteins, as well as proper Treg/Th17 cells. Additionally, they demonstrated that A. muciniphila protected basal-out and/or apical-out intestinal organoids against ETEC infection via Wnt signaling. While a large body of work has been performed in this study, there are quite a few questions to be addressed.
Major comments:
- The similar protective effect of FMT with fecal samples, A. muciniphila or B. fragilis is perhaps not that surprising, considering that FMT likely restores microbiota-mediated colonization resistance against ETEC infection. While FMT with fecal samples increases SCFAs, it is unclear whether/how FMT with A. muciniphila or B. fragilis alter the microbiota composition/abundance as well as metabolites in the current models in a way that offers protection.
We examined changes in the gut microbiota of mice treated with A. muciniphila and B. fragilis through 16s rRNA, and results showed that both A. muciniphila and B. fragilis improved the alpha and beta diversities of the microbiota, while these results were not included in this manuscript.
- Does ETEC infection in piglets/mice cause histological damage in the intestines? These data should be shown.
The results of scanning electron microscopy (Figure 3A) showed the intestinal damage of piglets after ETEC infection. H&E staining and transmission electron microscopy (Figure 5A and 5B) showed the intestinal damage of mice after ETEC infection.
- Line 447, "ETEC adheres to intestinal epithelial cells". However, there is no data showing the adherence (or invasion) of ETEC to intestinal epithelial cells, irrespective of piglets/mouse/organoids.
The scanning electron microscope (Figure 3A bottom) showed that ETEC K88 infected piglets existed obvious rod-shaped bacterial adhesion on the surface of microvilli. Figure 2C showed the colonization of ETEC K88 in the jejunum and colon of piglets. Figure S2A showed the E. coli colonization in intestines and other tissues of mice.
- In both basal-out and apical-out intestinal organoid models, A. muciniphila protects organoids against ETEC infection. Did ETEC enter into intestinal epithelial cells at all after only one hour of infection? Is the protection through certain A. muciniphila metabolites?
It has been reported that the duration of the co-culture for studying the host-microbiota cross-talk by apical-out organoids model is 1 hour (Poletti et al., 2021). In addition, Co et al. (2019) used apical-out organoids model to study host-pathogen interactions, with Salmonella enterica serovar Typhimurium or Listeria monocytogenes invading organoids for an hour.
References:
Poletti M, Arnauts K, Ferrante M, et al. Organoid-based Models to Study the Role of Host-microbiota Interactions in IBD. J. Crohns Colitis. 2021, 15(7): 1222-1235.
Co JY, Margalef-Catala M, Li XN, et al. Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions. Cell Reports. 2019, 26(9): 2509-2520.
Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.
Strengths:
The strengths of this manuscript include the use of multiple model systems and follow-up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.
Weaknesses:
The major weakness is that, as presented, the manuscript is quite difficult to follow, even for someone familiar with the field. The lack of detail in figure legends, organization of the text, and frequent use of non-intuitive abbreviated group names without a clear key (ex. EP/EF, or C E A B) make comprehension challenging. The results section is perhaps too succinct and does not provide sufficient information to understand experimental design and interpretation without reading the methods section first or skipping to the discussion (as an example: WNT-c59 treatment). Extensive revisions could be encouraged to aid in communicating the potentially exciting findings.
The abbreviations of experimental groups are firstly defined in the Methods and Materials, and we have supplemented the experimental design in the results section on line 397-399, 439-442 and 516-520.
The bioinformatics section of the methods requires revision and may indicate issues in the pipeline. Merging the forward and reverse reads may represent a problem for denoising. Also since these were sequenced on a NovaSeq, the error learning would have to be modified or the diversity estimates would be inappropriately multiplied. "Alpha diversity and beta diversity were calculated by normalized to the same sequence randomly." Not sure what this means, does this mean subsampled? "Blast was used for sequence alignment", does this mean the taxonomic alignment? This would need to be elaborated on and database versions should be included. The methods, including if any form of multiple testing was included, for LEFSE was also not included.
