An in vitro functional characterization of cholesterol-transforming Blautia hominis strain HA2291 isolated from the human gut

Cholesterol is an essential lipid required for membrane structure and normal physiologi-cal functions. However, dysregulation of cholesterol homeostasis, manifesting as hyper-cholesterolemia, can precipitate a range of metabolic and cardiovascular diseases. Blautia species are important gut commensals, but their role in cholesterol metabolism re-mains poorly defined. Methods: Blautia strains were isolated from human fecal sam-ples, and their activity of cholesterol conversion was screened using the o‑phthalaldehyde colorimetric assay after growth in cholesterol-containing media with or without oxgall. The cholesterol removal capacity of live and heat‑inactivated cells was compared. Metabolomic, transcriptomic, and proteomic analyses were em-ployed to investigate molecular mechanisms and involved genes. Results: Among 63 Blautia strains screened, both live and heat-killed Blautia cells of nine strains signifi-cantly lowered cholesterol levels (31–78% and 8–64%, respectively), with B. hominis strain HA2291, Blautia sp. strain HA3515, and B. coccoides strain HA4419 showing the strongest activity. Metabolomic profiling revealed that B. hominis HA2291 transformed cholesterol into cholest‑4‑en‑3‑one and epicholestanol. An SCP2‑like protein, namely RS03310, was identified as a candidate cholesterol‑interacting factor; its recombinant form catalyzed measurable NAD⁺‑dependent redox reactions in vitro. Conclusions: Blautia hominis HA2291 employs multiple cholesterol‑lowering mechanisms, including cell‑surface adsorption, bile-enhanced removal, and enzymatic transformation. This dual capacity highlights the potential of B. hominis HA2291 as a next-generation probiotic candidate and provides foundational insights and candidate gene targets for developing Blautia-based interventions against hypercholesterolemia and cardiovascular risk.