Limitation of amino acid availability by bacterial populations during enhanced colitis in IBD mouse model

ABSTRACT Members of the Enterobacteriaceae and Enterococcus are associated with persistent gut inflammation due to rapid colonization combined with pathogenic tendencies. Here, we investigated the functions of gut microbial populations resulting in persistent gut inflammation. In this study, we utilized the IL-10 knockout mouse model and induced colitis using dextran sulfate sodium (2%) after development. Dams during gestation were provided cefoperazone to induce vertically transmitted dysbiosis in the pups that were monitored in this study. We characterized the dysbiotic gut microbial community and potential crosstalk of these microbes, and host gene expression changes to identify bacterial populations and potential functions that were involved in gut inflammation. We isolated Enterobacteriaceae populations from mice to validate the utilization of sulfur-containing amino acids. Members of Enterobacteriaceae and Enterococcus were highly detected in inflamed mice. Enterobacteriaceae populations containing L-cysteine dioxygenase were strongly correlated with the upregulation of host gene CSAD, responsible for cysteine breakdown. We observed that bacterial isolates from dysbiotic mice displayed increased growth rates when supplemented with L-cysteine, highlighting the use of sulfur metabolism. Our results show that microbial populations use alternate metabolisms and sequester host nutrients for growth, associated with inflammation in the gut. IMPORTANCE Inflammatory bowel disease is associated with an increase in Enterobacteriaceae and Enterococcus species; however, the specific mechanisms are unclear. Previous research has reported the associations between microbiota and inflammation, here we investigate potential pathways that specific bacteria populations use to drive gut inflammation. Richie et al. show that these bacterial populations utilize an alternate sulfur metabolism and are tolerant of host-derived immune-response products. These metabolic pathways drive host gut inflammation and fuel over colonization of these pathobionts in the dysbiotic colon. Cultured isolates from dysbiotic mice indicated faster growth supplemented with L-cysteine, showing these microbes can utilize essential host nutrients.

• Comment (Line 267 of the resubmitted manuscript): Thanks for changing the numerical representation to follow the standards about significative figures, but my questions were not answered.Please clarify in the text, as it's the first but not the single appearance of this construction, whether the value after the plus-minus sign (in this and other occasions) is the standard error of the mean.
2) My comment was: *** 29.Lines 275 to 286: There are a lot of issues with numerical representations in this long paragraph: please correct numbers with too many figures clearing non-significative ones and correct errors with more precision than the precision of the magnitude.

Authors' reply is:
>>> This area of the text has been modified for simplicity and easy readability.It read "(control/no-CPZ: 0.894 {plus minus} 0.0496; CPZ/no-gavage: 0.997 {plus minus} 0.0005; CPZ/gavage-with-PBS: 0.924 {plus minus} 0.0505; CPZ/FMT: 0.998 {plus minus} 0.0005), regardless of whether the mice were dysbiotic or not.Although control mice had significantly lower MAG 001 detection compared with other groups (p<0.0421, Figure 4B)," and now reads "(control/no-CPZ: 0.89; CPZ/no-gavage: 0.99; CPZ/gavage-with-PBS: 0.92; CPZ/FMT: 0.99)," with the error removed.(PAGE 17,LINE 410) • Comment (Page 18 of the resubmitted manuscript): Thanks for improving the readability of the entire section.However, my comment was about correcting the numerical representation regarding the number of significative digits of each magnitude with its uncertainty, not to keep the former and remove the latter.Finally, the lines 429 and 432 contain the construction "p ={space} 3) My comment was: >>> Line 495: As general comments about the "Metagenomics" subsection, (i) details about the different databases used are missing (e.g., date of build, size), and they are as important as the details about the codes used; (ii) I miss some discussion about possible contamination sources, how they may affect the analysis, and the procedures and techniques that have been used to eliminate or mitigate them (as an example, the use of end-to-end negative control samples for this purpose).*** We have clarified the "Metagenomics" section to point out to the readers clearly that all versions of the databases used are detailed in following paragraphs in the "Metagenomics" subsection.The sentence now reads "The workflows use Snakemake (37), and implemented numerous tasks detailed in the following sections, " (PAGE 7,LINE 182) We added the following line in the Methods section "A negative-control sample was also included in the process to ensure there was no contamination from molecular DNA extraction and sequencing." to improve clarity for the readers.(PAGE 7,LINE 161) We also added the following in the Discussion section "While our findings provide a compelling link between the Enterobacteriaceae and Enterococcus MAGs and sulfur metabolism, these are potential functions resulting from MAGs that were further validated using cultured isolates from the mice in this project by a nutrient dependency assay with L-cysteine indicating the use of these amino acids.We have shown these MAGs were similar to the isolates used for the nutrient dependency assay, but are likely not identical."to show the limitations in our study.(PAGE 24,LINE 567) • Comment (Bioinformatics section on the resubmitted manuscript): thank you very much for addressing the point (ii) of my comment.However, despite authors' claims, the point (i) has not been addressed: details of the databases (DBs) used are still missing.The details about the DBs are as important as the details about the software used.Both software and data should be detailed as requirement for any attempt of replication.
Reviewer #2 (Comments for the Author): Thank you to the authors for their careful responses.The authors have addressed the majority of my concerns and the new growth curve and sanger sequencing results are helpful.However, I continue to find that certain areas of the manuscript 'overconclude', and would therefore change the phrasing in the following areas: 1. Abstract lines 32-33 and importance lines 40-41.The manuscript never directly shows that Enterobacteriaceae metabolism drives host inflammation.(To do so would have required an intervention in the mice with specific strains and mutants).Please change the phrasing to emphasize this is only a likely mechanism based on association, rather than a definite conclusion from this paper.E.g. replace 'driving' with 'associated with'.2. Line 369-370 conclusion here does not fit results re: cytokines; FMT mice did not display less host-associated cytokine modulation, they displayed the same or more.Please re-phrase the conclusion to make it clear that only histological inflammation, not cytokines, decreased.3. line 732-734.This conclusion is misleading since isolates from healthy mice display similar cysteine-based growth boost as isolates from dysbiotic mice.Also, there was never a direct statistical comparison between FMT isolates + cysteine and dysbiotic isolates + cysteine.It would be more accurate to say that "Enterobacteriaceae from dysbiotic mice use cysteine highly efficiently for growth", without any attempt at comparison between groups.

