Neurotransmitter Profiles Are Altered in the Gut and Brain of Mice Mono-Associated with Bifidobacterium dentium

Round 1

Reviewer 1 Report

The work is original: an interesting idea, database analysis, experimental research conducted at a high level, the results are clearly demonstrated. I would especially like to mention the beautiful informative illustrations. The work will undoubtedly arouse great interest among readers and will be cited.

The only comment / suggestion: after the presentation of the results and a lively discussion, the authors ' Conclusion is missing, and I would like to hear it in more detail than in a short Abstract.

Author Response

Reviewer Comments:

We appreciate the opportunity to clarify and improve our work. All our edits in the revision manuscript as highlighted in blue text.

 

Reviewer #1

1. The only comment/suggestion: after the presentation of the results and a lively discussion, the authors ' Conclusion is missing, and I would like to hear it in more detail than in a short Abstract.

We appreciate the suggestion and we have now added a final conclusion paragraph to the discussion.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

In this study, the authors analyzed the enzymes involved in GABA and tyrosine biosynthesis pathways in Bifidos and B. dentium genomes, and then studied the capability of B dentium in producing GABA and tyrosine in vitro and in vivo. Although it was shown B dentium contains the enzymes to produce GABA via glutamine, glutamate and even succinate, the in vitro and in vivo results only confirmed that B dentium can produce GABA, but how B dentium produced GABA in the experimental settings in this study was unknown as glutamine/glutamate was unchanged, while succinate was not tested. Moreover, the authors’ previous study has shown that B dentium can produce GABA. Therefore, there seems to be no new findings in this part regarding GABA. For the tyrosine part, it was claimed that B. dentium could synthesize tyrosine by analyzing tyrosine/dopamine pathway. However, according to the existing enzymes, tyrosine can actually only be produced from chorismate, which seems not something directly contained in foods. Collectively, the in vitro and in vivo results do not support the genome analysis findings in this study. There are some other comments for the authors’ consideration in improving the manuscript:

 

More detailed background information of B dentium should be included in the Introduction session. The Results session 3.1 contains mainly information that should be put to Introduction session.

 

The statement ‘Some neuro-active compounds like GABA cannot cross the blood brain barrier and have localized effects within the gut’ is not correct. It was previously thought that GABA could not cross the blood-brain barrier, but new research suggests that it may be able to.

 

More details should be provided for ‘cell culture assays’.

 

It was stated ‘….are described in the Supplemental Materials Section. See Tables S1-S2 in Supplemental Materials for…..’ in session 2.4.2, but no such files were provided.

 

It is confusing that ATCC 27678 and ATCC 27679 were used in Methods and Results sessions inconsistently.

 

‘Calibration standards were prepared at concentrations of 0.98, 3.9, 15.6, 62.5, and 1,000 ng/mL for all metabolites measured in each method.’ However, some of the data shown in figures were far out of the range of these standards, such as Figure 3D.

 

Figure 1B and 1C, better indicate what genes are contained and lacked in the respective genomes, but not the gene number. Gene names should be denoted but not the enzyme IDs for easy understanding.

 

Figure 1C was not fully annotated. It would be better revised to show ‘B. dentium and 10 other Bifidobacteria species contained all genes required to convert glutamine, glutamate and succinate into GABA.’ This part should be moved before the results describing Figure 1B.

Table 1 is not important and can be put as a supplementary table, with inclusion of genes contained/lacked in the corresponding species/genome.

 

The description on page 5 about B dentium strain ATCC27679 and culturing conditions are different from the Methods session.

 

The descriptions on the change in glutamate concentrations, ‘a slight decrease’ and ‘relatively unchanged, are confusing. The changes in glutamate, GABA and glutamine should be shown in figures, as compared with the levels at time 0 of culturing. It is important to show how efficient is B dentium in producing GABA.

Session 3.2, no need to introduce gnotobiotic mice here in Results session.

Where the bottom right area of Figure 2A was enlarged from should be indicated.

 

In mice, B dentium increased fecal level of GABA but did not change glutamine and glutamate. This suggests that B dentium can produce GABA, but not via glutamine and glutamate? It has been previously reported that B dentium can produce GABA. So what is the new finding in this study?

 

‘31 (49%) Bifidobateria species did not have tyrosine aminotransferase (EC 2.6.1.5), which converts 4-hydroxy-phenylpyruvate into tyrosine, indicating that tyrosine production is species dependent.’ Why this pathway and enzyme were not indicated in Figure 3? Does B dentium contains this enzyme?

 

‘Analysis of the KEGG pathway revealed that B. dentium could convert chorismate into tyrosine through a three-step process (Figure 3A).’ ‘we observed that B. dentium had the molecular machinery to produce tyrosine’ These statements are confusing. B dentium only has enzyme to convert chorismate into tyrosine, but not all enzymes involved in the ‘three-step process’. In vitro assays showed B dentium could not produce tyrosine. Why culture medium at baseline with high concentration of tyrosine was used? Did the medium contain material that B dentium can make use to produce tyrosine, such as chorismate?

 

What is the point of analyzing all Bifido species to see if they contain the necessary enzymes involved in producing GABA and tyrosine in this study? Many other Bifido species contain the relevant genes and B dentium is not an outstanding one.

What is the point in explaining enteric neurons can convert tyrosine to other neurotransmitters, but then none of these neurotransmitters converted from tyrosine was elevated in brain of the mice?

 

Figure 5 contains very little information. SLC7A9 and SLC16A10 were not included in this study and should not be shown here.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Great paper, overall very clear, nice graphics!

Line 24 "GABA in a fully-defined microbial media and elevated fecal GABA is in B. dentium mono-associated..."

Line 74 , 178,...: "ZMBI" --> reference "ZMB1" (as in reference paper)

 Line 80: "Germ-free mice were had ad ..." 

Line 91: dehydrated as decsribed in line 93?

Line 119:  In each instance, a consistent concentration of 119 method-specific deuterated IS compounds were added to each. --> please rephrase/precise

Line 122: "...was used generate graphs " --> used to 

Line 127: machinergy?

Line 300: "...and these levels"

Line 304: "...acid that can be transported from ..."

Line 323: dopapmine

 

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf