Eiichi Satoh

A Mucus Adhesion Promoting Protein, MapA, Mediates the Adhesion of Lactobacillus reuteri to Caco-2 Human Intestinal Epithelial Cells

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.

Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis

Abstract A recombinant Lactobacillus casei expressing a flagellar antigen from Salmonella enterica serovar Enteritidis was constructed and evaluated as a mucosal vaccine. Intragastric immunization of the recombinant strain conferred protective immunity against Salmonella infection in mice. This immunization did not result in antigen-specific antibody in either feces or sera but induced the release of IFN-γ on restimulation of primed lymphocytes ex vivo. The results suggested that the protective efficacy provided by flagellin-expressing L. casei is mainly attributable to cell-mediated immune responses. In addition, an adjuvant-type effect of the antigen delivery system with L. casei was also observed.

Structures of Two Monomeric Units of Teichoic Acid Prepared from the Cell Wall of Lactobacillus plantarum NRIC 1068

The cell wall of Lactobacillus plantarum contains large amounts of cell wall teichoic acid (WTA). WTA was isolated from the cell wall of L. plantarum NRIC 1068 (= ATCC 8014 and 17-5) by extraction with trichloroacetic acid, and two monomeric units (F1 and F2) were prepared from the alkaline hydrolysate of WTA. Componential analysis by HPLC showed that these monomers were composed of ribitol, glucose, and phosphoric acid. Structural analyses of the monomers were performed by NMR spectroscopy with comparison to chemically synthesized monomers. The structures of F1 and F2 were determined to be 3,4-α-D-diglucosyl-2-phosphoryl ribitol and 3,4-α-D-diglucosyl-1-phosphoryl ribitol respectively. The unique structure of WTA in L. plantarum results from modification of the main chain with multiple glucose residues.

Comparison of Components and Synthesis Genes of Cell Wall Teichoic Acid among Lactobacillus plantarum Strains

The contents, components, and synthesis genes of cell wall teichoic acid (WTA) in 18 strains of Lactobacillus plantarum were compared. The WTA of each strain was classified by its components as being either the glycerol- or the ribitol-type. The different strains in the WTA type showed marked differences also in two gene regions, tagD1–tagF2 and lp_1816–tagB2, as for the presence or absence, nucleotide sequences, and transcriptional activities. Our results clearly showed that the tagD1–tagF2 and lp_1816–tagB2 regions contained the synthesis genes of the WTA backbone of L. plantarum. We verified that the genes in the tagD1–tagF2 region were involved in the synthesis of the glycerol-type backbone. Furthermore, we propose that the genes in the lp_1816–tagB2 region were tarI, tarJ, tarK, and tarL, which are involved in the synthesis of the ribitol-type backbone.

Lactobacillus equigenerosi sp. nov., a coccoid species isolated from faeces of thoroughbred racehorses

Two strains of lactic acid bacteria were isolated from faeces of two actively racing thoroughbred horses. The isolates formed a subcluster in the Lactobacillus reuteri phylogenetic group, closely related to Lactobacillus fermentum, L. gastricus, L. ingluviei and L. mucosae, by phylogenetic analysis based on 16S rRNA gene sequences. Levels of DNA-DNA relatedness revealed that the isolates belonged to the same taxon and were genetically separated from their phylogenetic relatives. Biochemical and physiological characteristics also distinguished the isolates from their phylogenetic relatives. The isolates produced spherical or oval cells, and tetrad-like cells were rarely seen. To the best of our knowledge, this is the first report of this morphological characteristic within the genus Lactobacillus. Thus, the isolates represent an atypical novel species of the genus Lactobacillus, for which the name Lactobacillus equigenerosi sp. nov. is proposed. The type strain is NRIC 0697T (=JCM 14505T =DSM 18793T).

Metabolic engineering of Lactobacillus plantarum for succinic acid production through activation of the reductive branch of the tricarboxylic acid cycle.

Biosynthesis of succinic acid is an alternative method from conventional chemical synthesis. For this application, several bacteria and fungi have been employed and genetically modified. Lactic acid bacteria (LAB) are gaining recognition as novel producers of useful compounds by metabolic engineering. Among LAB, Lactobacillus plantarum NCIMB 8826 is an interesting candidate for succinic acid production by metabolic engineering since it has an incomplete tricarboxylic acid (TCA) cycle and naturally produces small amounts of succinic acid. In this study, we constructed recombinant LAB and evaluated them as hosts of succinic acid production. We examined the enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and malic enzyme for their potential to improve metabolic flux from glycolysis to the reductive TCA cycle in a lactate dehydrogenase-deficient strain of L. plantarum NCIMB 8826 (VL103). We investigated the effects of overexpression or coexpression of each enzyme on succinic acid production. Our results suggested that PC is the key enzyme for succinic acid production by L. plantarum VL103, whereas PEPCK is critical for increasing biomass. The highest yield of succinic acid was obtained through coexpression of PC and PEPCK in L. plantarum VL103. This recombinant strain produced a 22-fold higher amount of succinic acid than the wild-type and converted 25.3% of glucose to succinic acid.

Bidirectional cell-surface anchoring function of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403.

With the aim of constructing an efficient protein display system for lactic acid bacteria (LABs), the effect of fusion direction on the cell-surface binding activity of the C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 was studied. CPH fused to the alpha-amylase (AMY) of Streptococcus bovis 148 either at its C-terminus (CPH-AMY) or at its N-terminus (AMY-CPH) was expressed intracellularly in Escherichia coli. This domain was able to direct binding of AMY to the surface of L. lactis ATCC 19435 in both constructs. However, the number of bound molecules per cell and the specific activity for starch digestion in the case of CPH-AMY were 3 and 14 times greater than those in the case of AMY-CPH, respectively. Of the LABs tested, L. lactis ATCC 19435 showed the highest binding capability for CPH-AMY, up to 6 x 10(4) molecules per cell, with a dissociation rate constant of 5.00 x 10(-5) s(-1). The binding of CPH-AMY to the surface of Lactobacillus delbrueckii ATCC 9649 cells was very stable with a dissociation rate constant of 6.96 x 10(-6) s(-1). The production of CPH-AMY in the soluble form increased 3-fold as a result of coexpression with a molecular chaperone, trigger factor. The results of this study suggest the usefulness of CPH as a bidirectional anchor protein for the production of cell-surface adhesive enzymes in E. coli. Furthermore, the importance of the fusion direction of CPH in determining cell-surface binding and enzymatic activities was shown.

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.