Human gut bacteria produce structurally related monoglycolipids with contrasting immune functions

Abstract

Gut symbiont Bacteroides fragilis can produce α-galactosylceramides (BfaGCs), sphingolipids with immunomodulatory functions that regulate colonic natural killer T (NKT) cells. However, their synthesis pathway and whether other human gut bacteria can produce them are unclear. Here, using genetic and metabolomic approaches, we mapped the sphingolipid biosynthesis pathway of B. fragilis and determined that α-galactosyltransferase (agcT) is essential and sufficient for colonic NKT cell regulation in mice. The distribution of agcT is restricted to only a few species among Bacteroidales. However, structural homologues of AgcT, such as BgsB, are widely distributed in gut microbiota and produce α-glycosyldiacylglycerols (aGDGs), particularly in Enterococcus. Analysis of infant gut metagenomes revealed that B. fragilis predominantly accounts for agcT abundance regardless of the cohort, but bgsB-encoding bacteria were taxonomically diverse and showed dynamic changes with host age. In addition, aGDGs from bgsB-encoding species act as antagonistic ligands for BfaGC-mediated NKT cell activation in vitro and in vivo. Our findings highlight the distinct natures of immunoactive glycolipid-producing symbionts and their relevance in the human gut microbiome, particularly in early life.

Extended data 1

Extended Data 1. Characterization of screened gene targets and generation of isogenic knockout and transformant strains. a) Loss-of-function screening identified a transposon insertion mutant (Tn8A7) with depleted BfaGC production. The relative abundance was quantified by LC-MS/MS based on peak area. b) BfaGC production was abrogasted in the B. fragilis Δ3069 mutant. The abundance of BfaGC (C34:0) was quantified by LC-MS/MS based on peak area. c) RNA-seq volcano plot comparing B. fragilis Δ3069 to wild type revealed multiple genes downregulated in Δ3069 mutant. d) The expression of agcT is reduced in B. fragilis Δ3069. Transcript levels of agcT, normalized to leuB, were measured by qRT-PCR. Significance was determined by an unpaired two-tailed t-test. e) Gain-of-function screening identified P. vulgatus transformant producing aGCs. The relative abundance of aGC/Cer was quantified by LC-MS/MS based on peak area. f) agcT-transformed P. vulgatus produced molecules identical to BfaGC. XICs of aGCs from B. fragilis, P. vulgatus wild type and P. vulgatus expressing agcT were displayed. Chromosome-integrated expressing system pNBU2-tetR was used to expressing agcT in P. vulgatus. g) MS/MS spectra mirror plot demonstrated a match between aGCs from P. vulgatus expressing agcT and those from B. fragilis. h) B. thetaiotaomicron and P. vulgatus heterologously expressing agcT produced aGCs. Chromosome-integrated expressing system pNBU2-tetR was used to expressing agcT in B. thetaiotaomicron and P. vulgatus. i) Relative abundance of KDS, ketoCer, Cer, and BfaGC in B. fragilis wild type, Δspt, ΔcerS, ΔcerR, and ΔagcT were shown. The abundance was quantified by LC-MS/MS based on peak area.

Extended data 2

Extended Data 2. Tandem mass spectra analysis determined sphingolipid intermediate structures synthesized by B. fragilis. To assign the structures unambiguously, we chose chain length variants of major sphingolipid species, whose synthetic version was available. a) MS/MS spectra of B. fragilis KDS (C18:0) matched those of the synthetic standard. b) MS/MS spectra of synthetic C35:1 ceramide (m/z 568.53) was displayed. c) The parent ion with an m/z value of 570.53 from B. fragilis was structurally assigned as C35:0 dihydroceramide. d) The parent ion of m/z 568.53 from B. fragilis ΔcerR was assigned as C35:0 ketoCer. Comparison with isobaric C35:1 ceramide (Panel b) exhibits a distinct fragmentation pattern, as shown by the presence and absence of m/z 264 (300-2H²O). e-f) MS/MS spectra of C34:0 ketoCer and dhCer from B. fragilis were shown.

