Protective effect of Enterococcus faecium against ethanol-induced gastric injury via extracellular vesicles

ABSTRACT Recently, Enterococcus has been shown to have gastric protective functions, and the mechanisms by which Enterococcus modulates gastric function are still being investigated. Herein, we investigated how Enterococcus faecium (Efm) and E. faecium-derived extracellular vesicles (EVs) (EfmEVs) exert protective effect against ethanol-induced gastric injury by investigating the effect of EfmEVs on gastric mucosal ulcer scoring, histological lesion, mucosal glycoprotein production, acidity, anti-oxidative function, and inflammatory responses in rat. Pretreatment with Efm showed significant reduction of ethanol-induced gastric injury, as evidenced by the lowering of ulcer index, histological lesion, gastric pH, and inflammatory responses and the enhancement of mucosal glycoprotein production and anti-oxidative function. Further functional studies on three bioactive components [inactivated Efm, EfmEVs (EVs), and EV-free supernatants] of the bacterial culture showed that EVs are mostly responsible for the gastroprotective effect. Moreover, EV secretion is beneficial for the gastroprotective effect of Efm. Hence, EVs mediated the protective effect of Efm against ethanol-induced gastric injury by lowering inflammatory responses and enhancing anti-oxidative function and may be a potent anti-inflammatory and anti-oxidative strategy to alleviate hyperinflammatory gastrointestinal tract conditions. IMPORTANCE This study indicated that Enterococcus faecium provided a protective effect against rat gastric injury, which involved improvement of the mucosal glycoprotein production, anti-oxidative function, and inflammatory responses. Furthermore, we confirmed that three bioactive components (inactivated Efm, extracellular vesicles, and EV-free supernatants) of E. faecium culture also contributed to the gastroprotective effect. Importantly, E. faecium-derived EVs showed an effective impact for the gastroprotective effect.

