1. Evolutionary Biology
  2. Immunology and Inflammation

Glycan-based shaping of the microbiota during primate evolution

  1. Sumnima Singh
  2. Patricia Bastos-Amador
  3. Jessica Ann Thompson
  4. Mauro Truglio
  5. Bahtiyar Yilmaz
  6. Silvia Cardoso
  7. Daniel Sobral
  8. Miguel P Soares  Is a corresponding author
  1. Instituto Gulbenkian de Ciência, Portugal
https://doi.org/10.7554/eLife.67450
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Cite this article
  1. Sumnima Singh
  2. Patricia Bastos-Amador
  3. Jessica Ann Thompson
  4. Mauro Truglio
  5. Bahtiyar Yilmaz
  6. Silvia Cardoso
  7. Daniel Sobral
  8. Miguel P Soares
(2021)
Glycan-based shaping of the microbiota during primate evolution
eLife 10:e67450.
https://doi.org/10.7554/eLife.67450
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4 figures, 1 table and 1 additional file

Figures

Figure 1 with 4 supplements
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Ggta1 deletion alters the gut microbiota.

(A) Linear discriminant analysis (LDA) scores generated from LEfSe analysis, representing taxa enriched in the fecal microbiota of Ggta1+/+ (green) (n = 15) and Ggta1-/- (red) (n = 14) mice. (B) Breeding strategy where F0 Ggta1-/- males were crossed with Ggta1+/+ females to generate F1 Ggta1+/- mice, which were bred to generate F2 Ggta1+/+ vs. Ggta1-/- littermates. These were subsequently bred to generate F3 to F5 Ggta1+/+ vs. Ggta1-/- mice. Microbiota Principal Coordinate Analysis of Unweighted Unifrac distance in fecal samples from (C) F0 Ggta1+/+ (n = 15), F1 Ggta1+/- (n = 15), F2 Ggta1+/+ (n = 11) and F2 Ggta1-/- (n = 10) mice, (D) F3 Ggta1+/+ (n = 9) and Ggta1-/- (n = 8) mice, (E) F4 Ggta1+/+ (n = 13) and Ggta1-/- (n = 7) mice and (F) F5 Ggta1+/+ (n = 7) and Ggta1-/- (n = 12) mice generated as in (B). Data from one experiment with two to three independent breedings/cages per genotype. Symbols (C–F) are individual mice. p Values (C–F) calculated using PERMANOVA test.

Figure 1—figure supplement 1
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Analyses of gut microbiota composition of Ggta1+/+ and Ggta1-/- mice.

Relative abundance of bacteria at all levels of taxonomy, present at >2% frequency, in the same mice as in Figure 1A. Symbols represent individual mice. Red bars correspond to mean values. Error bars correspond to SD. Adjusted p values calculated using Benjamini-Hochberg correction. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Figure 1—figure supplement 2
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Analyses of gut microbiota composition of Ggta1+/+ vs. Ggta1-/- mice.

Relative abundance of bacteria at all levels of taxonomy, present at >2% frequency in the same mice as in Figure 1A. Stacked bars represent the mean of the bacterial taxa. Colors represent the relative fraction of each taxon. Data from one experiment.

Figure 1—figure supplement 3
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Ggta1 deletion does not cause inflammation at steady state.

(A) Representative H/E sections of the small intestine, large intestine, liver, spleen, lung and kidney of Ggta1+/+ (n = 5) and Ggta1-/- (n = 5) mice at steady state. (B) Fecal Lcn-2 concentrations in Ggta1+/+ (n = 10) and Ggta1-/- (n = 10) mice at steady state. Symbols represent individual mice. Red bars correspond to mean values. Error bars correspond to SD, ns: not significant.

Figure 1—figure supplement 4
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Ggta1 deletion shapes the gut microbiota composition.

Relative abundance of bacteria at all levels of taxonomy, present at >2% frequency in the same mice as in Figure 1C–F. Stacked bars represent the mean of the bacterial taxa. Colors represent the relative fraction of each taxon. Data from one experiment.

Figure 2 with 2 supplements
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Ggta1 deletion enhances IgA responses to the gut microbiota.