Extended data 3

Extended Data 3. In vitro and in vivo assays for NKT cell modulation by BfaGCs. a) Schematic process illustrates the in vitro antigen presentation assay. Bacterial lipid extracts were loaded onto biotinylated CD1d molecules, which were subsequently immobilized on streptavidin-coated plates. After washing, NKT cell hybridomas (24.7) were added and incubated overnight. IL-2 secretion was measured by ELISA to assess NKT cell activation. b) Representative gating strategies for colonic NKT cell analysis by flow cytometry are shown. NKT cell refers to the CD1d tetramer⁺ cells within the CD3⁺CD45⁺TCRβ⁺ T cell population. c) Representative flow cytometry plots of unloaded CD1d tetramer controls are shown.

Extended data 4

Extended Data 4. B. fragilis and B. salyersiae produce aGCs and induce NKT cell activation. a) A MS/MS mirror plot of B. fragilis and B. salyersiae C34:0 aGC species showed an exact match. b) All B. fragilis and B. salyersiae strains, including type strains and clinical isolates, synthesize aGC, in contrast, no tested B. nordi, P. gordornii, and P. copri strains produce aGCs. c) Only aGC-producing bacterial lipid extract can elicit NKT cell activation. NKT cell hybridomas (24.7) were incubated with CD1d-lipid complex and IL-2 secretion was measured by ELISA. Each dot indicates a biologically independent replicate. Bars and error bars depict the mean ± s.e.m. Data are representative of three independent experiments showing consistent trends. Statistical analysis was performed using one-way ANOVA. d) Symbiotic Bacteroidales species did not produce aGlcACers, whereas Sphingomonas species served as positive controls and produced aGlcACers.

Extended data 5

Extended Data 5. Homologs of AgcT share conserved structures. a) The histogram displays the distribution of e-values from the search for proteins containing cd03817 domain. The red dashed vertical line indicates the cutoff threshold used for identifying cd03817 family hits by HMMER search. b) An AgcT homolog from B. salyersiae and a cd03817 family protein from E. faecalis belong to the same protein family and have comparable structures.

Extended data 6

Extended Data 6. Targeted metabolomic analysis confirms aGDG production by cd03817-encoding bacteria. a) Mirror plot of MS/MS spectra of E. faecalis aGDGs and a commercially available standard (Avanti #840522p) showed a match. b) The retention time of aGDGs of E. faecalis matches that of standard molecule (Avanti #840522p). c) aGDGs from cd03817-domain-containing gut bacterial species exhibit chain-length variation. XICs of aGDGs from Streptococcus mitis, Lacticaseibacillus rhamnosus, and E. faecalis were shown. d) Various Enterococcus strains produce aGDGs. XICs of C34:1 aGDGs were displayed. e) E. faecalis WT and bgsB mutant exhibit comparable colonization abilities in C57BL/6 mice. f) aGDG levels in stool samples from C57BL/6 SPF, conventionalized, and GF mice were shown. “Conventionalized” indicates GF mice conventionalized via cohousing with SPF mice.

Extended data 7

Extended Data 7. Metagenomic profiles of agcT and bgsB in multiple cohorts. a) Skimming process to search species with homologs of AgcT or BgsB was shown. b) Histograms show sequence identity distributions from the search for homologs of AgcT or BgsB, with vertical dashed lines indicating identity cutoffs. c) In the DIABIMMUNE cohort, B. fragilis consistently dominates agcT abundance, while bgsB abundance shows dynamic changes and is contributed by a taxonomically diverse set of species. Pie charts show the proportion of species abundance in the dataset. d-e) Across multiple infant and adult cohorts, agcT abundance is consistently driven by B. fragilis, whereas bgsB is distributed among a broader array of species.

Extended data 8

Extended Data 8. aGDGs species inhibit the effects of BfaGCs on NKT cells. a) Bacterial lipid extracts from various Lactobacillales bgsB-encoding species exhibit dose-dependent antagonism to SB2217-induced NKT cell activation. b) aGDGs competed with BfaGCs for CD1d binding in a dose-dependent manner. After loading the lipids onto CD1d, CD1d-lipids complexes were purified, and the bound lipids were extracted and analyzed by LC-MS. c) Representative flow cytometry plots show the gating strategy used for analysis of splenic dendritic cells.