Denoising was conducted using UNOISE3 to correct for sequencing errors. Subsequent analysis of alpha diversity and beta diversity were all performed based on the output normalized data. Multiple sequence alignment was performed using MUSCLE (v3.8.31) software to obtain the phylogenetic relationships of all OTUs sequences. We have supplemented the method of multiple testing on line 323-328.
Reviewer #1 (Recommendations For The Authors):
At some points, the rationale for using both porcine and murine models was unclear, and it would be helpful for the reader to elaborate on the benefits of these models and why they were used in the introduction. Similarly, it would be helpful to describe the benefits of basal-in organoids versus injecting standard organoids with bacteria.
The main subject of this study was piglets, supplemented by a mouse model for validation. Interpretation of measurements from organoid microinjection experiments must account for multiple confounding variables such as heterogeneous exposure concentrations and durations, as well as impacts of disrupting the organoid wall. We have added the description in the introduction on line 88-90.
Line 165 -- The number of piglets used seems high, is it correct approximately 100 pigs were used?
Nine litters were selected for processing, while only 18 piglets were finally slaughtered.
There is very little discussion of the preliminary experiment that the authors used to determine how much bacteria to use. I recommend either discussing the data and how the doses were chosen or omitting it. It was not clear if the authors used pasteurized or live bacteria in the experiments. It would also be interesting to include a discussion of the observation that relatively low levels of Akkermansia (10^6 CFU) appeared more beneficial than the higher doses, typically used in these types of experiments.
We removed these results. The experiments used live bacteria.
Microscopy methods for both light microscopy and EM would be stronger with added details including how many sections and fields of view were imaged and how the numbers of goblet cells normalized across samples. Without having a clear cross-section of a crypt, it is not clear to me how the images can be used to accurately quantify the number of cells per crypt. Additional details in the methods on how many total crypts were counted should also be included.
For light microscopy, 3-4 fields were selected from each mouse to count about 30 crypts. We have removed the data of the mucus layer and goblet cells.
Line 236 -- missing which gene was used.
The Genbank Accession was added on line 232-233.
Line 310 -- OTU nomenclature.
We have supplemented the OTU nomenclature on line 314.
Line 413 -- This line seems inconsistent with the data analysis described in the methods section. The authors may need to expand their description of the 16S data analysis to be clear and reproducible.
We have redescribed the 16S data analysis on line 312-328.
Line 413 -- it is not surprising that 16s analysis did not capture species, it will have limited resolution beyond the genus level.
We deleted this sentence.
Methods are missing some details on the data analysis, eg. methods/programs and statistical analysis of PCoA and NMDS, LefSe.
The methods and statistical analysis of PCoA, NMDS and LEfSe were supplemented on line 323-328.
Fig 4C -- The images do not clearly capture the mucus layer or how it was analyzed. The sections appear to be cut at a slight angle, with multiple partial sections of crypts. I think this might make it challenging to count goblet cells, especially if the counts are normalized over the number of crypts or villi. The mucus layer does not appear well preserved. For example, I would expect to see an intact mucus layer lining the colon in the PBS control group. Re-cutting sections with a clean cross-section through the tissue will make data analysis easier.
We have removed data of the mucus layer.
Fig 4D -- The images appear to be of the mouse proximal colon, whereas the mucus layer and most muc2 will be in the distal colon. If the authors have tissue sections of the distal colon, this may give a clearer image of the mucus layer and might be more consistent with the TEM images in Fig. 4B.
We apologize for the absence of the distal colon sections.
To fully preserve the mucus layer, in addition to fixing in Carnoy's solution, the embedding process must be run without the standard washes in 70% ethanol (see: Johansson and Hansson. Methods Mol Biol. (2012) 229; doi: 10.1007/978-1-61779-513-8_13). The mucus will wash away during standard paraffin embedding if the tissue is washed with 70% ethanol, and I wonder if that has occurred in these samples.
