Bacteroides ovatus alleviates dysbiotic microbiota-induced intestinal graft-versus-host disease

Abstract

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.

Keywords: Bacteroides ovatus; Bacteroides thetaiotaomicron; allogeneic hematopoietic stem cell transplantation; carbapenem; graft-versus-host disease; intestinal microbiome; mucus layer; polysaccharide utilization loci; polysaccharides; xylose.

Extended Data Fig. 1.. aGI-GVHD patients showed a higher proportion of carbapenem exposure

. (A) PCoA of fecal samples collected from healthy volunteers or aGI-GVHD patients. (B) Volcano plot of differentially abundant genera analyzed by 16S rRNA gene sequencing of fecal samples compared between healthy volunteers and aGI-GVHD patients. (C) Relative abundance of genera that were significantly different between steroid-responsive and -refractory aGI-GVHD.

Extended Data Fig. 2.. Bacteroides ovatus did not show mucus-degrading functionality like B. theta.

(A) Circular plot of open reading frames (ORFs) derived from the complete genome (MDA-HVS BO001). Blue and green bars represent ORFs on the plus strand and the minus strand, respectively. Inner purple-olive ring depicts degree of GC skewing. (B) Experimental schema of in vitro bacterial culture assay of B. theta (MDA-JAX BT001) or B. ovatus (MDA-HVS BO001) in media with porcine gastric mucin-containing medium. (C) Relative concentrations of porcine gastric mucin in medium following culture with B. theta (MDA-JAX BT001) or B. ovatus (MDA-HVS BO001). B. theta or B. ovatus was first introduced to porcine gastric mucin-containing medium. At 24 hours of culture, levels of mucin glycans in the culture supernatant were determined using a colorimetric assay.

Extended Data Fig. 3.. Introduction of Bacteroides ovatus did not alter abundance and functionality of B. theta in meropenem-untreated allo-HSCT mice.

(A) Experimental schema of murine GVHD model with oral gavage of 20 million colony-forming units of B. ovatus daily from days 16 to 18. (B) Overall survival after allo-HSCT. Data are combined from two independent experiments. (C) Bacterial densities of mouse stool samples collected on day 21. Bacterial densities were measured by 16S rRNA gene qPCR. (D) Alpha diversity, measured by the Shannon index, was quantified in fecal samples. (E) Bacterial genera composition of fecal samples. (F) Relative abundance of B. ovatus (left) and B. theta (right). (B-F) Combined data from two independent experiments. (G) PAS staining of histological colon sections collected on day 23. Bar, 100 μm. The areas inside dotted lines indicate the inner dense colonic mucus layer. (H) Mucus thickness on day 23. Data are shown from one representative experiment.

Extended Data Fig. 4.. Introduction of Bacteroides ovatus increased fecal levels of soluble monosaccharides in mice monocolonized with B. ovatus.

(A) Heatmap showing scaled relative expression levels of polysaccharide utilization loci (PULs) in B. theta RNA transcripts sequenced from stool collected from meropenem-treated allo-HSCT mice with or without administration of B. ovatus on day 21. Right: PULs and their modularity and substrate names. (B) Relative expression levels of PULs in B. theta RNA transcripts sequenced from stool collected on day 21 from meropenem-untreated allo-HSCT mice with or without administration of B. ovatus. Right: PULs and their modularity. (C) Relative abundances of monosaccharides of supernatants from colonic luminal content collected from germ-free (GF) mice with or without administration of B. ovatus on day 14 measured by IC-MS. Data are shown from one representative experiment. (D) Relative abundances of monosaccharides of supernatants from colonic luminal content collected from meropenem-untreated allo-HSCT mice with or without administration of B. ovatus on day 23 measured by ion chromatography-mass spectrometry (IC-MS). Data are shown from one representative experiment. (E) Absolute abundances of tryptophan metabolites of supernatants from colonic luminal content collected from meropenem-treated allo-HSCT mice with or without administration of B. ovatus on day 23 measured by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Data are combined from two independent experiments and are shown as means ± SEM.

Extended Data Fig. 5.. PULs of Bacteroides ovatus were significantly altered in meropenem-treated allo-HSCT mice compared to meropenem-untreated mice.

(A) Relative expression levels of PULs in B. ovatus RNA transcripts sequenced from stool collected from allo-HSCT mice treated or untreated with meropenem on day 28. Right: PULs and their modularity and substrate names. (B) The correlation network analysis of B. ovatus RNA transcripts and B. theta RNA transcripts sequenced from stool collected on day 21 from meropenem-treated and -untreated allogeneic mice with administration of B. ovatus. Only negatively correlated networks are shown.

Fig. 1.. The high abundance of Bacteroides was associated with steroid-responsive GVHD.

