The interactions between gut microbes and immune cells play an important role in the health and disease of hosts. Ligilactobacillus agilis is a flagellated commensal bacterium found in the gut of mammals and birds. However, the flagellin proteins of L. agilis are immunologically attenuated and barely induce TLR5-dependent inflammation, unlike the flagellins of several pathogenic bacteria.

Many flagellated bacteria possess multiple flagellins, but the roles and the compositions of each flagellin are diverse and poorly understood. In Ligilactobacillus agilis BKN88, there are two active flagellin gene paralogues but their function and composition in its flagellar filaments have not been described. The aim of this study is to find the function and composition of the flagellins by employing mutant strains each of which expresses a single flagellin or a modified flagellin. Two single flagellin-expressing strains were both flagellated while the number of flagella per cell in the single flagellin-expressing derivatives was lower than that in the wild type. Nonetheless, these derivative strains were apparently equally motile as the wild type. This indicates that either flagellin is sufficient for cell motility. The immunological activity via Toll-like receptor 5 of the single flagellin-expressing strains or purified single flagellins was readily detectable but mostly variably weaker than that of the wild type. The flagellar filaments of wild type L. agilis BKN88 were more acid-/thermo-stable than those of single flagellin-expressing derivatives. Using a combination of immunoprecipitation and flagellin-specific staining, wild type BKN88 appeared to possess heteropolymeric flagellar filaments consisting of both flagellins and each flagellin appeared to be equally distributed throughout the filaments. The results of this study suggest that the two flagellins together form a more robust filament than either alone and are thus functionally complementary.