Claim-aware prioritization of microbiome-associated metabolite-target hypotheses using bifidobacterial genome context

Microbiome-associated variation in immune checkpoint blockade response has created interest in microbial metabolites, but taxonomic association, strain-level production, immune-pathway relevance, structural plausibility, and causal activity are often conflated. We developed a claim-aware workflow that keeps these evidence layers separate. Complete Reference Sequence (RefSeq) genomes from five Bifidobacterium species were integrated with antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) and Biosynthetic Genes Similarity Clustering and Prospecting Engine (BiG-SCAPE) genome mining, exopolysaccharide (EPS)-like operon scanning, targeted literature curation, weighted metabolite-target scoring, AutoDock Vina docking, and GROningen MAchine for Chemical Simulations (GROMACS) molecular dynamics (MD). The candidate space resolved into matched inhibitory, innate-support, direction-conflict, and exploratory branches. Matched inhibitory hypotheses were concentrated in the butyrate-histone deacetylase (HDAC) axis, with structure-facing hypotheses involving indole-3-lactic acid (ILA)-indoleamine 2,3-dioxygenase 1 (IDO1), indole-3-aldehyde (I3Ald)-IDO1, and conjugated linoleic acid (CLA)-prostaglandin-endoperoxide synthase 2 (PTGS2). Genome mining supported coding potential but did not establish expression or product identity. No metabolomic, transcriptomic, flux, or concentration data demonstrated rumenic acid production by any analyzed strain; rumenic acid was therefore tested as an exogenous candidate compound rather than a confirmed bifidobacterial immunometabolite. Independent-seed 100 ns MD repeats supplied replicate structural diagnostics: non-CLA systems were summarized across three trajectories, and CLA-PTGS2 was reassessed in two corrected Fe-heme trajectories while the historical cofactor-free trajectory was retained only for provenance. In technical replicate wells, exogenous rumenic acid showed differential HCT116 and NCM460 viability, coincided with greater anti-programmed cell death protein 1 (anti-PD-1)-associated tumor-cell killing, and reduced crude-lysate total cyclooxygenase (COX)-associated prostanoid output. These experiments did not measure prostaglandin E2 (PGE ₂ ), PTGS2 dependency, T-cell activation, cytokines, exhaustion, memory phenotype, or antigen presentation. The study therefore provides a transparent prioritization map and an experimental agenda, not evidence that the analyzed bifidobacteria produce therapeutically active rumenic acid or that the observed phenotype is PTGS2-mediated.