Immunogenic Modification of Ligilactobacillus agilis by Specific Amino Acid Substitution of Flagellin.

Abstract

Ligilactobacillus agilis is a flagellated motile commensal microbe that resides in the gastrointestinal tract of mammals and birds. Flagellin, the major subunit protein of flagellar filament, from pathogenic bacteria is generally a proinflammatory molecule that stimulates immune cells via Toll-like receptor 5 (TLR5). Interestingly, the flagellins of L. agilis are known to be immunologically attenuated despite the fact that the structure of the proteins, including the TLR5 recognition site, is highly conserved among bacteria. The results of our previous study suggested that this is attributed to the differences in three specific amino acids within the conserved TLR5 recognition site; however, this hypothesis remains to be confirmed. In this study, a series of recombinant L. agilis flagellins, with amino acid substitutions at the TLR5 recognition site, were constructed, and their immunogenic activity was evaluated in vitro. Then, an L. agilis strain with an active immunogenic TLR5 recognition site was generated. In vitro and in vivo immunological studies revealed that the mutant L. agilis strain with the modified flagellin was more immunogenic than the wild-type strain. In conclusion, the specific amino acid residues in L. agilis flagellins likely contribute to the discrimination between pathogens and commensals by the host defense system. Additionally, the immunogenically potent L. agilis mutants may serve as a useful platform for oral vaccine delivery. IMPORTANCE 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. This study demonstrated that three specific amino acids in the flagellin protein are responsible for this low immunogenicity in L. agilis. The results obtained herein improve our understanding of the symbiosis between gut microbes and their hosts.