The tissue wasn’t washed with 70% ethanol.
Fig 6A and 6B -- Although the legend indicates that the data is representative of two independent experiments, it is not clear how many fields of view or cells were imaged. In the bar graphs, it is not clear how many crypts were analyzed and from how many fields of view.
3-4 fields were selected from each mouse to count about 30 crypts.
**For all of the bar graphs, this could be addressed by displaying all of the data points, rather than just the mean, to give the reader a sense of how many cells were counted. (as was done in Fig 7B).
We have changed the bar graphs with data points.
498-501 -- The text says that the gene expression patterns in the organoids are consistent with the in vivo data, but the data patterns of gene expression appear to be different. For example, patterns for Wnt3 and B-catenin expression in mice, appear to be the opposite of what was observed in the organoid?
Lines 509-512 mean that the expression patterns of mice in organoids and in vivo is consistent. Figure 7C was incorrectly written as Figure 8C, we have changed it.
Since Akkermansia does not grow under aerobic conditions, it should be made clear that the organoid co-culture treatment does not involve actively growing bacterial cultures.
Reunanen et al. found that Akkermansia can tolerate oxygen, more than 90% Akkermansia can keep for 1 h under oxic, 5% CO2 conditions.
Reference:
Reunanen J, Kainulainen V, Huuskonen L, et al. Akkermansia muciniphila Adheres to Enterocytes and Strengthens the Integrity of the Epithelial Cell Layer. Appl. Environ. Microbiol. 2015, 81(11): 3655-3662.
Minor points
Line 50 -"evidence".
We have changed to “evidence” on line 49.
Line 64, 422 - italicize, check italics throughout.
We have checked italics throughout the manuscript.
Line 64 - may need to be reworded.
We have changed to “Clostridioides difficile” on line 66.
Line 77 - pathogen.
We have changed to “pathogen” on line 77.
Line 161 - the.
We have removed “the” on line 161.
Line 178 - mouse.
We have changed to “mouse” on line 179.
Line 313 -- wording is confusing.
We have changed the description on line 319-320.
Line 318 -- Silva version #.
The version is Silva 132. We have added it on line 316.
Line 334 - Manufacturer for Live/Dead cell stain?
The Live/Dead cell stain was used BD Biosciences FVS510. We have added it on line 345.
Line 433 -- FD4 not defined until here.
We have refined the FD4 on line 218-219.
Line 512 -- but did not promote.
We have changed to “but did not promote” on line 526.
Line 517 -- Looks like this should be "basal-in organoids" instead of basal-out?
We have changed the "basal-out" to "apical-to" on line 531.
Line 546 -- induced neonatal should be protected?
They are in separate pens.
Jumps from Fig 7B to Fig 8C in the text.
We apologize for the wrong writing, and we have change it.
Reviewer #2 (Recommendations for The Authors):
The title itself is a bit misleading. Please consider changing it. The authors meant that A. muciniphila prevents pathogen invasion, but does not function in pathogen invasion.
We have changed the title.
Major comments:
- Figures 4A, 4D, and 6B should include presentation of cross-section pictures.
We provided cross-section pictures to the journal.
- Figures 7, 8, and 9 should indicate clearly whether mouse or piglet organoids are used. For instance, in the main text, line 490, it indicates piglet organoids, but in Figure 7A legend, it indicates mouse tissue.
We apologize for the misspelling, and have changed to “mice” on line 501-502.
- In Figure 7A, the 3rd row, 2nd panel, crypts formed into spherical organoids; whereas in Figure 8, ETEC infection of basal-out organoids formed budding organoids. This needs to be better explained.
Mouse intestinal organoids were cultured ex vivo from crypts isolated from mice infected with ETEC, while porcine intestinal organoids were co-cultured with ETEC in vitro.
