Research ResourceHost-Microbe Interactions

A dynamic mouse peptidome landscape reveals probiotic modulation of the gut-brain axis

See allHide authors and affiliations

Science Signaling  28 Jul 2020:
Vol. 13, Issue 642, eabb0443
DOI: 10.1126/scisignal.abb0443
  • Fig. 1 Experimental design and integrated peptidomics workflow.

    (A) Seven groups of mice (n = 12 animals in each group) were differentially treated with plain water (C), L. helveticus NS8 (NS8), L. fermentum NS9 (NS9), or heat-killed NS8 (NS8h) for 1 or 2 months as shown. After treatment, gut microbiome composition (16S rRNA sequencing) and brain peptidomics data were generated for each group. The n value for each omics dataset is indicated next to the symbol for that dataset. For data analysis, all the data in each group were combined. The n value for data in some omics sets is less than the number of animals in each experiment (n = 12) because of failed sample preparation or data acquisition. C, 1-month control; NS8, 1-month NS8 treatment; NS8h, 1-month heat-killed NS8 treatment; NS9, 1-month NS9 treatment; CC, 2-month control; NS8C, 1-month NS8 treatment and 1-month nontreatment; NS8NS8, 2-month NS8 treatment. (B) Integrated workflow for in-depth analysis of brain peptidomes.

  • Fig. 2 Benchmarking the 1-hour peptidomics approach and peptidome changes upon probiotics administration.

    (A) Dynamic range of brain peptides based on peptide intensities across four brain regions in control animals given plain water for 1 month (group C). Values for some peptides are noted, and the complete list of identified peptides is in data file S1. (B) Cumulative abundance of peptides in the hypothalamus of group C from the highest to the lowest abundance. The box plot inset shows the change in somatostatin-28(1-12) abundance induced by probiotics treatment. (C) Families to which the top 75% of neuropeptides (by accumulative intensity) in the hypothalamus of group C animals belong. (D) Histogram illustrating the quantifiable and significantly altered peptides in hypothalamus after 1-month of NS8 treatment compared to 1 month of no treatment (NS8/C) including the prohormone-derived (left) and nonprohormone-derived peptides (right). Unique sequences and abundance data are shown in data file S2. (E) Logo plot illustrating the motifs present in the terminal regions flanking peptides corresponding to the quantifiable peptides in (D) and data file S2. (F) Rate of NS8-induced change of neuropeptide-derived peptides compared to control. CCK, cholecystokinin; NP, neuropeptide; LPV, longest peptide variants. Significance of altered peptides was calculated by a two-tailed t test (P < 0.05).

  • Fig. 3 Correlation between changes in the brain peptidome and gut microbiome upon 1-month treatment with the probiotic NS8 and heat-killed NS8.

    (A) PCoA plot of gut microbiome and PCA plots of the hypothalamus and hippocomapus peptidomes after 1 month of treatment with live (NS8) or heat-killed (NS8h) NS8 compared to control animals (C). (B) Relative abundance of four bacterial genera in groups C, CS8, and NS8h. Two-tailed t test. (C) Heatmap showing the matrix of Spearman’s correlation coefficient (r) of significantly altered bacterial genera and representative brain peptides in hypothalamus. r < −0.5 or r > 0.5 for at least one of the four bacterial genera. Details are available in data file S3. (D) Scatter plot showing the relationship between the abundance of vasoactive intestinal peptide (VIP) against the relative abundance of Anaerotruncus in the gut microbiome. (E) Hierarchical clustering of changes in the hypothalamus peptidome C, NS8h, and NS8 treatment groups. One-way analysis of variance (ANOVA), P < 0.05. The peptides exclusively altered in the NS8 group as compared to the C and NS8h groups are boxed, and complete data is included in data file S4. (F) Neuropeptide families of the hypothalamus peptidome that were exclusively altered in the NS8 group.

  • Fig. 4 Regional brain distribution and change of peptides in NS8-treated versus control animals.

    (A) Circular brain peptidome maps depict the qualitative, quantitative, and region-specific information in the peptidomes from control (group C) animals (details in data file S5). The outer ring indicates the neuropeptide families; the middle four rings are qualitative information of brain peptides in the four regions of the brain; the inner four rings are quantitative information for each of the four brain regions. The 26 neuropeptide families are listed in table S1. (B) Heatmap shows the change rate (altered/quantifiable peptides in a family) of brain peptide families in NS8-treated mice compared to control mice. (C) Histograms illustrate the quantifiable brain peptides that were altered in the four brain regions in NS8-treated mice compared to group C. Box plots show the fold change of the altered brain peptides (modification-specific peptides). (D) Bar graphs show the number of peptides that were identified, increased, or decreased in each brain region. Subsets of peptides that were present in multiple regions (colocalized) or showed similar changes in abundance in multiple regions (colocalization) are noted in the matrix below the graphs. The pie charts summarize the percentage of brain peptides that were coregulated in one, two, three, or all four brain regions. The details are shown in data file S6. (E) Heatmap shows the matrix of Spearman’s correlation coefficient (r) of significantly altered bacterial genera and representative brain peptides across four brain regions. r < −0.5 or r > 0.5 for at least one of the four bacterial genera. (F) Scatter plots show the relationships between the abundances of neuropeptide Y (NPY) and orexin-B and the [Eubacterium] xylanophilum group and Anaerotruncus in the gut microbiome. The data were processed from quantitative data of NS8-treated versus control (NS8/C) in four brain regions. Significance of altered peptides was calculated by a two-tailed t test (P < 0.05), and peptides that increased or decreased in abundance were determined by mean differences.

  • Fig. 5 Changes in hypothalamus peptides across treatment conditions.