Dear Dr. Methe,
We are very thankful for the helpful suggestions and comments to further improve this manuscript.These suggestions were incorporated in the revised version of the manuscript with our responses included (colored blue text).In brief, we: • Addressed all statistical variances in the manuscript as standard error of the mean, and replaced all variances in the manuscript that fit the significant figures.• Improved the clarity of several areas including the Abstract, serum data and growth curve results.• Checked for accuracy of all programs and databases used in this study to ensure they are included in the "Materials and Methods" section, and added all versions used.
In our revision we have included a revised "CLEAN" and "MARKED-UP" version for transparency of the edits made.All page and line numbers will correspond with the "CLEAN" version of the manuscript.We hope that we have clarified the remaining comments the reviewers brought up and thank you again for the improvements that you and the reviewers have offered.
Comment (Line 267 of the resubmitted manuscript): Thanks for changing the numerical representation to follow the standards about significative figures, but my questions were not answered.Please clarify in the text, as it's the first but not the single appearance of this construction, whether the value after the plus-minus sign (in this and other occasions) is the standard error of the mean.Thank you to the reviewer for the thorough clarification.We have added the term "mean ± SE" in both the "Methods" and "Results" sections in the first appearance for better clarity.We have noted this in the manuscript as follows: (Page 11 Line 265) "Shotgun sequencing of the fecal samples resulted in a total of over 623 million sequences with an average of 12.2 ± 1.6 (mean ± SE) million reads per metagenome."(Page 10 Line 234) as well as mentioning this in detail under the "Statistical analyses" section of the "Materials and Methods" "P-values of less than 0.05 were considered statistically significant, with standard error of the mean (mean ± SE) utilized where appropriate."

2.
Comment (Page 18 of the resubmitted manuscript): Thanks for improving the readability of the entire section.However, my comment was about correcting the numerical representation regarding the number of significative digits of each magnitude with its uncertainty, not to keep the former and remove the latter.Finally, the lines 429 and 432 contain the construction "p ={space}<number" several times.It's unclear to me if the intended meaning is "equal or less than" (then the space should be moved to the right, between the sign and the number), or the equal sign was included by mistake.We thank the reviewer for clarification in this section.The magnitude of uncertainty (SE) was added back in the correct lines using fewer significant figures for the values (mean) matching the magnitude of the error.It is shown in bold in this reply.