(a) Cluster dendrogram analyzed using H-clustering of weighted UniFrac. (b) The microbiome composition shown as stacked bar graphs. (c) PCoA of fecal samples collected from healthy volunteers or each cluster of aGI-GVHD patients. (d) Distances from healthy volunteers in weighted UniFrac. (e) Numbers of patients with steroid-responsive and -refractory GVHD. (f) Proportions of patients with steroid-responsive and -refractory GVHD. (g) Volcano plot of differentially abundant genera between clusters 1 and 2.

Fig. 2.. Steroid-refractory aGI-GVHD patients showed significantly dysbiotic intestinal microbiome than steroid-responsive aGI-GVHD patients.

(A-E) The intestinal microbiome analyzed by 16S rRNA sequencing in patient stool samples collected at presentation with acute intestinal graft-versus-host disease (aGI-GVHD). (A) Alpha diversity shown as Shannon index. (B) Principal coordinates analysis (PCoA) of fecal samples collected from healthy volunteers or steroid-responsive or steroid-refractory patients. (C) Distances from healthy volunteers in weighted UniFrac. (D) Volcano plot of differentially abundant genera. (E) The composition of the intestinal microbiome.

Fig. 3.. The high abundance of Bacteroides ovatus and B. ovatus-derived pathways were associated with steroid-responsive GVHD.

(A) Graphical summary of antibiotics used in individual patients. (B) Numbers of patients with antibiotic exposure between hematopoietic stem cell transplant (HSCT) and the onset of GVHD. (C-F) Data analyzed by shotgun sequencing of fecal samples collected from aGI-GVHD patients (steroid-responsive; n=11, steroid-refractory; n=12). (C) Volcano plot of differentially abundant species between steroid-responsive and - refractory GVHD. (D) PCoA of genes in the genus Bacteroides. (E) Volcano plot of differentially abundant pathways of the genus Bacteroides. (F) The top 50 subclasses of differentially abundant pathways of the genus Bacteroides.

Fig. 4.. Bacteroides ovatus improved GVHD-related mortality in meropenem-aggravated colonic GVHD via suppressing the abundance of B. theta.

(A) Experimental schema of murine GVHD model using meropenem treatment followed by oral gavage of 20 million colony-forming units of B. ovatus daily for 3 days. (B) Overall survival after allo-HSCT. Data are combined from two independent experiments. (C) Bacterial densities of mouse stool samples collected on day 21 after administering meropenem by drinking water. Bacterial densities were measured by 16S rRNA gene qPCR. (D) Alpha diversity, measured by the Shannon index, was quantified in fecal samples. (E) Bacterial genera composition of fecal samples. (F) Relative abundance of B. ovatus (left) and B. theta (right). (G) Periodic acid-Schiff (PAS) staining of histological colon sections collected on day 23. Bar, 100 μm. The areas inside dotted lines indicate the inner dense colonic mucus layer. (H) Mucus thickness on day 23. Data are shown from one representative experiment.

Fig. 5.. Mucolytic activity of Bacteroides thetaiotaomicron is suppressed in meropenem-treated mice by administration of Bacteroides ovatus.

(A) Heatmap showing scaled relative expression levels of polysaccharide utilization loci (PULs) in B. theta RNA transcripts sequenced from stool collected from meropenem-treated allo-HSCT mice with or without administration of B. ovatus on day 21. Right: Significantly altered PULs and their substrates. (B) Relative abundances of monosaccharides of supernatants from colonic luminal content collected from meropenem-treated allo-HSCT mice with or without administration of B. ovatus on day 23 measured by ion chromatography-mass spectrometry (IC-MS). Combined data from two independent experiments are shown as means ± SEM.

Fig. 6.. Degradation of xylose-comprising polysaccharides by Bacteroides ovatus suppressed mucus-degrading functionality by Bacteroides thetaiotaomicron.

(a) The correlation network analysis of B. ovatus RNA transcripts and B. theta RNA transcripts sequenced from stool collected on day 21 from meropenem-treated and -untreated allogeneic mice with administration of B. ovatus. Only negatively correlated networks are shown. (b) Experimental schema of in vitro bacterial culture assay using B. ovatus (MDA-HVS BO001) cultured in minimum nutrition medium with each polysaccharide and B. theta (MDA-JAX BT001) cultured in BYEM10 with porcine gastric mucin. (c) Normalized concentrations of porcine gastric mucin in the culture supernatant were determined using a PAS-based colorimetric assay. Combined data from two independent experiments are shown as means ± SEM. (d) Heatmap showing scaled relative expression levels of polysaccharide utilization loci (PULs) in B. theta RNA transcripts sequenced from stool collected from B. theta (ATCC 29148)-colonized gnotobiotic mice with or without co-administration of B. ovatus. Transcripts were evaluated on day 14 after bacterial introduction to germ-free mice. Right: Significantly altered PULs and their substrates.