(A) Relative binding of IgA in the serum of Ggta1+/+ (n = 10) and GF Ggta1+/+ (n = 5) mice to fecal extract from Ggta1+/+ mice, and Ggta1-/- (n = 6) and GF Ggta1-/- (n = 6) mice to fecal extract from Ggta1-/- mice; one experiment. (B) Relative binding of IgA in the serum of GF Ggta1+/+ (n = 7) and GF Ggta1-/- (n = 10) mice to cecal extract from Ggta1-/- mice at indicated time-points after colonization with cecal extract from Ggta1-/- mice; two experiments. (C) Median Fluorescence Intensity (MFI) of αGal+ bacterial strains stained with BSI-B4 lectin relative to unstained control; seven experiments. (D) Representative flow cytometry plots showing bacteria stained for IgA and αGal in the small intestinal content of Ggta1+/+ (n = 5) and Ggta1-/- (n = 11) mice; four independent experiments. (E) Quantification of αGal+, IgA+, and IgA+αGal+ bacteria in the same samples as in (D). (F) Concentration of anti-αGal IgM in serum of Ggta1+/+ (n = 2), Ggta1-/- (n = 12), Igha-/-Ggta1+/+ (n = 10), and Igha-/-Ggta1-/- (n = 12) mice before and after streptomycin treatment, two experiments. (G) Concentration of anti-αGal IgG, in the same mice as (F). Symbols (A, B, E, F, G) are individual mice. Red bars (A, B, E, F, G) correspond to mean values. Error bars (A, B, C, E, F, G) correspond to SD. p Values in (A, B, F, G) calculated using Kruskal-Wallis test using Dunn’s multiple comparisons test and in (E) using Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

Figure 2—figure supplement 1
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Ggta1 deletion enhances IgA responses to the gut microbiota.

(A) Concentration of total IgA in serum of Ggta1+/+ (n = 9), GF Ggta1+/+ (n = 5), Ggta1-/- (n = 5) and GF Ggta1-/- (n = 6) mice. (B) Concentration of total IgA in the small intestinal content of Ggta1+/+ (n = 10), GF Ggta1+/+ (n = 5), Ggta1-/- (n = 10) and GF Ggta1-/- (n = 5) mice. (C) Relative binding of IgA in the serum of GF Ggta1+/+ (n = 5) and GF Ggta1-/- (n = 5) mice to fecal extract from Ggta1+/+ mice at indicated time-points after colonization with cecal extract from Ggta1+/+ mice; two experiments. (D) Concentration of total IgA in serum of Ggta1-/- (n = 7), Tcrb-/-Ggta1-/- (n = 12) mice. (E) Representative plots showing the gating strategy for staining of intestinal content with Syto41, BSI-B4, and Anti-IgA. (F) Quantification of αGal+, IgA+, and IgA+αGal+ bacteria in the cecum, colon, and feces of the same mice as in Figure 2D–E. Symbols (A, B, C, D, F) are individual mice. Red bars (A, B, C, D, F) correspond to mean values. Error bars (A, B, C, D, F) correspond to SD. P values in (A, B, C) calculated using Kruskal-Wallis test using Dunn’s multiple comparisons test and in (D, F) using Mann-Whitney test. *p<0.05, **p<0.01, ns: not significant.

Figure 2—figure supplement 2
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αGal expression by probiotic bacteria.

Representative flow cytometry plots of the data in Figure 2C showing probiotic bacterial strains unstained or stained with BSI-B4 lectin.

Figure 3 with 1 supplement
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Ggta1 deletion shapes the gut microbiota via an Ig-dependent mechanism.

(A) Breeding strategy where F0 Igh-J-/-Ggta1-/- males were crossed with Igh-J+/+Ggta1+/+ females to generate F1 Igh-J+/-Ggta1+/-mice, which were bred to generate F2 and F3 Igh-J+/+Ggta1+/+, Igh-J-/-Ggta1+/+, Igh-J+/+Ggta1-/-, and Igh-J-/-Ggta1-/- mice. Microbiota Principal Coordinate Analysis of (B) Weighted and (C) Unweighted Unifrac and (D) Distance of Weighted and Unweighted Unifrac of 16S rRNA amplicons, in fecal samples from F3 Igh-J+/+Ggta1+/+ (n = 13) vs. Igh-J-/-Ggta1+/+ (n = 16) mice and F3 Igh-J+/+Ggta1-/- (n = 13) vs. Igh-J-/-Ggta1-/- (n = 11) mice generated as in (A). (E) Cladogram and (F) Linear discriminant analysis (LDA) scores generated from LEfSe analysis, representing taxa enriched in the fecal microbiota of the same mice as in (B–D). Data from one experiment with two to three independent breedings/cages per genotype. Symbols (B, C, D) are individual mice. Red bars (D) correspond to mean values. Error bars (D) correspond to SD. p Values in (B, C) calculated using PERMANOVA and in (D) using Mann-Whitney test. **p<0.01, ns: not significant.