Extended data 9

Extended Data 9. Graphical overview of forward genetics-based metabolomic screening to characterize AgcT and explore related metabolites in the gut microbiome. Unannotated metabolites were linked to their biosynthetic gene agcT through forward genetics-based metabolomic screening. Protein structural homologs were identified in gut symbionts, revealing structurally related glycolipids with contrasting immunological activities. Species-level contributions of agcT and its homologs bgsB were profiled in human gut metagenomes.

Figure 1.. BfaGC is necessary and sufficient to modulate colonic NKT cells.

a) Sphingolipid biosynthesis pathway of B. fragilis confirmed with individual gene knockout strains. CerS produces 3-keto-4,5-dihydro-ceramide (ketoCer) as a transient intermediate, which is further reduced by CerR to produce dihydroceramide (Cer) and complex sphingolipids such as BfaGCs. Extracted ion chromatograms (XICs) of 3-keto-dihydrosphingosine (KDS) (17:0), ketoCer (34:0), Cer (34:0), and alpha-galactosylceramides (aGC) (34:0) from B. fragilis wild type (WT), Δspt, ΔcerS, ΔcerR, and ΔagcT were shown. b) Deleting individual genes involved in BfaGC biosynthesis abolishes CD1d-mediated IL-2 induction in NKT cells elicited by B. fragilis total lipids. NKT cell hybridomas (24.7) were incubated with CD1d-lipid complex and IL-2 secretion was measured by ELISA (biologically independent replicates, n = 4 per group). KRN7000 (n=3) was included as a positive control. c) Heterologous expression of agcT in ceramide-producing species (biologically independent replicates, n=4 for each group) enabled induction of IL-2 in NKT cell hybridomas (24.7). d) BfaGC production in vivo depends on agcT. C34:0 BfaGC levels were measured in stool samples from 5-week-old female C57BL/6 mice monocolonized with B. fragilis wild-type (n=3) or agcT mutant strains (n=4) and compared to GF (n=4). f) Selective loss of BfaGC by deletion of agcT or its upstream regulator Δ3069 abrogated colonic NKT cell modulation by B. fragilis. At 6 weeks of age, colonic NKT cell frequencies were analyzed in male and female Swiss Webster mice monocolonized from birth with B. fragilis wild type (n=5), Δ3069 (n=4), or ΔagcT (n=5), alongside SPF (n=13) and GF (n=6) mice. The representative gating strategy is shown in Fig. S3c-d. a-d) Each dot indicates a biologically independent replicate. Results represent two independent experiments showing similar trends. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test in a-c, andone-way ANOVA followed by Tukey’s multiple comparisons test in d. Bars and error bars represent the mean ± s.e.m.

Figure 2.. Lipidomic and genomic analyses revealed a narrow distribution of agcT among gut Bacteroidales.

a) Most Bacteroidales species in the human gut produce ceramides. The heatmap shows LC-MS/MS intensities of ceramide species detected in lipid extracts from individual bacterial cultures with background values determined from blank samples. b) aGC was only found in B. fragilis and B. salysiae. c) Sequence homology analysis revealed distinct conservation patterns between upstream ceramide synthesis enzymes (Spt, CerS, CerR) and AgcT. The sequence identity was determined by searching species genomes against UniRef90 proteins; Spt (A0A0K6BUE2), CerS (E1WSI8), CerR (A0A2M9VAV5) and AgcT (A0A380YRQ3). d) Analysis of agcT homologs across 24,562 strain genomes in the UHGG catalogue revealed a dichotomous distribution. B. fragilis and B. salyersiae consistently carried agcT, while other species showed little to no conservation. The bar plot shows the proportion of strains harboring agcT homologs relative to the total number of strains per species.

Figure 3.. Structural homologs of B. fragilis AgcT are widely distributed in gut microbiota.

a) Workflow for identifying sequence and structural homologs of B. fragilis AgcT was presented. Protein homology searches were conducted using DIAMOND BLAST for primary sequence similarity and HMMER hmmsearch for domain-based detection of homologs. b) Taxonomic cladogram with homolog search results showed the distribution of AgcT homologs among prominent infant gut bacterial species. c) Phylogeny of 42 cd03817 proteins from gut bacterial species showed a clear separation between Lactobacillales and Bacteroidales clusters. The maximum likelihood tree was generated using IQ-TREE2. d) The superimposed structures revealed structural similarity between AgcT (A0A380YRQ3) and cd03817 proteins of various Lactobacillales bacteria.

