Metabolomic profiling of oxalate-degrading probiotic Lactobacillus acidophilus and Lactobacillus gasseri

Casey A. Chamberlain; Marguerite Hatch; Timothy J. Garrett

doi:10.1371/journal.pone.0222393

Peer Review History

Original SubmissionJuly 17, 2019
18 Aug 2019 Decision Letter - Andrea Motta, Editor PONE-D-19-20190 Metabolomic Profiling of Oxalate-Degrading Probiotic Lactobacillus acidophilus and Lactobacillus gasseri PLOS ONE Dear Dr. Garrett, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers reported that the manuscript is interesting and well written. The authors are required to discuss the minor points raised by the reviewers. We would appreciate receiving your revised manuscript by Oct 02 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In Methods The authors should better clarify the storage times, were the bacterial lysates stored at -80 ° C for how long before the analysis? Was the storage time the same for all the samples analyzed in this work? The authors compare the metabolic profiles between the two bacterial strains grown in a medium supplemented with oxalate (20 mM), they believe that it is not necessary to carry out controls on cultures not supplemented with oxalate? Or have they already performed these experiments in the past? The description of lipid extraction should be improved Reviewer #2: In the manuscript by Chamberlain et al, the authors deal focus on the metabolomes and lipidome of the oxalate-degrading Lactobacillus acidophilus and Lactobacillus gasseri. These studies might be important to define the metabolic properties of Lactobacillus that allow these bacteria to interact with the host intestine and influence overall health The studies are well designed and data presented clearly. There are some issues that will need to be addressed prior to publication, however The authors should explain for how long the bacterial lysates were stored at -80 ° C? before analysis Have the authors performed the metabolic profiles also on cultures not supplemented with oxalate? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0222393.r001
Revision 1
22 Aug 2019 Author Response Reviewer 1: Comment: Comment #1: The authors should better clarify the storage times, were the bacterial lysates stored at -80 ° C for how long before the analysis? Thank you for your feedback. The lysates were only stored for a brief time (approximately 1 month) before extraction, a time which was consistent across all samples. In our experience, lysates of this nature suspended in KH2PO4-based lysis buffer are stable for metabolomics analyses for several years, so we are confident that this short storage period is inconsequential to the experiment. We have adjusted the text (highlighted below) as follows to make this more clear to the reader. “Cell lysates were immediately frozen at -80 °C to ensure their stability and were briefly held frozen (approximately 1 month) until needed for extraction, all samples being stored for an equal period of time. In our experience, lysates of this nature suspended in KH2PO4-based lysis buffer are stable for metabolomics analyses for several years.” Comment #2: Was the storage time the same for all the samples analyzed in this work? Thank you for your feedback. Yes, the storage time was the sample for all samples analyzed in this work. Please see our response to Comment #1 in which we clarify this point in the adjusted text. Comment #3: The authors compare the metabolic profiles between the two bacterial strains grown in a medium supplemented with oxalate (20 mM), they believe that it is not necessary to carry out controls on cultures not supplemented with oxalate? Or have they already performed these experiments in the past? Thank you for your feedback. The purpose of this experiment was to profile and compare the metabolomes of L. acidophilus and L. gasseri, not necessarily to compare the metabolomes in an oxalate versus non-oxalate environment. Oxalate was included in the media simply because that is how we have historically cultured Lactobacillus, but it will grow in many different types of media so it is difficult to assign one media as its native environment. Therefore, we did not perform an analysis on Lactobacillus grown without oxalate since such a control was outside of the focus of this experiment. It would be interesting for future studies to compare how the metabolome of Lactobacillus changes in different types of media, but being that it grows in many different matrices, it would be difficult to establish what is the “native” media since there is no designated media that puts it into its native state. Comment #4: The description of lipid extraction should be improved. Thank you for your feedback. We believe there may be some confusion that the lipid extraction was not explained given the placement of references to the Folch method and our previous work at the beginning of the Lipid Extraction section. Immediately following this first sentence, the lipid extraction is explained in significant detail. To make this more clear to the reader, we have inserted text (highlighted below) that indicates the beginning of the description of the lipid extraction. “Lipids were extracted using a modified version of the Folch method (41) similarly to our previous work (38) using the following process which we describe in detail here. From each normalized cell lysate sample, 150 µL was transferred to a 12 mL glass vial. Extraction blanks were included for downstream data filtering using 150 µL lysis buffer and were treated identically to biological samples. To each sample, 20 μL of internal standard mix (Avanti Polar Lipids, Alabaster, AL, USA) was added followed by brief vortexing – LPC(17:0), PC(17:0/17:0), PG(14:0/14:0), PE(15:0/15:0), PS(14:0/14:0), TG(15:0/15:0/15:0), PI(8:0), SM(d18:1/17:0), CER(d18:1/17:0), DG(14:0/14:0), CL(15:0(3)-16:1), SO(d17:1), PAzePC, CER(Glycosyl(β) C12), BMP(14:0 (S,R)), LSM(d17:1), 5 μg/mL each in 2:1 chloroform:methanol. Next, 400 μL methanol was added to each sample followed by vortexing, then 800 μL chloroform was added. Samples were vortexed and incubated on ice for 20 min, with vortexing at 10 and 20 min, followed by addition of 200 μL water. Samples were briefly vortexed and incubated on ice for 10 min with vortexing at 5 and 10 min. Separation of the organic and aqueous layers was achieved by centrifugation at 3260×g, 4°C for 10 min. The organic (bottom) layer containing lipid content was transferred to a new 12 mL glass vial in two steps: first with removal of 800 μL of the original organic layer, followed by another removal of 400 μL after re-extracting the remaining aqueous layer with 400 μL 2:1 chloroform:methanol by incubating on ice for 10 min and centrifugation at 3260×g, 4°C for 10 min. Lipid extracts were dried under nitrogen at 30°C and reconstituted in 300 μL isopropanol. Samples were centrifuged at 3260×g, 4°C for 10 min to pellet any residual protein, and 250 μL supernatants were transferred to glass LC vials for UHPLC-HRMS analysis.” Reviewer 2: Comment #1: The authors should explain for how long the bacterial lysates were stored at -80 ° C before analysis? Thank you for your feedback. Please see our response to Reviewer 1 Comments #1 and #2, which addresses these same questions. Comment #2: Have the authors performed the metabolic profiles also on cultures not supplemented with oxalate? Thank you for your feedback. Please see our response to Reviewer 1 Comment #3, which addresses this same question. https://doi.org/10.1371/journal.pone.0222393.r002
29 Aug 2019 Decision Letter - Andrea Motta, Editor Metabolomic Profiling of Oxalate-Degrading Probiotic Lactobacillus acidophilus and Lactobacillus gasseri PONE-D-19-20190R1 Dear Dr. Garrett, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Andrea Motta Academic Editor PLOS ONE Additional Editor Comments (optional): All comments have been addressed. Reviewers' comments: https://doi.org/10.1371/journal.pone.0222393.r003
Formally Accepted
13 Sep 2019 Acceptance Letter - Andrea Motta, Editor PONE-D-19-20190R1 Metabolomic Profiling of Oxalate-Degrading Probiotic Lactobacillus acidophilus and Lactobacillus gasseri Dear Dr. Garrett: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Andrea Motta Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0222393.r004

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