Figure 3—figure supplement 1
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Ggta1 deletion shapes the gut microbiota via an Ig-dependent mechanism.

Microbiota Principal Coordinate Analysis of (A) Weighted and Unweighted Unifrac and (B) Distance of Weighted and Unweighted Unifrac of 16S rRNA amplicons, in fecal samples from F2 Igh-J+/+Ggta1+/+ (n = 8) vs. Igh-J-/-Ggta1+/+ (n = 11) mice and F2 Igh-J+/+Ggta1-/- (n = 5) vs. Igh-J-/-Ggta1-/- (n = 8) mice generated as described in Figure 3A. (C–D) Cladogram and linear discriminant analysis (LDA) scores generated from LEfSe analysis, representing taxa enriched in the fecal microbiota of the same (C) F3 Igh-J+/+Ggta1+/+ vs. Igh-J+/+Ggta1-/- mice and (D) F3 Igh-J-/-Ggta1+/+ vs. Igh-J+/+Ggta1+/+ mice as in Figure 3B–D. Symbols (A, B) are individual mice. Red bars (B) correspond to mean values. Error bars (B) correspond to SD. p Values in (A) calculated using PERMANOVA and in (B) using Mann-Whitney test. ns: not significant.

Figure 4 with 1 supplement
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Ggta1 deletion reduces microbiota pathogenicity.

(A) Schematic showing infection of Rag2-/-Ggta1-/- mice with a cecal inoculum from either Ggta1-/- or Rag2-/-Ggta1-/- mice. (B) Survival of Rag2-/-Ggta1-/- (n = 9) mice after infection with a cecal inoculum from Ggta1-/- mice, and from Rag2-/-Ggta1-/- (n = 9) mice after infection with a cecal inoculum from Rag2-/-Ggta1-/- mice; two experiments. (C) Colony-forming units (CFU) of aerobic (Ae) and anaerobic (An) bacteria in Rag2-/-Ggta1-/- mice (n = 5 per group), 24 hr after infection as in (B); two experiments. (D) Schematic showing infection of Rag2-/-Ggta1+/+ mice with a cecal inoculum from either Ggta1+/+ or Rag2-/-Ggta1+/+ mice. (E) Survival of Rag2-/-Ggta1+/+ (n = 5) mice after infection with a cecal inoculum from Ggta1+/+ mice, and Rag2-/- Ggta1+/+ (n = 5) mice after infection with a cecal inoculum from Rag2-/-Ggta1+/+ mice; one experiment. (F) CFU of Ae and An bacteria in Rag2-/-Ggta1-/- mice (n = 4 per group), 24 hr after infection as in (E); one experiment. (G) Survival of Ggta1-/- mice receiving vehicle (PBS) (n = 9) or Anti-Gr1 Ab (n = 8), 24 hr before infection with cecal inoculum from Ggta1-/- mice; two experiments. (H) CFU of Ae and An bacteria in Ggta1-/- mice receiving vehicle (PBS) (n = 4–5) or Anti-Gr1 Ab (n = 4–5), 24 hr after infection as in (G); five experiments. Symbols (C, F, H) represent individual mice. Red lines (C, F, H) correspond to median values. p Values in (B, E, G) calculated with log-rank test and in (C, F, H) with Mann-Whitney test. Peritoneal cavity (PC). *p<0.05, **p<0.01, ***p<0.001, ns: not significant.

Figure 4—figure supplement 1
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Ggta1 deletion reduces microbiota pathogenicity.

