Background: Mucus-digesting Ruminococcus gnavus (Rg), adherent-invasive E. coli (AIEC) and hydrogen sulfide (H₂S) are enriched in active Crohn’s disease (CD) compared to healthy subjects. We established a link between Rg and ileal CD AIEC LF82 by showing that dual colonization of ex-germ-free (GF) IL10^-/- mice enhanced colitis and cecal H₂S production. Rg pre-digested mucus increased growth and H₂S production of LF82, H₂S supported Rg survival in minimal medium in vitro, and H₂S enhanced LF82 adhesion and invasion both in vitro and in vivo (DDW 2023). These results suggest that interactions and “cross-feeding” between AIEC and Rg might exacerbate intestinal dysbiosis and inflammation. However, it is unclear if this interplay is selective for other CD AIEC strains vs. non-AIEC.
Hypothesis: Rg pre-digested mucus, mucus-derived monosaccharides and sulfur substrates, and Rg capsular components selectively enhance growth and H₂S production by CD-associated AIEC strains.
Methods: We studied the in vitro functional characteristics of E. coli isolated from CD ileal mucosa (AIEC 541-15 (phylogroup A), 541-1 (B1), LF82 and NRG857c (B2), and non-AIEC T75 (A)), and lab strain K12 (A). We supplemented M9 minimal media with porcine gastric mucin (PGM) or ileal/colonic mucus from GF mice (MIM/MCoM) and cultured human 2D enterocytes (HIM/HCoM). We measured aerobic and anaerobic growth and H₂S production by AIEC and non-AIEC in sterile supernatants of mucin/mucus +/- Rg pre-digestion or mucus monosaccharides and capsular constituents liberated by Rg.
Results: Rg pre-digested mucus enhanced AIEC growth relative to non-AIEC vs. mucus alone especially in aerobic conditions (Fig.1A-C). Rg digested PGM released mucin monosaccharides and Rg capsular glucose (Fig.2A) that selectively enhanced growth of AIEC in aerobic and anaerobic conditions (Fig.2B). Amongst gut-relevant sulfur substrates, only L-cysteine non-selectively enhanced H₂S production by AIEC and non-AIEC strains (Fig.2C). Results of RNA-seq experiments to determine the transcriptional basis of these findings are pending.
Conclusions: The selectively enhanced growth of AIEC on Rg pre-digested mucus, mucus derived monosaccharides and capsular components liberated by Rg may enable them to outcompete non-AIEC and other proteobacteria in the dysbiotic inflamed ileum. H₂S production by AIEC, which was maximal on Rg pre-digested mucus and mucus component cysteine, may modulate the mucosal microenvironment, e.g. inflammation, oxygen tension and H₂S inhibition of butyrate oxidation, to favor AIEC growth and virulence and enhance Rg survival. Enhanced AIEC growth on ileal mucus from intact mice vs. cultured human enterocytes may reflect luminal environmental factors. Elucidating the mechanisms underpinning the synergy between AIEC and Rg may reveal novel approaches to combat microbially driven inflammation in CD.

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Bill Thelin

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