Minor comments:
- In the result section, the numbering of Figures or supplementary Figures is problematic, i.e it should start with Figure 1..., Figure S1, but not directly go to Figure S2A etc.
The Figure 1 was in Materials and Methods.
- Line 458, please add the gating strategy used in the flow cytometry study.
The gating strategy was added on line 351-356.
- The effect of A. muciniphila on the proliferation of intestinal epithelium through the Wnt/β-catenin signaling pathway is well known (such as PMID: 32138776). The authors should discuss this in detail.
We have supplemented the discussion on line 637-639.
Reviewer #3 (Recommendations For The Authors):
It is somewhat unusual that the results from the piglets are in the supplement as this is a major strength of the manuscript (Fig S2).
We have put these results into Figure 2 of the manuscript.
"Collectively, our results may provide theoretical basis that FMT is a promising mitigation method for pathogenic bacteria infection and a new strategy for precise application of FMT in clinical and livestock production"- This is somewhat of an odd statement as the introduction of the manuscript completely skips over most of what is known about FMTs in the context of C. difficile. Also if anything, does the authors' own data not point mostly at using A. muciniphila on its own? Clinical trials are well underway in humans.
We have changed the sentences to “Collectively, our results may provide theoretical basis that A. muciniphila is a promising method to repair intestinal barrier damage and a new strategy for the precise application of A. muciniphila in livestock production.” on line 98-100.
Line 26: I am not sure probiotic is the right word here given its strict scientific definition. Perhaps beneficial or protective would be more appropriate.
We have changed “probiotic” to “beneficial” on line 25.
Line 27: I believe AIMD is antibiotic-induced microbiome-depletion in most usages which may be more accurate and informative than dysregulated.
The type, dosing, and time of antibiotic we used were applied to induce microbiota disorder.
It would appear that there are issues in the reference formatting where a number of journal names are missing.
We have re-edited the reference formatting.
Line 64- I believe eLife requires the standard practice of italicizing genus and species names. Also Clostridium difficile should now be referred to as Clostridioides difficile.
We have changed to “Clostridioides difficile” and italicized it on line 66 and 569. The italicizing genus and species names were checked throughout the manuscript.
Figure S2C: is it not clear why the melt curve was included here, but the legend should make it more clear what is being shown. I assume this is to provide evidence of specificity?
The melting curve was used to demonstrate that only the ETEC K88 could be amplified by the primers we used. We have added an illustration in the figure legend.
Figure 2D: there should be a quantitative analysis done on the staining of Muc2.
We have quantified the staining of MUC2 in Figure 3D.
Figure 3: The legends are not sufficient. For example: it is not clear what Figure 3A actually shows as the y-axis is not labelled and it is not clear what the relationship is between this and the anosim which is a function for permanova.
Anosim analysis was performed using the R software with anosim package function based on the rank order of Bray-Curtis distance values to test the significance of differences between groups. The y-axis is the rank of the distance between samples.
Line 416- OTU not OUT.
We have changed to “OTU” on line 428.
Figure 4- the naming key needs to be included in the figure legend. C, E, A, and B are immediately obvious.
The naming key was included in the figure legend.
Methods: additional information on the flow cytometry gating strategy/controls should be included.
The gating strategy was added on line 351-356.
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eLife assessment
The work by Han and collaborators describes valuable findings on the role of Akkermansia muciniphila during ETEC infection. If confirmed, these findings will add to a growing list of beneficial properties of this organism. However, as it stands, the strength of the evidence used to justify the conclusions in the manuscript is incomplete.