(A) Schematic showing infection of Ggta1-/- mice with a cecal inoculum from either Ggta1-/- mice or Rag2-/-Ggta1-/- mice. (B) Survival of Ggta1-/- (n = 14) mice after infection with a cecal inoculum from Ggta1-/- mice and of Ggta1-/- (n = 13) mice after infection with a cecal inoculum from Rag2-/-Ggta1-/- mice; two experiments. (C) Survival of Igh-J+/+Ggta1-/- (n = 9) and Igh-J-/-Ggta1-/- (n = 8) mice after infection with a cecal inoculum from Ggta1-/- mice; two experiments. (D) Survival of Tcrb+/+Ggta1-/- (n = 8) and Tcrb-/-Ggta1-/- (n = 12) mice infected as in (C); two experiments. (E) Survival of Rag2+/+Ggta1-/- (n = 9) and Rag2-/-Ggta1-/- (n = 23) mice infected as in (C); five experiments. (F) Representative plots showing depletion of CD11b+Ly6G+ cells in the blood 24 hr after Anti-Gr1 Ab injection (i.p.) in the same mice as in Figure 4G. (G) Representative plots showing depletion of CD11b+F4/80+ and CD11b+Ly6C+ cells in the peritoneal lavage 72 hr after injection (i.p.) with Clodronate liposomes. (H) Survival of Ggta1-/- mice receiving PBS liposomes (n = 7) or Clodronate liposomes (n = 8), 72 hr before infection (i.p.) with cecal inoculum from Ggta1-/- mice; two experiments. p Values in (B, C, D, E, H) calculated with log-rank test. Peritoneal cavity (PC). ns: not significant.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)B6.C57BL/6/J: Ggta1+/+Obtained originally from Jackson Laboratory and maintained at IGCN/A
Strain, strain background (Mus musculus)B6. Ggta1-/-Obtained originally from Tearle et al., 1996 and maintained at IGCN/A
Strain, strain background (Mus musculus)B6. Igha-/-Ggta1-/-Singh et al., 2021N/A
Strain, strain background (Mus musculus)B6. Igh-J-/-Ggta1-/-Yilmaz et al., 2014N/A
Strain, strain background (Mus musculus)B6. Rag2-/-Ggta1-/-Singh et al., 2021N/A
Strain, strain background (Mus musculus)B6. Tcrb-/-Ggta1-/-Yilmaz et al., 2014N/A
Strain, strain background (Bifidobacterium bifidum)Bifidobacterium bifidum BB-G90BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Bifidobacterium breve)Bifidobacterium breve BB-G95BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Bifidobacterium infantis)Bifidobacterium infantis BI-G191BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Bifidobacterium infantis)Bifidobacterium infantis BI-G201BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Bifidobacterium lactis)Bifidobacterium lactis BL-G101BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Bifidobacterium longum)Bifidobacterium longum BL-G301BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Escherichia coli)Escherichia coli K12ATCCATCC10798See Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Escherichia coli)Escherichia coli O86:B7ATCCATCC12701See Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus acidophilus)Lactobacillus acidophilus LA-G80BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus acidophilus)Lactobacillus acidophilus LA-G436BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus bulgaricus)Lactobacillus bulgaricus LB-G4058BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus bulgaricus)Lactobacillus bulgaricus LB-G117BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus bulgaricus)Lactobacillus bulgaricus LB-G40BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus casei)Lactobacillus casei LC-G11BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus cremoris)Lactobacillus cremoris LLC-G42BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus helveticus)Lactobacillus helveticus LH-G444BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus casei)Lactobacillus casei LC-G402BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus lactis)Lactobacillus lactis LLL-G25BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus plantarum)Lactobacillus