BACTERIAL CROSS FEEDING: EXPLORING EFFECTS OF R. GNAVUS RELEASED MUCUS CARBOHYDRATE SUBSTRATES ON ADHERENT-INVASIVE E. COLI LF82 GROWTH AND EXPRESSION OF RELEVANT MOLECULAR PATHWAYS IN AEROBIC AND ANAEROBIC CONDITIONS

Mucus-digesting Ruminococcus gnavus (Rg), adherent-invasive E. coli (AIEC) and fecal hydrogen sulfide (H2S) are increased in active Crohn’s disease (CD) compared to healthy subjects. We established a link between Rg and ileal CD AIEC strain LF82 by showing that dual colonization of ex-germ-free (GF) IL10-/- mice potentiated colitis and cecal H2S production. Rg precultured mucus increased LF82 growth and H2S production, H2S supported in vitro Rg survival in minimal medium, and H2S enhanced LF82 adhesion and invasion both in vitro and in vivo (DDW 2023). Rg’s capsule contains glucose and has an enzyme that cleaves off sialic acid (Neu5Ac), one of the terminal carbohydrates of mucus. Because inflamed colons become microaerophilic to some extent, we focused on Rg-derived glucose and mucus-derived Neu5Ac to explore expression of LF82 molecular pathways under aerobic and anaerobic conditions. AIEC LF82 genes regulating growth, H2S production and virulence are differentially expressed with glucose or Neu5Ac substrates in aerobic vs. anaerobic conditions. LF82 was cultured in M9 minimal medium supplemented with Rg-/mucus-derived carbohydrates or gut-relevant sulfur substrates for 16 hours in aerobic or anaerobic conditions. LF82 RNA cultured with glucose or Neu5Ac medium in each condition was isolated for RNA sequencing. We compared aerobic vs. anaerobic results for each medium with |Log2FC| > 3.0 considered significant change. Rg associated mucus carbohydrates and gut-relevant sulfur substrates variably enhanced LF82 growth. LF82 grew better in Neu5Ac than glucose in aerobic and vice versa in anaerobic conditions (Fig.1). Neu5Ac metabolism pathways involving nanE (N-acetylmannosamine-6-phosphate 2-epimerase) and dnaA (chromosomal replication initiator), directly involved in cell replication, were overexpressed in aerobic vs. anaerobic conditions in Neu5Ac medium. Additionally, expression of other Neu5Ac metabolic pathway genes, nanT (sialic transporter) and nanA (N-acetylneuraminate lyase) were slightly increased. Porin OmpC, a non-specific glucose transporter, and dnaA were more highly expressed in anaerobic than aerobic conditions in glucose medium (Table 1A). Moreover, LF82 genes involved in H2S production, biofilm formation and antimicrobial drug resistance were overexpressed in glucose vs. Neu5Ac with aerobic culture (Table 1B). AIEC LF82 differentially uses R. gnavus released mucus components for growth in aerobic vs. anaerobic conditions, and sialic acid and glucose activate distinct molecular pathways in the two conditions. Confirmatory functional experiments are underway. Glucose influences on LF82 H2S production and biofilm formation would provide strong evidence of cross-feeding between LF82 and R. gnavus, leading to potential diagnostic/treatment clues for further investigation.