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Reviewer #1 (Public Review):
Summary:
In this paper, the authors investigate the impact of fecal microbiota transfer (FMT) on intestinal recovery from enterotoxigenic E. coli infection following antibiotic treatment. Using a piglet model of intestinal infection, the authors demonstrate that FMT reduces weight loss and diarrhea and enhances the expression of tight junction proteins. Sequencing analysis of the intestinal microbiota following FMT showed significant increases in Akkermansia muciniphila and Bacteroides fragilis. Using additional mouse and organoid models, the authors examine the impact of these microbes on intestinal recovery and modulation of the Wnt signaling pathway. Overall, the data support the notion that FMT following ETEC infection is beneficial, however, additional investigation is required to fully elucidate the …Reviewer #1 (Public Review):
Summary:
In this paper, the authors investigate the impact of fecal microbiota transfer (FMT) on intestinal recovery from enterotoxigenic E. coli infection following antibiotic treatment. Using a piglet model of intestinal infection, the authors demonstrate that FMT reduces weight loss and diarrhea and enhances the expression of tight junction proteins. Sequencing analysis of the intestinal microbiota following FMT showed significant increases in Akkermansia muciniphila and Bacteroides fragilis. Using additional mouse and organoid models, the authors examine the impact of these microbes on intestinal recovery and modulation of the Wnt signaling pathway. Overall, the data support the notion that FMT following ETEC infection is beneficial, however, additional investigation is required to fully elucidate the mechanisms involved.Strengths:
Initial experiments used a piglet model of infection to test the value of FMT on recovery from E. coli. The FMT treatment was beneficial and the authors provide solid evidence that the treatment increased the diversity of the microbiota and enhanced the recovery of the intestinal epithelium. Sequencing data highlighted an increase in Akkermansia muciniphila and Bacteroides fragilis after FMT.The mouse data are consistent with the observations in pigs, and reveal that daily gavage with A. muciniphila or B. fragilis enhances intestinal recovery based on histological analysis, expression of tight junction proteins, and analysis of intestinal barrier function.
The authors demonstrate the benefit of probiotic treatment following infection using a range of model systems.
Weaknesses:
Without sequencing the pre-infection pig microbiota or the FMT input material itself, it's challenging to firmly say that the observed bloom in Akkermansia muciniphila and Bacteroides fragilis stemmed from the FMT.The lack of details for the murine infection model, such as weight loss and quantification of bacterial loads over time, make it challenging for a reader to fully appreciate how treatment with Akkermansia muciniphila and Bacteroides fragilis is altering the course of infection. Bacterial loads of E. coli were only quantified at one time point, and the mice that received A. muciniphila and B. fragilis had very low levels of E. coli. Therefore, it is not clear if all mice were subjected to the same level of infection in the first place. The reduced translocation of E. coli to the organs and enhanced barrier function may just reflect the low level of infection in these mice. Further, the authors' conclusion that the effect is specific to A. muciniphila or B. fragilis would be more convincing if the experiments included an inert control bacterium, to demonstrate that gavage with any commensal microbe would not elicit a similar effect.
Many of the conclusions in the study are drawn from the microscopy results. However, the methods describing both light microscopy and electron microscopy lack sufficient detail. For example, it is not clear how many sections and fields of view were imaged or how the SEM samples were prepared and dehydrated. The mucus layer does not appear to be well preserved, which would make it challenging to accurately measure the thickness of the mucus layer.
Gene expression data appears to vary across the different models, for example, Wnt3 expression in mice versus organoids. Additional experiments may be required to clarify the mechanisms involved. Considering that both of the bacteria tested elicited similar changes in Wnt signaling, this pathway might be broadly modulated by the microbiota.
The unconventional choice to not include references in the results section makes it challenging for the reader to put the results in context with what is known in the field. Similarly, there is a lack of discussion acknowledging that B. fragilis is a potential pathogen, associated with intestinal inflammation and cancer (Haghi et al. BMC Cancer 19, 879 (2019) ), and how this would impact its utility as a potential probiotic.
