Background and Aims: We previously identified a fecal bacterial gene marker ‘m3’ from an unclassified bacterium to be significantly increased in patients with colorectal cancer and adenoma (Liang et al. Gut 2020). This study isolated and characterized this m3-carrying bacterium (M3) and investigated its role in colorectal tumorigenesis.
Methods: Fresh frozen stools with high m3 abundances were used for isolation of M3. Columbia agar with 5% sheep blood was employed by referring to Lachnoclostridium/Clostridium, as Lachnoclostridium sp. YL32 contains m3 homolog. C57BL/6J-Apc^Min/+mice were administered M3, E. coli, or broth to compare effects on colon tumor development. Colon cancer cells (HT-29, HCT116, and Caco-2) were treated with M3/E. coli supernatant, or broth for in vitro functional assays. M3 metabolites were analyzed by non-targeted LC-MS. Gene expression profiles were analyzed by RNA-seq.
Results: We isolated M3 using a target-enrichment culturing strategy from a 73-year-old woman with colonic adenoma, which carries the full-length m3 gene as confirmed by PCR-Sanger sequencing and nanopore sequencing. M3 is a strictly anaerobic, nonsporulating, nonhemolytic, gram-negative, rod-shaped (0.5–0.8 μm × 2–5 μm) bacterium with no flagella. Colonies are circular, convex, smooth, shiny, opaque to white, and 1–3 mm in diameter after 72h at 37°C. 16s rDNA and MALDI Biotyper analysis showed M3 is closest to Enterocloster aldenensis (Ea), but Ea does not contain the m3 gene. M3 and Ea genomes show an average nucleotide identity of 96.7% but alignment fractions <50%. We therefore propose that M3 is a new species of Enterocloster. Administration of M3 significantly increased colon tumor incidence and tumor size in mice compared with E. coli or broth controls (P<0.05). Moreover, fecal m3 abundance at sacrifice positively correlated with tumor number (r=0.785, P<0.01) and tumor size (r=0.524, P=0.06) in M3-treated mice (n=10). M3 supernatant significantly promoted colon cancer cell growth, clonogenicity, migration, and invasion, and accelerated cell cycle G1-S transition while reducing apoptosis as compared with E. coli or broth controls (all P<0.05). The effective components of M3 supernatant were non-proteins as shown by heat and protease K inactivation. Metabolomics identified known carcinogens (styrene and 2,6-Dimethylaniline) and potential oncogenic metabolites (chenodeoxycholic acid, norvaline, pyroglutamic acid, etc) to be produced by M3. GO and KEGG pathway enrichment analysis identified DNA repair-related processes to be dysregulated by M3 treatment, involving genes in DNA damage response (ATM, ATR, H2AX, GEN-1) and DNA damage repair (BRCA1, BRCA2, BIVM-ERCC5, XRCC2, SLX1B, and BRIP1).
Conclusions: This study isolated and characterized a new Enterocloster sp. M3, which may promote colorectal tumorigenesis by producing carcinogenic metabolites.

Yao HUANG

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