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.

PCR-based screening, isolation, and partial characterization of motile lactobacilli from various animal feces.

BACKGROUND: Most lactobacilli found in animal intestines are generally non-motile, but there are few exceptions. Our previous work showed that Lactobacillus agilis BKN88, which is a highly motile strain originating from a chicken, takes advantage of motility in gut colonization in murine models, and thus motile lactobacilli likely have unique ecological characteristics conferred by motility. However, the ecology and habitat of gut-derived motile lactobacilli are still rarely understood. In addition, the limited availability of motile Lactobacillus isolates is one of the major obstacles for further studies. To gain insight into the ecology and habitat of the motile lactobacilli, we established a routinely applicable detection method for motile lactobacilli using PCR and subsequent selective isolation in semi-solid MRS medium for the collection of additional motile lactobacilli from animal feces. RESULTS: We applied the PCR detection using motile lactobacilli-specific primers, based on the motor switch protein gene (fliG) of flagella, to 120 animal feces, followed by selective isolation performed using 45 animal feces. As a result, motile lactobacilli were detected in 44 animal feces. In the selective isolation, 29 isolates of L. agilis and 2 isolates of L. ruminis were obtained from 8 animal species. CONCLUSIONS: These results indicated that motile lactobacilli are distributed in different animal species. Moreover, phylogenetic analysis of the L. agilis isolates suggests co-evolution with the host, and adaptation to a particular environmental niche.

Adsorption of Tolaasins, the Toxins Behind Mushroom Bacterial Blotch, by Microbacterium spp. is Insufficient for Its Detoxification.

Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of bacterial blotch on several kinds of cultivated mushrooms. Our previous study reported on tolaasin detoxification by Microbacterium sp. K3-5 as a potential biocontrol of the disease. In this study, the tolaasin-detoxifying activities of various type strains of Microbacterium spp. were evaluated through chemical and biological assays. The bacterial cells of all tested strains of Microbacterium spp. showed tolaasin I-elimination from liquid phase. However, the toxin activities of tolaasins were still retained on the tolaasin-treated bacterial cells of all Microbacterium strains except M. foliorum NBRC 103072T. Furthermore, intact tolaasin I was recovered from the tolaasin-treated bacterial cells of all tested strains except M. foliorum NBRC 103072T. Our data reveal that Microbacterium spp. can be characterized as effective tolaasin I-eliminating bacteria through cell adsorption, but that this adsorption alone is insufficient for actual tolaasin detoxification. The biological degradation process must be needed to carry out the detoxification.

Comparative analysis of immunological properties of S-layer proteins isolated from Lactobacillus strains.

Previous studies have suggested that some Lactobacillus S-layer proteins could modulate immune responses. Primary structures of the S-layer proteins are variable, and their immunological differences are poorly understood. In this study, we evaluated the immunological properties of eight distinct S-layer proteins from different Lactobacillus species. We found that removal of the S-layer proteins from the cell surface reduced the immunological activities of Lactobacillus cells in THP-1 cells. Furthermore, the purified S-layer proteins induced the production of IL-12 p40, although their immunological activities varied between the different S-layer proteins. The production of IL-12 p40 was notably induced by the S-layer protein SLP(aly) from Lactobacillus amylolyticus NRIC 0558T. Multiple sequence alignment revealed that the percent identity of the S-layer proteins of the eight strains vary from 10 to 90 %. In particular, N-terminal regions showed high levels of diversity. To obtain more information about their structure and the immunogenicity, truncated and chimeric S-layer proteins were constructed in recombinant E. coli. Profiling of cytokine production in THP-1 cells by truncated and chimeric S-layer proteins suggested that the intact conformation of the N-terminal region of SLP(aly) contributes to high immunogenicity.

Pseudofructophilic Leuconostoc citreum Strain F192-5, Isolated from Satsuma Mandarin Peel.