1st Editorial Decision Re: Spectrum03894-23 (Protective effect of Enterococcus faecium against ethanol-induced gastric injury via Extracellular Vesicles) Dear Dr. Qien Qi: Thank you for the privilege of reviewing your work.I have received comments from two reviewers.They all think your manuscript need extensive improving from various aspects.I would reconisder your muanscript if you could revise the manuscript extensively as suggested by the two reviewers.Below you will find the comments and instructions from the Spectrum editorial office.
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Reviewer #1 (Comments for the Author): In this manuscript, the authors present data suggesting that extracellular vesicles derived from the gram-positive bacteria E. faecium provide some protection against ethanol-induced gastric ulcer in rats.The authors measure multiple parameters, as well as gene expression of some mRNAs relevant to gastric ulcer.They then go on to show that increasing the number of extracellular vesicles artificially using LZD also increase this protective effect.
Generally, the manuscript provides data to support the main conclusions, but little is done to put these results any context.The results section reads more like a figure legend and is challenging to assess due to limited information.The authors use the discussion section to give the results some context, but they do not present any potential limitations of the study.A few ideas on additional challenges that could be discussed are suggested below: Major Comments 1.The text of the results is very fragmented.I appreciate the direct approach to relaying only the findings, but it is too simplistic.It makes understanding this section very difficult for the reader.Additional effort to explain the rationale and setup for each experiment would clarify the authors' motivations.2. Line 112-114, it is unclear how the mass of EfmEVs delivered compares to the mass of EVFS or iaEfm used.Is it possible that the effects observed are dependent on the total material delivered?Can the authors comment on whether these amounts are physiologically relevant to the EVs naturally released by the bacteria or would this only be relevant in the case of artificial delivery.Relatedly, the scoring for Efm and iaEfm (Figure 1B and Figure 4B), appear to be similar.Does this argue against EV release as an active process?Similarly, when comparing Figure 1B vs 7B, the Efm scoring is quite different.I understand that there is some inherent variability in the assay between experiments, but this should be brought up in the discussion section as a potential limitation of the study.3. Figure 1A (and 4A, 7A), I find this schematic a little confusing.What are the numbered items?Why is PBS listed twice?A few extra words in the legend might help.4. Line 187-195, why were these genes chosen for measurement of mRNA expression?This information is at least partially included in the discussion, but it would help to have some rationale for the experimental setups with the results.5. Line 258-269, it is difficult to assess what is happening in these experiments from these results descriptions.See comment 1 above.6. Figure 5B, the error bars appear to be cut off by the y-axis break.7. Can the authors rule out that they are not just delivering protein aggregates with their EV isolation method?Would they see the same effect if another EV isolation method like size-exclusion chromatography was used to isolate EVs?In this regard, Figure 6A would be strengthened by an alternative measure of EVs, such as NTA or TRPS that directly measures particles.8.A related potential experiment would be to first treat the EVs with a low amount of detergent to see if breaking the EVs prevents the effect on gastric ulcer.A proper control of a small amount of detergent alone would also be required to make this meaningful.Have the authors considered such an experiment?Maybe it is not possible with rats.9. What is the mechanism of action of LZD? Are there other effects on the bacteria that could be contributing to protection?Again, this could be discussed in the potential limitations of this study.10.Are all EVs resistant to low pH or is this specific to gram positive bacterial EVs? Relatedly, if you provided EVs in high numbers from other gram-positive bacteria, would you also expect protection, or is this effect unique to E. faecium?
Minor Comments 1.The manuscript has a lot of acronyms, and many are first defined in the methods, which makes understanding the results section quite challenging at times.It would be helpful to reduce the total number of acronyms, or at least strategically redefine some in the results section.Some are only defined in the list of acronyms at the end (NIR, UI, etc).2. Line 48-50, it could be beneficial to distinguish between bacterial-derived EVs and host derived EVs (exosomes, microvesicles).3. Line 87, Linezolid mechanism of action should be defined at first description.4. Line 170, The positive control group is denoted OME in the methods.Maybe it would be helpful to the reader to define OME alongside omeprazole?Some sort of description of omeprazole should also be included.5. Figure 2B, the authors should consider switching to using colors that are friendly to color-blind readers.
Reviewer #2 (Comments for the Author): In the manuscript entitled "Protective effect of Enterococcus faecium against ethanol-induced gastric injury via Extracellular Vesicles" Luo and co-authors studied the protective effect of the Extracellular vesicles produced by E. faecium compared the organism itself, and omeprazole, in the gastric injury model caused by ethanol.The observations made in the study are interesting, as this organism is part of our microbiota and could play an important role in preventing damage to the gastrointestinal tissue.The authors used histological data and transcript expression to determine the level of injury observed in the stomach after different treatments and controls were used.The study draws attention to the role of EVs in this scenario.The authors used PBS and Omeprazole as controls, as well as the use of the supernatant after EV isolation.The EVs characterization also showed the MET and LDS for measurement and phenotype analysis.Major comments: The English needs extensive revision.Lines 30 and 31 -"Gastric ulcer is the most common gastrointestinal disorder affects 10% of the world population with different etiologies [1].It is mucosal erosions caused by many factors such as..." Disorder THAT affects."It is mucosal erosions" -lacks a connector.
Line -"with an important functions", remove the AN Line -"Extracellular vesicles (EVs) are lipid-bilayers produced by all domains of life" -after bilayers it lacks a noun.

Line -"However role of EV"
These are some examples found only on page one.So, the authors need to review the language throughout the entire text.
Line -How many technical and biological replicates were performed?
Results subsections -The results section titles are presented as a methodology.The authors should call attention to the main result presented here.
Line -How many EV isolations did the authors prepare?Was this reproducible?
Line -What does "reasonable request" mean?Open science currently does not support and even discourages this type of statement.It lacks transparency and reproducibility.
Reviewer #1 (Comments for the Author): In this manuscript, the authors present data suggesting that extracellular vesicles derived from the gram-positive bacteria E. faecium provide some protection against ethanol-induced gastric ulcer in rats.The authors measure multiple parameters, as as gene expression of some mRNAs relevant to gastric ulcer.They then go on to show that increasing the number of extracellular vesicles artificially using LZD also this protective effect.
Generally, the manuscript provides data to support the main conclusions, but little is done to put these results any context.The results section reads more like a figure and is challenging to assess due to limited information.The authors use the discussion section to give the results some context, but they do not present any potential limitations of the study.A few ideas on additional challenges that could be discussed are suggested below: Reply: We thank the reviewer for recognizing our work.
Based on the comments of the reviewer, We have made a comprehensive logical arrangement and series of the results section and presented some potential limitation of the study in the discussion section.

