Shunya Suzuki

Ligilactobacillus ruminis is a flagellated lactic acid bacterium found in the intestines of various mammals, including humans. Although this species harbors a complete flagellar gene cluster, flagella formation has not been observed in human-derived strains, and the underlying regulatory mechanisms remain unknown. Here, we isolated a motility-acquired mutant of L. ruminis ATCC 25644 that exhibited full flagellation and a measurable chemotactic response under acidic conditions (pH 3.0). Whole-genome sequencing revealed a ~35 kb deletion encompassing multiple regulatory genes. Functional complementation identified a single response regulator, designated FpsR (flagellation-piliation switchover regulator), as a central switch that suppresses flagella formation while promoting pilus expression. The motility-acquired mutant displayed reduced pilus production, diminished adhesion to murine intestinal mucus and fibronectin, and increased susceptibility to acid (pH 3.0) and bile (0.25-0.5%), resulting in a complete loss of intestinal colonization in a murine model. Furthermore, while flagellin from the motile strain activated TLR5 and induced proinflammatory responses comparable to those of pathogenic bacteria, no such inflammation was observed in vivo, likely due to the strain's colonization failure. These findings reveal FpsR as a previously unrecognized genetic mechanism that coordinates motility and mucosal colonization in a human commensal bacterium and provide insight into how flagella are regulated and silenced in the gut environment to support host-microbe symbiosis.

A novel lactic acid bacterium, OKN36^T, was isolated from the bark of Entada phaseoloides on Amami Island in Japan. Strain OKN36^T is characterized as a Gram-stain-positive, non-motile, catalase-negative, heterofermentative, and rod-shaped bacterium. The strain grew at temperatures ranging from 10 to 37 °C, in a pH range of 4.0–8.0, and in the presence of 0–6.5% (w/v) NaCl. Phylogenetic analysis using the 16S rRNA gene analysis revealed that strain OKN36^T belongs to the Furfurilactobacillus group. The average nucleotide identity (ANI) and the digital DNA–DNA hybridization (dDDH) values between strain OKN36^T and type strains of validly published Furfurilactobacillus species were 71.83–73.85% and 19.7–20.6%, respectively. These values are below the cutoff of species differentiation, which is 95–96% for ANI and 70% for dDDH. Based on these data, we propose a novel species, named Furfurilactobacillus entadae sp. nov., with the type strain OKN36^T (= JCM 37107^T = DSM 118293^T).

Lactic acid bacteria (LAB) are promising mucosal vaccine vectors due to their safety, immunostimulatory properties, and the availability of genetic tools for certain strains. Bacterial flagellin has attracted attention as both a versatile scaffold for antigen surface display and a potent adjuvant via Toll-like receptor 5 (TLR5) activation. Despite these advantages, LAB-derived flagellin remains largely unexplored as an antigen display platform. In this study, we demonstrate the potential of flagellin (FliC2) from Ligilactobacillus agilis , one of the few flagellated LAB species, as a scaffold for the surface display of the HIV-1 membrane-proximal external region (MPER) epitope. Recombinant L. agilis strains were engineered to express FliC2 with the MPER epitope inserted at various positions within its hypervariable domain, identifying optimal sites for effective surface display. To enhance the adjuvant activity of FliC2, specific amino acid substitutions were introduced into the TLR5 recognition site, resulting in improved TLR5-stimulating activity in vitro . Immunization of mice with MPER-displaying L. agilis strains induced MPER-specific IgA and IgG responses, demonstrating the efficacy of the L. agilis flagellin-based display platform in eliciting both mucosal and systemic immune responses. This study is the first to demonstrate LAB-derived flagellin as an antigen display scaffold, highlighting L. agilis flagellin as a promising platform for mucosal vaccine development. IMPORTANCE: Lactic acid bacteria (LAB) are promising delivery vehicles of active molecules, and surface display systems are gaining interest for efficiently displaying heterologous peptides and proteins. Flagellin, a TLR5 agonist, has been widely used as an adjuvant and an antigen scaffold, making it a potentially valuable platform for such systems. However, the potential of LAB-derived flagellin as a surface display scaffold remains largely unexplored. This study demonstrates that flagellin from Ligilactobacillus agilis , a flagellated LAB species, effectively functions as an antigen display platform, eliciting mucosal and systemic immune responses. Our findings highlight the feasibility of LAB-derived flagellin as a versatile tool for surface display of heterologous peptides, expanding its potential applications in vaccine development and mucosal immunotherapy.