    (A) Ternary PCA plot of the brain peptidome changes across the seven experimental groups C, NS8, NS8h, NS9, CC, NS8C, and NS8NS8. The colored arrows indicate treatment groups through which pairwise comparisons can identify changes specific to probiotic treatment, different probiotic strains, the 1-month age difference between animals, live versus heat-killed probiotic, and recovery from 1-month treatment with live NS8. (B) Volcano plots of altered brain peptides for 1- and 2-month treatment with NS8 compared to water-only controls (NS8/C and NS8NS8/CC). Two-tailed t test, P < 0.05. (C) PCoA plot of gut microbiome and PCA plot of hypothalamus peptidome for groups C, NS8, and NS9. (D) Screening curve showing putative probiotics-altered peptides that increased (red) or decreased (green) with NS8 treatment compared to control. CRH is highlighted in blue (data file S7, first sheet). (E) Dot blot validation of CRH abundance under the six indicated conditions. (F) Changes in the abundances of representative neuropeptides after 1-month (NS8) and 2-month (NS8NS8) NS8 treatment regimens relative to (/) the control groups as indicated (data file S7, second sheet). Peptides were selected by significance of a two-tailed t test for NS8NS8/CC, P < 0.05. AVP, arginine vasopressin; α-MSH, α-melanocyte-stimulating hormone.

  • Fig. 6 The MINIbar algorithm for text mining of behaviors related to specific neuropeptides in published abstracts.

    (A) Schematic showing how PubMed abstracts were mined to identify physiological processes and behaviors regulated by specific neuropeptides altered by probiotic treatment. (B) Radar diagram showing possible connections between probiotic treatments and behaviors predicted from publication mining. The five probiotic treatment conditions are linked with the six behaviors on the basis of numbers of publications reporting regulatory roles of neuropeptides on behaviors. Details are shown in data file S9.

  • Fig. 7 Monitoring the brain peptide change of HPA axis upon prenatal stress and NS8 treatment.

    (A) Column scatter plot shows peptidomic intensities of CRH in the hypothalamus of control (C#2, n = 12) and NS8-treated (NS8#2, n = 12) offspring of untreated mothers and in the hypothalamus of control (V#2, n = 6) and NS8-treated (VNS8#2, n = 6) offspring of VPA-treated mothers. Below the graph, dot blots for CRH in peptides extracted from the hypothalamus of the indicated mice are shown. Blots are representative of independent experiments with samples from three different mice. (B) Column scatter plot shows peptidomic intensities of ACTH in the pituitary of control (C#2, n = 7) and NS8-treated (NS8#2, n = 5) offspring of untreated mothers and in the hypothalamus of control (V#2, n = 6) and NS8-treated (VNS8#2, n = 5) offspring of VPA-treated mothers. Below the graph, dot blots for ACTH in peptides extracted from the hypothalamus of the indicated mice are shown. Blots are representative of independent experiments with samples from three different mice. Data are means ± SEM. Significance was calculated by a two-tailed t test.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/642/eabb0443/DC1

    Fig. S1. Data quality of the 1-hour peptidomics approach in analysis of hypothalamus peptides.

    Fig. S2. Quality control of the quantitative peptidomics.

    Fig. S3. Gut microbiome compositions resulting from 16S rRNA analysis.

    Fig. S4. Histogram illustrates the total quantifiable and significantly altered hypothalamus peptides (unique sequences) of the CC/C dataset resulting from 1-month development of mice.

    Fig. S5. Hierarchical clustering shows the brain peptide change in hypothalamus among the C, NS8, and NS9 groups.

    Table S1. Neuropeptide families.

    Data file S1. Identified peptides from four brain regions (corresponding to Fig. 2A) (Excel file).

    Data file S2. Detailed information of peptides shown in Fig. 2D (Excel file).

    Data file S3. Detailed information of correlation analysis shown in Fig. 3C (Excel file).

    Data file S4. Detailed information of clustering analysis shown in Fig. 3E (Excel file).

    Data file S5. Detailed information of peptidome shown in Fig. 4A (Excel file).

    Data file S6. Detailed information of peptidome shown in Fig. 4D (Excel file).

    Data file S7. Detailed information of peptidome shown in Fig. 5, D and F (Excel file).

    Data file S8. Percentage of basic-cleavage-site–containing peptides (Excel file).

    Data file S9. Detailed information of MINIbar script (Excel file).

  • The PDF file includes:

    • Fig. S1. Data quality of the 1-hour peptidomics approach in analysis of hypothalamus peptides.
    • Fig. S2. Quality control of the quantitative peptidomics.
    • Fig. S3. Gut microbiome compositions resulting from 16S rRNA analysis.
    • Fig. S4. Histogram illustrates the total quantifiable and significantly altered hypothalamus peptides (unique sequences) of the CC/C dataset resulting from 1-month development of mice.
    • Fig. S5. Hierarchical clustering shows the brain peptide change in hypothalamus among the C, NS8, and NS9 groups.
    • Table S1. Neuropeptide families.
    • Legends for data files S1 to S9

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1. Identified peptides from four brain regions (corresponding to Fig. 2A) (Excel file).
    • Data file S2. Detailed information of peptides shown in Fig. 2D (Excel file).
    • Data file S3. Detailed information of correlation analysis shown in Fig. 3C (Excel file).
    • Data file S4. Detailed information of clustering analysis shown in Fig. 3E (Excel file).
    • Data file S5. Detailed information of peptidome shown in Fig. 4A (Excel file).
    • Data file S6. Detailed information of peptidome shown in Fig. 4D (Excel file).
    • Data file S7. Detailed information of peptidome shown in Fig. 5, D and F (Excel file).
    • Data file S8. Percentage of basic-cleavage-site–containing peptides (Excel file).
    • Data file S9. Detailed information of MINIbar script (Excel file).

Stay Connected to Science Signaling

Navigate This Article