Major Comments
1.The text of the results is very fragmented.I appreciate the direct approach to relaying only the findings, but it is too simplistic.It makes understanding this section very difficult for the reader.Additional effort to explain the rationale and setup for each experiment would clarify the authors' motivations.
Reply: We greatly appreciate this reviewer's above comments.The results section are revised according to the comment that make our manuscript much more easy-to-read.
2. Line 112-114, it is unclear how the mass of EfmEVs delivered compares to the mass of EVFS or iaEfm used.Is it possible that the effects observed are dependent on the total material delivered?Can the authors comment on whether these amounts are physiologically relevant to the EVs naturally released by the bacteria or would this only be relevant in the case of artificial delivery.Relatedly, the scoring for Efm and iaEfm (Figure 1B and Figure 4B), appear to be similar.Does this argue against EV release as an active process?Similarly, when comparing Figure 1B vs 7B, the Efm scoring is quite different.I understand that there is some inherent variability in the assay between experiments, but this should be brought up in the discussion section as a potential limitation of the study.

Reply:
We greatly appreciate this reviewer's above comments.Due to our carelessness, we made mistakes in the calculation and description of the mass of EfmEVs delivered compares to the mass of EVFS or iaEfm.After the calculation confirmed, the original manuscript is revised to "In our experiment, each 1 mL overnight cultures contained about 2 × 10 9 CFU Efm, and 20 μg EfmEVs.
Therefore, 1 mL EVFS was comparable to 2 × 10 9 CFU inactived Efm and 20 μg EfmEVs in the subsequent tests.",Line 115-116.These amounts of Efm and EfmEVs in a given volume was the experimental results of the author in vitro culture.In the natural environment, due to differences in nutritional conditions, bacterial density, and other environmental conditions, this may have an impact.
We strongly agree with the reviewers' understanding of the differences of scoring in different experiments.Following the reviewer's suggestion, the author brought up this limitation in the discussion section, Line 365-368.

Reply:
The results section are revised according to the comment 1 that make our manuscript much more easy-to-read.6. Figure 5B, the error bars appear to be cut off by the y-axis break.
Reply: Revised.7. Can the authors rule out that they are not just delivering protein aggregates with their EV isolation method?Would they see the same effect if another EV isolation method like size-exclusion chromatography was used to isolate EVs?In this regard, Figure 6A would be strengthened by an alternative measure of EVs, such as NTA or TRPS that directly measures particles.
3. Figure1A(and 4A, 7A), I find this schematic a little confusing.What are the numbered items?Why is PBS listed twice?A few extra words in the legend might help.Reply: Three animal tests were conducted in this study, with a total of 11 treatment groups.The item number represents the treatment group number in the full text.PBS listed twice in Figure1a.In the first animal test, rats in the normal control group were orally administered with 5 mL/kg body weight (BW) of PBS every other day for a total of three times, and received 5 mL/kg BW of PBS to induce gastric mucosal injury at 2 h after the last treatment.The author has improved the description of the Figure legends.4.Line 187-195, why were these genes chosen for measurement of mRNA expression?This information is at least partially included in the discussion, but it would help to have some rationale for the experimental setups with the results.Reply: Endothelin-1 (ET-1) is a potent vasoconstrictor,The gastric mucin can protect the gastric epithelium from diverse chemical or physical stimuli.Twelve genes encoding mucins have been reported.MUC1, MUC5AC, and MUC6 are included.The nuclear transcription factor Nfkb1 plays a vital role in regulating the immune responses.IL-1b, IL-6, IL-10 are important inflammation-related cytokine in the body, mainly regulate the cell function and involved in the body immunity.These genes are known to be activated in EtOH-induced gastric ulcer in mice.The results section are revised according to the comment, Line 201-202. 5. Line 258-269, it is difficult to assess what is happening in these experiments from these results descriptions.See comment 1 above.