plantarum Lp-G18BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus paracasei)Lactobacillus paracasei LpC-G110BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus plantarum)Lactobacillus plantarum Lp-G425BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus plantarum)Lactobacillus plantarum Lp-G429BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus plantarum)Lactobacillus plantarum Lp-G443BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus reuteri)Lactobacillus reuteri Lr-G100BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus rhamnosus)Lactobacillus rhamnosus Lr-G14BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus rhamnosus)Lactobacillus rhamnosus Lr-G26BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Lactobacillus salivarus)Lactobacillus salivarus LS-G60BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
Strain, strain background (Streptococcus thermophilus)Streptococcus thermophilus ST-G30BioGrowing ProbioticsN/ASee Materials and methods, Section Detection of αGal expression by probiotic bacteria
AntibodyAnti-Mouse Monoclonal Gr1 (Clone: RB6-8C5)Bio-RadN/A300 µg in 200 µL PBS per mouse
AntibodyAnti-Mouse Monoclonal IgA (mA-6E1)eBioscience, Thermo Fisher ScientificCat# 12-4204-82, RRID:AB_4659171:100 (Flow cytometry)
AntibodyGoat Anti-Mouse Polyclonal IgA-HRPSouthern BiotechCat# 1040–05;
RRID:AB_2714213		1:4000 (ELISA)
AntibodyGoat Anti-Mouse Polyclonal IgA-UnlabelledSouthern BiotechCat# 1040–01, RRID:AB_23146692 µg/mL (ELISA)
AntibodyGoat Anti-Mouse Polyclonal IgG, Human ads-HRPSouthern BiotechCat# 1030–05, RRID:AB_26197421:4000 (ELISA)
AntibodyGoat Anti-Mouse Polyclonal IgM, Human ads-HRPSouthern BiotechCat# 1020–05, RRID:AB_27942011:4000 (ELISA)
AntibodyAnti-α-gal Mouse Monoclonal IgG1Ding et al., 2008; Yilmaz et al., 2014N/AStandard (ELISA). 0,5 μg/mL, 1:2 serial dilutions, 50 μL
AntibodyAnti-α-gal Mouse Monoclonal IgG2aYilmaz et al., 2014N/AStandard (ELISA). 0,5 Originally o/mL, 1:3 serial dilutions, 50 μL
AntibodyAnti-α-gal Mouse Monoclonal IgG2bDing et al., 2008; Yilmaz et al., 2014N/AStandard (ELISA). 0,5 μg/mL, 1:3 serial dilutions, 50 μL
AntibodyAnti-α-gal Mouse Monoclonal IgG3Ding et al., 2008; Yilmaz et al., 2014N/AStandard (ELISA). 0,5 μg/mL, 1:2 serial dilution
AntibodyAnti-α-gal Mouse Monoclonal IgMYilmaz et al., 2014N/AStandard (ELISA). 2 μg/mL, 1:2 serial dilutions, 50 μL
AntibodyMouse IgA-UnlabelledSouthern BiotechCat# 0106–01, RRID:AB_2714214Standard 0.5 µg/mL(ELISA)
Commercial assay or kitKAPA Mouse Genotyping KitKAPA BiosystemsCat# KK7352
Commercial assay or kitLEGEND MAX Mouse NGAL (Lipocalin-2) ELISA KitBiolegendCat# 443707
Commercial assay or kitQIAamp Fast DNA Stool Mini KitQiagenCat# 50951604
Chemical compound, drμgClodronate liposomes and control liposomes (PBS)https://clodronateliposomes.com/SKU: CP-005–00510 µL/g (in vivo depletion)
Chemical compound, drμgGalα1-3Galβ1-4GlcNAc-BSA (14 atom spacer)DextraCat# NGP13345 µg/mL (ELISA)
Chemical compound, drμgLectin from Bandeiraea simplicifolia (Griffonia simplicifolia) Isolectin B4 (BSI-B4), FITC conjμgate, lyophilized powderSigma-Aldrich#L2895-1MG40 µg/mL (Flow cytometry)
Chemical compound, drμgSYTO41 Blue Fluorescent Nucleic Acid StainThermofisher ScientificCat# S113521:200 (Flow cytometry)
Software, algorithmGreengenesDeSantis et al., 2006http://greengenes.secondgenome.com/v0.13.8
Software, algorithmQIIMECaporaso et al., 2010http://qiime.org/v0.1.9.1

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  1. Sumnima Singh
  2. Patricia Bastos-Amador
  3. Jessica Ann Thompson
  4. Mauro Truglio
  5. Bahtiyar Yilmaz
  6. Silvia Cardoso
  7. Daniel Sobral
  8. Miguel P Soares
(2021)
Glycan-based shaping of the microbiota during primate evolution
eLife 10:e67450.
https://doi.org/10.7554/eLife.67450