Human gut flagellome profiling using FlaPro reveals TLR5-related phenotype-specific alterations in IBD.

Flagellin, the structural protein of bacterial flagella, activates the innate immune receptor Toll-like receptor 5 (TLR5). However, the ability of different flagellins to bind and stimulate TLR5 varies widely, suggesting that the composition of an individual's flagellin repertoire, defined as flagellome, may influence host-microbiome interactions and inflammation. Here, we developed FlaPro, a computational pipeline for quantification and functional annotation of human gut flagellomes. Functional categories in FlaPro are derived from a machine learning model trained on experimentally characterized flagellins with defined TLR5-binding and stimulatory activities. Application of FlaPro to a multi-omics inflammatory bowel disease (IBD) cohort revealed a marked depletion of flagellome diversity and a reduced ratio of silent to stimulatory flagellins in Crohn's disease and ulcerative colitis. These alterations were consistent across genomic and transcriptional layers, indicating a disease-associated shift toward more stimulatory flagellome profiles. Our findings suggest that specific features of the gut flagellome contribute to TLR5-mediated immune activation and may serve as functionally interpretable microbiome markers for future microbiome-wide association studies in health and disease. The workflow implemented in Snakemake is openly available at https://github.com/leylabmpi/FlaPro.