Fructophilic lactic acid bacteria (FLAB), composed of Fructobacillus spp., Lactobacillus kunkeei, and Lactobacillus apinorum, are unique in that they prefer d-fructose over d-glucose as a carbon source. Strain F192-5, isolated from the peel of a satsuma mandarin and identified as Leuconostoc citreum, grows well on d-fructose but poorly on d-glucose and produces mainly lactate and acetate, with trace amounts of ethanol, from the metabolism of d-glucose. These characteristics are identical to those of obligate FLAB. However, strain F192-5 ferments a greater variety of carbohydrates than known FLAB. Comparative analyses of the genomes of strain F192-5 and reference strains of L. citreum revealed no signs of specific gene reductions, especially genes involved in carbohydrate transport and metabolism, in the genome of F192-5. The bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) is conserved in strain F192-5 but is not transcribed. This is most likely due to a deletion in the promoter region upstream of the adhE gene. Strain F192-5 did, however, ferment d-glucose when transformed with a plasmid containing the allochthonous adhE gene. L. citreum F192-5 is an example of a pseudo-FLAB strain with a deficiency in d-glucose metabolism. This unique phenotypic characteristic appears to be strain specific within the species L. citreum This might be one of the strategies lactic acid bacteria use to adapt to diverse environmental conditions.IMPORTANCE Obligate fructophilic lactic acid bacteria (FLAB) lack the metabolic pathways used in the metabolism of most carbohydrates and differ from other lactic acid bacteria in that they prefer to ferment d-fructose instead of d-glucose. These characteristics are well conserved at the genus or species level. Leuconostoc citreum F192-5 shows similar growth characteristics. However, the strain is metabolically and genomically different from obligate FLAB. This is an example of a strain that evolved a pseudo-FLAB phenotype to adapt to a fructose-rich environment.

The impact of motility on the localization of Lactobacillus agilis in the murine gastrointestinal tract.

BACKGROUND: While the overall composition of the mammalian gut microbiota has been intensively studied, the characteristics and ecologies of individual gut species are incompletely understood. Lactobacilli are considered beneficial commensals in the gastrointestinal mucosa and are relatively well-studied except for the uncommon species which exhibit motility. In this study, we evaluate the importance of motility on gut colonization by comparing motile and non-motile strains of Lactobacillus agilis in mice models. RESULTS: A flagellated but non-motile L. agilis strain was constructed by mutation of the motB gene. Colonization of the wild type and the mutant strain was assessed in both antibiotic-treated female Balb/c mice and gnotobiotic mice. The results suggest that the motile strain is better able to persist and/or localize in the gut mucosa. Chemotaxis assays indicated that the motile L. agilis strain is attracted by mucin, which is a major component of the intestinal mucus layer in animal guts. CONCLUSIONS: Motility and chemotactic ability likely confer advantages in gut colonization to L. agilis. These findings suggest that the motile lactobacilli have unique ecologies compared to non-motile commensals of the lactic acid bacteria.

Intracellular localization of sirtuin and cell length analysis of Lactobacillus paracasei suggest possible role of sirtuin in cell division and cell shape regulation.

Sirtuin has been associated in prolonging lifespan of different model organisms. It has been shown to have an enzymatic activity of NAD+-dependent protein deacetylation targeting acetylated proteins. To determine targets and possible roles of sirtuin (LpSirA) in the Lactobacillus paracasei BL23, deletion (ΔsirA), sirtuin overexpressor (highsirA) and GFP fusion (highsirA-Venus) strains were generated, and microscopic localization and cell length analysis were done. Microscopic analysis revealed localization of LpSirA at cell division plates, at cell poles and all throughout the cell length in a spiral manner. Cell length analysis revealed that 46.9% of the ΔsirA cells were observed to be shorter (<2 µm), whereas 12.6% of the highsirA cells were observed to be longer (>4 µm) in comparison with the wild-type with only 17.1% short cells and 5.3% long cells. Our results suggest that sirtuin may have an essential role in cell division and cell shape regulation.

Detoxification process of tolaasins, lipodepsipeptides, by Microbacterium sp. K3-5.

Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.

Characterization of flagellins isolated from a highly motile strain of Lactobacillus agilis.

BACKGROUND: Most lactic acid bacteria are non-motile but some of them are flagellated and exhibit motility. So far, motile lactobacilli have rarely been studied, and characteristics of their flagellins are poorly understood. In this study, a highly motile strain of Lactobacillus agilis was recruited for transcriptional analysis and characterization of its flagellins. RESULTS: Unlike another motile lactic acid bacteria of intestinal isolate, Lactobacillus ruminis, flagellar filaments of the L. agilis strain probably consist of two homologous but distinct flagellins. Glycosylation of the flagellar filaments and their resistance to heat, acid and SDS were also observed. The immunological activity of the flagellins was evaluated through the stimulation of Caco-2 cells. The results show that TLR5-stimulating activity of the protein is attenuated, likely due to an incomplete TLR5-recognition site. CONCLUSIONS: The flagella filaments of L. agilis BKN88 consist of two homologous glycosylated flagellins, which likely have an incomplete TLR5-recognition site. The characteristics of the flagellin are presumably a consequence of adaptation as a commensal microbe in the gastrointestinal tract.

