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

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.

Ligilactobacillus agilis BKN88 possesses thermo-/acid-stable heteropolymeric flagellar filaments.

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.

Electrochemical Synthesis of Poly(3,4-ethylenedioxythiophene) Film Having Dot Structures for Diffraction Grating.

Poly(3,4-ethylenedioxythiophene) (PEDOT) dot micro/nanostructures are synthesized by electrochemical polymerization in a concentrated hydroxypropyl cellulose (HPC) liquid crystal electrolyte solution. Surface observations by scanning electron microscopy and atomic force microscopy reveal micro/nanostructures having hemisphere-like dots on the surface of the PEDOT film, which causes light diffraction at ultraviolet and visible light wavelengths. The size of the dots depends on the concentration of the HPC electrolyte solution, decreasing with increasing the HPC concentration. Electrochemical oxidation and reduction causes changes in the color of the PEDOT film and the diffracted light. Moreover, Au coating on the surface of the PEDOT film enhances the diffracted light reflection intensity by more than tenfold compared to the noncoated PEDOT film.