3.
Comment (Bioinformatics section on the resubmitted manuscript): thank you very much for addressing the point (ii) of my comment.However, despite authors' claims, the point (i) has not been addressed: details of the databases (DBs) used are still missing.The details about the DBs are as important as the details about the software used.Both software and data should be detailed as requirement for any attempt of replication.(Page 7 Line 179) Please find the list below of databases and programs utilized for this study taken directly from the methods section.Please note that "anvi-gen-contigs-database" utilizes the contig database we assembled from the metagenome short-reads in this study using MEGAHIT, and is available in the figshare file https://doi.org/10.6084/m9.figshare.21753623.v2.This database represents all contigs that were assembled which we then mapped our metagenomes to resolve our MAGs (full details in methods of the manuscript).
Abstract lines 32-33 and importance lines 40-41.The manuscript never directly shows that Enterobacteriaceae metabolism drives host inflammation.(To do so would have required an intervention in the mice with specific strains and mutants).Please change the phrasing to emphasize this is only a likely mechanism based on association, rather than a definite conclusion from this paper.E.g. replace 'driving' with 'associated with'.We thank the reviewer for the helpful suggestions to the Abstract and Importance sections.To improve clarity for the readers, we mentioned multiple times in the revised manuscript, that these are potential pathways.We have made the suggested edits to the sections from: "Our results show that microbial populations use alternate metabolisms and sequester host nutrients for growth, driving inflammation in the gut.

Importance Inflammatory bowel disease is associated with an increase in Enterobacteriaceae and
Enterococcus species, however, the specific mechanisms are unclear.Previous research has reported associations of the microbiota and inflammation, here we investigate potential pathways specific bacteria populations use to drive gut inflammation.Richie et al. shows that these bacterial populations utilize an alternate sulfur metabolism and are tolerant of host-derived immune-response products.These metabolic pathways drive host gut inflammation and fuels over colonization of these pathobionts in the dysbiotic colon.Cultured isolates from dysbiotic mice indicated faster growth supplemented with L-cysteine, showing these microbes can utilize essential host nutrients."(Line 14-39) To now read: Abstract "Our results show that microbial populations use alternate metabolisms and sequester host nutrients for growth, associated with inflammation in the gut."Importance "Inflammatory bowel disease is associated with an increase in Enterobacteriaceae and Enterococcus species, however, specific mechanisms are unclear.Previous research has reports associations of the microbiota and inflammation, here we investigate potential pathways specific bacteria populations may use to drive gut inflammation.Richie et al. shows that these bacterial populations utilize an alternate sulfur metabolism and are tolerant of host-derived immune-response products.These metabolic pathways are associated with host gut inflammation and over colonization of these pathobionts in the dysbiotic colon.Cultured isolates from dysbiotic mice indicated faster growth supplemented with L-cysteine, showing these microbes can utilize essential host nutrients."

2.
Line 369-370 conclusion here does not fit results re: cytokines; FMT mice did not display less host-associated cytokine modulation, they displayed the same or more.Please re-phrase the conclusion to make it clear that only histological inflammation, not cytokines, decreased.We thank the reviewer for catching this point in our summation.The FMT mice had a similar cytokine profile, which was made clear in the text, and to clarify we have adjusted the summary statement to better reflect the data presented above from; "In summary, IL-10 KO mice receiving FMT displayed less colonic inflammation and host-associated cytokine modulation, indicating a successful FMT." (Page 13 Line 300) To now read: "In summary, IL-10 KO mice receiving FMT displayed less colonic inflammation with little change in host-associated cytokine modulation compared with other groups, indicating an overall successful FMT."

3.
Line 732-734.This conclusion is misleading since isolates from healthy mice display similar cysteine-based growth boost as isolates from dysbiotic mice.Also, there was never a direct statistical comparison between FMT isolates + cysteine and dysbiotic isolates + cysteine.It would be more accurate to say that "Enterobacteriaceae from dysbiotic mice use cysteine highly efficiently for growth", without any attempt at comparison between groups.We would like to thank the reviewer for bringing attention to this statement.We have revised this statement to conclude that the isolates from FMT mice did not appear to utilize L-cysteine for growth, but all other groups showing statistically significant growth increases when supplemented.This statement reads "These results suggest that Enterobacteriaceae in these dysbiotic mice were using cysteine more efficiently for growth than Enterobacteriaceae from healthy mice or mice that received FMT.." (Page 23 Line 528) and now reads "These results suggest that Enterobacteriaceae isolated from dysbiotic mice were using cysteine more efficiently for growth, whereas Enterobacteriaceae from mice that received FMT did not indicate this increased growth."Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.For your reference, ASM Journals' address is given below.Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication.They will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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-23R1 (Limitation of Amino Acid Availability by Bacterial Populations During Enhanced Colitis in IBD Mouse Model) Dear Dr. Sonny T.M. Lee: Thank you for completing the additional changes as recommended by the reviewers.
Thank you for submitting your paper to mSystems.