Negative chemotaxis of Ligilactobacillus agilis BKN88 against gut-derived substances.

Ligilactobacillus agilis is a motile lactic acid bacterium found in the gastrointestinal tracts of animals. The findings of our previous study suggest that the motility of L. agilis BKN88 enables gut colonization in murine models. However, the chemotactic abilities of motile lactobacilli remain unknown. This study aimed to identify the gut-derived chemoeffectors and their corresponding chemoreceptors in L. agilis BKN88. Chemotaxis assays with chemotactic and non-chemotactic (ΔcheA) L. agilis strains revealed that low pH, organic acids, and bile salts served as repellents. L. agilis BKN88 was more sensitive to bile and acid than the gut-derived non-motile lactobacilli, implying that L. agilis might utilize motility and chemotaxis instead of exhibiting stress tolerance/resistance. L. agilis BKN88 contains five putative chemoreceptor genes (mcp1-mcp5). Chemotaxis assays using a series of chemoreceptor mutants revealed that each of the five chemoreceptors could sense multiple chemoeffectors and that these chemoreceptors were functionally redundant. Mcp2 and Mcp3 sensed all tested chemoeffectors. This study provides further insights into the interactions between chemoreceptors and ligands of motile lactobacilli and the unique ecological and evolutionary features of motile lactobacilli, which may be distinct from those of non-motile lactobacilli.

[Protection against Salmonella via immunization with recombinant lactic acid bacteria].

Mucosal immunization of hens may be effective to prevent contamination of Salmonella enterica serovar Enteritidis (SE) in eggs. Lactic acid bacteria have been considered potential vaccine delivery agents because they are safe, immunogenic, and inexpensive. Our research group has been investigating the development of oral vaccines against SE using a Lactobacillus casei strain as an antigen delivery vehicle. Recombinant lactobacilli expressing SE antigens FliC and SipC have been constructed and administered to mice. Antigen specific immune responses and protective immunity were elicited after the immunization. For adjuvant delivery, IL-1beta-secreting L. casei was also engineered and its efficacies were evaluated in vitro and in vivo. This article reviews a novel approach to develop SE vaccines using recombinant lactobacilli.

Dissimilar properties of two recombinant Lactobacillus acidophilus strains displaying Salmonella FliC with different anchoring motifs.

Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro. The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.

Development of a highly efficient protein-secreting system in recombinant Lactobacillus casei.

The available techniques for heterologous protein secretion in Lactobacillus strains are limited. The aim of the present study was to develop an efficient protein-secretion system using recombinant lactobacilli for various applications such as live delivery of biotherapeutics. For the construction of expression vectors, the Lactobacillus brevis slpA promoter, Lactobacillus casei prtP signal sequence, and mouse IL-10 sequences were used as a model system. Interestingly, the slpA promoter exhibited strong activity in L. casei contrary to previous observations. In order to stabilize replication of the plasmid in E. coli, a removable terminator sequence was built into the promoter region. For the improvement of secretion efficiency, a DTNSD oligopeptide was added to the cleavage site of signal peptidase. The resulting plasmids provided remarkably efficient IL-10 secretion. Accumulation of the protein in the culture supernatant varied widely according to the pH conditions. By analysis of the secreted protein, formation of homodimers and biological activity, IL-10 was confirmed to be functional. The presently constructed plasmids could be useful tools for heterologous protein-secretion in L. casei.

Introduction of bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) in Fructobacillus fructosus settled its fructophilic characteristics.