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Reviewer #2 (Public Review):
Ma X. et al proposed that A. muciniphila was a key strain that promotes the proliferation and differentiation of intestinal stem cells by acting on the Wnt/b-catenin signaling pathway. They used various models, such as the piglet model, mouse model, and intestinal organoids to address how A. muciniphila and B. fragilis offer protection against ETEC infection. They showed that FMT with fecal samples, A. muciniphila or B. fragilis protected piglets and/or mice from ETEC infection, and this protection is manifested as reduced intestinal inflammation/bacterial colonization, increased tight junction/Muc2 proteins, as well as proper Treg/Th17 cells. Additionally, they demonstrated that A. muciniphila protected basal-out and/or apical-out intestinal organoids against ETEC infection via Wnt signaling. While a large …
Reviewer #2 (Public Review):
Ma X. et al proposed that A. muciniphila was a key strain that promotes the proliferation and differentiation of intestinal stem cells by acting on the Wnt/b-catenin signaling pathway. They used various models, such as the piglet model, mouse model, and intestinal organoids to address how A. muciniphila and B. fragilis offer protection against ETEC infection. They showed that FMT with fecal samples, A. muciniphila or B. fragilis protected piglets and/or mice from ETEC infection, and this protection is manifested as reduced intestinal inflammation/bacterial colonization, increased tight junction/Muc2 proteins, as well as proper Treg/Th17 cells. Additionally, they demonstrated that A. muciniphila protected basal-out and/or apical-out intestinal organoids against ETEC infection via Wnt signaling. While a large body of work has been performed in this study, there are quite a few questions to be addressed.
Major comments:
- The similar protective effect of FMT with fecal samples, A. muciniphila or B. fragilis is perhaps not that surprising, considering that FMT likely restores microbiota-mediated colonization resistance against ETEC infection. While FMT with fecal samples increases SCFAs, it is unclear whether/how FMT with A. muciniphila or B. fragilis alter the microbiota composition/abundance as well as metabolites in the current models in a way that offers protection.
- Does ETEC infection in piglets/mice cause histological damage in the intestines? These data should be shown.
- Line 447, "ETEC adheres to intestinal epithelial cells". However, there is no data showing the adherence (or invasion) of ETEC to intestinal epithelial cells, irrespective of piglets/mouse/organoids.
- In both basal-out and apical-out intestinal organoid models, A. muciniphila protects organoids against ETEC infection. Did ETEC enter into intestinal epithelial cells at all after only one hour of infection? Is the protection through certain A. muciniphila metabolites?
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Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.Strengths:
The strengths of this manuscript include the use of multiple model systems and follow-up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.Weaknesses:
The major weakness is that, as presented, the manuscript is quite difficult to follow, even for someone familiar with the field. The lack of detail in figure legends, organization of …Reviewer #3 (Public Review):
Summary:
The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.Strengths:
The strengths of this manuscript include the use of multiple model systems and follow-up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.Weaknesses:
The major weakness is that, as presented, the manuscript is quite difficult to follow, even for someone familiar with the field. The lack of detail in figure legends, organization of the text, and frequent use of non-intuitive abbreviated group names without a clear key (ex. EP/EF, or C E A B) make comprehension challenging. The results section is perhaps too succinct and does not provide sufficient information to understand experimental design and interpretation without reading the methods section first or skipping to the discussion (as an example: WNT-c59 treatment). Extensive revisions could be encouraged to aid in communicating the potentially exciting findings.The bioinformatics section of the methods requires revision and may indicate issues in the pipeline. Merging the forward and reverse reads may represent a problem for denoising. Also since these were sequenced on a NovaSeq, the error learning would have to be modified or the diversity estimates would be inappropriately multiplied. "Alpha diversity and beta diversity were calculated by normalized to the same sequence randomly." Not sure what this means, does this mean subsampled? "Blast was used for sequence alignment", does this mean the taxonomic alignment? This would need to be elaborated on and database versions should be included. The methods, including if any form of multiple testing was included, for LEFSE was also not included.
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