Figure 4.. Characterization of cd03817 family proteins in gut symbionts producing aGDGs.

a) Multiple Lactobacillales bacteria produce aGDGs. The abundance of aGDGs in individual bacterial cultures was assessed by LC-MS analysis. Each dot represents biologically independent replicates (n=2 for each species). Data are presented as mean values +/− SEM. b) c) bgsB is essential for aGDG production in Enterococcus. XICs and abundance of aGDGs in Enterococcus WT and bgsB mutants were shown. d) bgsB is responsible for aGDG production in vivo. C34:1 aGDG levels were measured in stool samples from 4-week-old male and female C57BL/6 mice monocolonized with E. faecalis WT (n=10) or bgsB mutants (n=6) and compared to GF mice (n=7). Each dot represents an individual mouse. Data are presented as mean values +/− SEM. Data represent two independent experiments with consistent trends. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 5.. Longitudinal metagenomic landscapes of agcT and bgsB show distinct patterns in infant gut.

a-b) agcT and bgsB show differences in composition. B. fragilis consistently dominates agcT abundance regardless of host age, whereas bgsB abundance is distributed among diverse species that undergo significant compositional shifts over developmental time. The abundance plot shows the relative abundance of agcT- and bgsB-encoding species across time, visualized using LOESS smoothing in ggplot2.

Figure 6.. aGDGs inhibit the actions of BfaGCs on NKT cells.

a–c) Lipid extracts from B. fragilis, alone or in combination with lipid extracts from other bacterial species, were loaded onto CD1d molecules and NKT cell activation was assessed as described in Fig. S3b. Each dot represents a biologically independent replicate. Data are presented as mean values +/− SEM. Data are representative of three independent experiments showing consistent trends. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. d) Lipid extracts from bgsB-encoding species antagonized to B. fragilis-mediated NKT cell activation. e) E. faecalis lipid extracts suppressed BfaGC-induced NKT cell activation in a dose-dependent manner. f) The antagonistic effect of bacterial lipids on NKT cell activation is dependent on the presence of bgsB. d-e) Female 7-week-old C57BL/6 mice (n=5 per group) were intraperitoneally injected with 5 μg synthetic BfaGC (SB2217) with or without 10 μg synthetic aGDGs (BbGL-2). After 19 hours, serum and spleens were collected for analysis. Each dot represents an individual mouse, and data represent three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. e) aGDGs reduced BfaGC-induced serum IFN-γ levels. f) aGDGs suppressed BfaGC-induced upregulation of co-stimulatory molecules on splenic dendritic cells. g) Monocolonization of E. faecalis from birth failed to normalize colonic NKT cell development. Pregnant Swiss Webster mice were monocolonized with E. faecalis WT or bgsB mutant. Colonic NKT cells were analyzed in male and female offspring at 6 weeks of age (n=10 for E. faecalis WT, n=6 for E. faecalis bgsB mutant) alongside SPF (n=10) and GF (n=10) Swiss Webster mice of both sexes. h) Experimental overview for h–i. Swiss Webster neonates were gavaged with synthetic lipids (250 ng SB2217 and 500 ng BbGL-2) on days 3, 6, 9, 12, and 15 after birth. Colonic NKT cells were analyzed in male and female offspring at 6 weeks of age. i) BfaGCs reduced colonic NKT cell levels. Swiss Webster GF mice gavaged with synthetic BfaGCs (SB2217) (n = 4) exhibited reduced colonic NKT cells compared with GF mice (n = 8) and levels comparable to SPF mice (n = 7). j) aGDGs suppressed the BfaGC-mediated reduction of colonic NKT cell levels. Swiss Webster GF mice gavaged with synthetic BfaGCs (SB2217) plus synthetic aGDGs (BbGL-2) (n = 10) exhibited elevated colonic NKT cells compared with B. fragilis monocolonized mice (n = 11) and levels comparable to GF mice (n = 11). f–i) Each dot represents an individual mouse, and data represent two independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test.