Fructophilic lactic acid bacteria (FLAB) are unique in the sense that they prefer D-fructose over D-glucose as main carbon source. If D-glucose is metabolised, electron acceptors are required and significant levels of acetate are produced. These bacteria are found in environments rich in D-fructose, such as flowers, fruits and the gastrointestinal tract of insects feeding on fructose-rich diets. Fructobacillus spp. are representatives of this unique group, and their fructophilic characteristics are well conserved. In this study, the bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) from Leuconostoc mesenteroides NRIC 1541T was cloned into a plasmid and transferred to Fructobacillus fructosus NRIC 1058T. Differences in biochemical characteristics between the parental strain (NRIC 1058T) and the transformants were compared. Strain 1-11, transformed with the adhE gene, did not show any fructophilic characteristics, and the strain grew well on D-glucose without external electron acceptors. Accumulation of acetic acid, which was originally seen in the parental strain, was replaced with ethanol in the transformed strain. Furthermore, in silico analyses revealed that strain NRIC 1058T lacked the sugar transporters/permeases and enzymes required for conversion of metabolic intermediates. This may be the reason for poor carbohydrate metabolic properties recorded for FLAB.

Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform.

Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants-NCK2025 and NCK2031-elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1ß in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.

Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein.

Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.

Construction and immunological evaluation of dual cell surface display of HIV-1 gag and Salmonella enterica serovar Typhimurium FliC in Lactobacillus acidophilus for vaccine delivery.

Oral vaccines that elicit a mucosal immune response may be effective against human immunodeficiency virus type 1 (HIV-1) because its transmission occurs mainly at the mucosa. The aim of this study was to construct recombinant Lactobacillus for oral delivery of oral vaccines against HIV-1 and to evaluate their immunogenicity. A recombinant Lactobacillus acidophilus strain expressing the HIV-1 Gag on the bacterial cell surface was established by fusion with the signal peptide and anchor motif of a mucus binding protein (Mub) from L. acidophilus with or without coexpression of Salmonella enterica serovar Typhimurium flagellin (FliC) fused to a different Mub signal peptide and anchor. Using HEK293 cells engineered to express Toll-like receptor 5 (TLR5), the biological activity of FliC on the bacterial cell surfaces was determined. The surface-exposed flagellin retained its TLR5-stimulating activity, suggesting that the recombinant strain with Gag and FliC dual display might provide a different immunopotency than the strain expressing only Gag. The immunological properties of the recombinant strains were assessed by coculture with human myeloid dendritic cells (DCs). The heterologous antigens on the cell surface affected maturation and cytokine responses of DCs. Acquired immune responses were also investigated by intragastric immunization of mice. The enzyme-linked immunosorbent spot assay showed induction of gamma interferon-producing cells at local mucosa after immunization of mice with the Gag-producing strain. Meanwhile, the immunization with L. acidophilus displaying both Gag and FliC resulted in an increase of Gag-specific IgA-secreting cells. These results suggested that the Gag-displaying L. acidophilus elicited specific immune responses and the coexistence of FliC conferred an adjuvant effect on local IgA production.

Development of recombinant vaccines in lactobacilli for elimination of salmonella.

Many Lactobacillus and Lactococcus strains are generally regarded as safe for consumption because they are utilized for food fermentation or inhabit the intestinal mucosa as commensals. Recently, vaccine delivery systems using lactic acid bacteria (LAB) have been under development. Our research group has been investigating the development of oral mucosal vaccines against Salmonella enterica serovar Enteritidis (SE) using Lactobacillus casei IGM393 as an antigen delivery vehicle. Recombinant lactobacilli expressing SE antigens, FliC, SipC, and OmpC, have been constructed and orally administered to mice. Antigen specific immune responses and protective immunity were elicited after the immunization. For adjuvant-delivery, IL-1ß-secreting L. casei was also engineered and its effects evaluated in vitro and in vivo. This article reviews a novel approach to the elimination of Salmonella via the development of a vaccine in lactobacilli.

Establishment and Evaluation of an in vitro M Cell Model using C2BBe1 Cells and Raji Cells.

In vitro M cell models, consisting of co-cultures of Caco-2 cells and lymphoid cells, were developed and examined to observe bacterial transport. However, under our experimental conditions, the differentiation of Caco-2 cells into M cell-like cells could not be induced efficiently. To obtain a functionally stable M cell model based on human cells, C2BBe1 cells were screened and co-cultured with human Raji cells. In our co-cultures, increased sialyl Lewis A antigen expression and decreased Ulex europeaus agglutinin 1 binding were observed. Regarding the functional properties of the model, microsphere and lactic acid bacteria transport across the C2BBe1 co-cultures were increased compared with the levels seen in monocultures. The C2BBe1 monolayers that were co-cultured with Raji cells exhibited some M cell features; therefore, we consider our M cell model to be useful for investigating the interactions of bacteria with M cells.