An In Vitro Functional Characterization of the Cholesterol-Transforming Blautia hominis Strain HA2291 Isolated from...

Background/Objectives: Cholesterol is an essential lipid required for membrane structure and normal physiological functions. However, dysregulation of cholesterol homeostasis, manifesting as hypercholesterolemia, can precipitate a range of metabolic and cardiovascular diseases. Blautia species are important gut commensals, but their role in cholesterol metabolism remains poorly defined. Methods: A total of 63 Blautia strains isolated from human fecal samples were screened for cholesterol conversion using the o-phthalaldehyde colorimetric assay in cholesterol-containing media with or without oxgall. Cholesterol removal by live and heat-inactivated cells was compared. Metabolomic, transcriptomic, and proteomic analyses were employed to investigate molecular mechanisms and involved genes. Results: Nine strains significantly lowered cholesterol levels (live cells: 31-78%; heat-inactivated cells: 8-64%), with the B. hominis strain HA2291, the Blautia sp. strain HA3515, and the B. coccoides strain HA4419 showing the strongest activity. Oxgall increased cholesterol removal by live cells to 74-83%, indicating bile-tolerant metabolism activity. Metabolomic profiling revealed that B. hominis HA2291 transformed cholesterol into cholest-4-en-3-one and epicholestanol. An SCP2-like protein, RS03310, was identified as a candidate cholesterol-interacting factor; its recombinant form catalyzed measurable NAD⁺-dependent cholesterol oxidation in vitro. Conclusions: Blautia hominis HA2291 may employ multiple in vitro strategies for cholesterol-lowering, including cell-surface adsorption (heat-inactivated cells), bile-enhanced removal (oxgall effect), and enzymatic transformation, with the gene RS03310 implicated as the main contributor. These findings provide in vitro mechanistic insights into Blautia-mediated cholesterol metabolism, highlight RS03310 as a candidate gene associated with cholesterol biotransformation, and advance our understanding of the potential role of Blautia in host cholesterol homeostasis.