Novel bacterium Enterocloster sp. M3 promotes colorectal tumorigenesis via the production of the carcinogen styrene.

The m3-harboring bacterium (M3) was successfully isolated and is phylogenetically related to Enterocloster aldenensis but exhibits distinct genetic and phenotypic characteristics.

• M3 was isolated using a targeted enrichment strategy.
• Whole-genome sequencing was used to characterize M3's genetic features.
• M3 is described as a novel bacterium classified as Enterocloster sp. M3, distinct from known Enterocloster species.
• The bacterium carries the previously identified bacterial marker 'm3' associated with colorectal cancer diagnosis.

M3 significantly promoted colon tumor development in two mouse models of colorectal cancer.

• Tumor promotion was demonstrated in both ApcMin/+ mice and azoxymethane-treated mice.
• In vivo experiments confirmed the pro-tumorigenic role of M3 in colorectal tumorigenesis.
• These two models represent distinct mechanisms of colorectal tumor induction (genetic and chemical carcinogen-based).

M3 culture supernatant enhanced multiple oncogenic properties in colon cancer cells and related models.

• M3 culture supernatant enhanced colon cancer cell proliferation, migration, and cell cycle progression.
• M3 accelerated xenograft tumor growth in vivo.
• M3 stimulated intestinal organoid expansion.
• M3 disrupted DNA damage repair pathways, as profiled by RNA-seq of host gene expression.

M3 produced styrene—a recognized human carcinogen—in both in vitro cultures and mouse models, representing the first reported instance of bacterial styrene biosynthesis.

• Styrene production was detected in M3 bacterial cultures and confirmed in mouse models colonized with M3.
• Bacterial and fecal metabolites were analyzed by untargeted LC-MS and targeted LC/GC-MS.
• This is described as 'a function not previously reported in bacteria.'
• Styrene is classified as a recognized human carcinogen.

Fecal styrene levels were significantly elevated in CRC patients and exceeded WHO safety limits in the mouse gut.

• Fecal styrene levels were significantly elevated in feces of CRC patients compared to controls.
• Styrene levels in the mouse gut exceeded WHO safety limits: 12.5 vs. 7.7 μg/kg/day.
• Measurements were performed using targeted LC/GC-MS.

Two novel bacterial enzymes—aspartate ammonia-lyase and uroporphyrinogen decarboxylase—were identified as responsible for converting phenylalanine to styrene in M3.

• Key enzymes were identified through whole-genome sequencing and proteomics.
• Aspartate ammonia-lyase and uroporphyrinogen decarboxylase catalyze the biosynthetic pathway from phenylalanine to styrene.
• This represents a previously unrecognized mechanism by which gut bacteria may promote colorectal tumorigenesis.
• This is described as the 'first evidence of bacterial styrene biosynthesis.'

The bacterial marker 'm3' shows promise for the non-invasive diagnosis of colorectal cancer and adenomas.

• The m3 marker had been previously associated with colorectal cancer prior to this study.
• Prior to this study, the m3-harboring bacterium had not been successfully cultured.
• The ability to now culture M3 enables functional investigation of its role in CRC.