Lentilactobacillus hilgardii H-50 strongly inhibits lipopolysaccharide-induced inflammatory responses in mouse splenocytes via its specific surface layer proteins.

AIMS: Certain lactic acid bacteria (LAB) are known to have anti-inflammatory effects; however, hiochi bacteria, which are taxonomically classified as LAB and known to spoil a traditional Japanese alcoholic beverage, have not been studied in the same context. The aim of this study is to investigate the anti-inflammatory effects of hiochi bacteria strains and the underlying mechanisms. METHODS AND RESULTS: We screened 45 strains of hiochi bacteria for anti-inflammatory effects and found that Lentilactobacillus hilgardii H-50 strongly inhibits lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in mouse splenocytes. This inhibition is attributed to its specific surface layer proteins (SLPs), which directly bind to LPS. CONCLUSIONS: The L. hilgardii H-50 strain exerts anti-inflammatory effects through its SLPs.

Application of two glycosylated Lactobacillus surface layer proteins in coating cationic liposomes.

The ability of isolated surface layer proteins (SLPs) to reassemble on suitable surfaces enables the application of SLPs in various fields of nanotechnology. In this work, SLPs from Lactobacillus buchneri BNCC 187,964 and L. kefir BNCC 190,565 were extracted and verified as glycosylated proteins. They were applied to coat on the surface of cationic liposomes. The absorption of the two SLPs on liposomes induced the zeta potential reduction and particle size increase. The two kinds of SLP-coated liposomes demonstrated better thermal, light and pH stability than the control liposomes. And the L. kefir SLP showed better protective effects than the L. buchneri SLP. Moreover, both of the SLPs could endow liposomes with the function of binding ferritin as observed by transmission electron microscope. Fourier transform infrared spectroscopy illustrated that the interaction between the two SLPs and liposomes was similar. The recrystallization of the two SLPs on the liposomes might drive the lipid into a higher order state and hydrogen bonds were formed between the two SLPs and the liposomes. All the findings demonstrated that L. kefir SLP and L. buchneri SLP had great potential to be explored as effective coating agents to improve the stability and function of cationic liposomes.Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.Yes, all have been checked.

Surface layer protein A from hypervirulent Clostridioides difficile ribotype 001 can induce autophagy process in human intestinal epithelial cells.

Clostridioides difficile is the leading cause of nosocomial diarrhea with high morbidity and mortality worldwide. C. difficile strains produce a crystalline surface layer protein A (SlpA), which is an absolute necessity for its pathogenesis. However, its pathogenic mechanisms and its pro-inflammatory behavior are not yet fully elucidated. Herein, we report for the first time that SlpA extracted from C. difficile can induce autophagy process in Caco-2 cells. SlpA protein was purified from two C. difficile strains (RT001 and ATCC 700075). The cell viability of Caco-2 cells after exposure with different concentrations (15, 20, 25 µg/mL) of SlpA at various time points (3, 6, 12, 24 h) was measured by MTT assay. Acridine orange staining was used to visualize the hypothetical acidic vesicular organelles. The gene expression of autophagy mediators including LC3B, Atg5, Atg16L, and Beclin-1 was determined by quantitative real-time PCR assay. Western blotting assay was used to detect the expression of LC3B protein. MTT assay showed that different concentrations of SlpA did not induce significant changes in the viability of Caco-2 cells. SlpA at concentration of 20 µg/mL enhanced the formation of acidic vesicular organelles in Caco-2 cells after 12 h of exposure. Moreover, SlpA treatment significantly increased the expression of autophagy-associated genes, and increased the expression of LC3B protein in Caco-2 cells. In conclusion, our study demonstrated that SlpA is capable to induce autophagy in intestinal epithelial cells. These findings reveal a novel mechanism for the pathogenesis of C. difficile mediated by its SLPs.

Protective function of surface layer protein from Lactobacillus casei fb05 against intestinal pathogens in vitro.

Escherichia coli and Salmonella are common pathogenic bacteria in human intestine, which can infect epithelial cells and cause diseases. Adhesion to intestinal tissue is the first step of pathogen infection. This work was to investigate the protective function of surface layer protein (SLP) from Lactobacillus casei fb05 against the harmful effects of E. coli and Salmonella on intestinal tissue (collagen and HT-29 cells). The SLP of L. casei fb05 was identified by transmission electron microscopy and SDS-PAGE. The purified SLP could reduce the adhesion of E. coli and Salmonella to collagen and HT-29 cells as observed by light microscope. The flow cytometry results showed that the L. casei fb05 SLP decreased the two pathogens-induced apoptosis of HT-29 cells by about 45%-49%. In addition, the activation of caspase-9 and caspase-3 caused by the two pathogens was significantly declined by the interference of the L. casei fb05 SLP. All the findings demonstrated that the L. casei fb05 SLP could decrease the deleterious effects of E. coli and Salmonella on intestinal tract in two ways: reducing pathogen adhesion and inhibiting pathogen-induced apoptosis. The potential of L. casei fb05 SLP in the treatment of intestinal diseases might be explored in this work.

Protection of surface layer protein from Enterococcus faecium WEFA23 against Listeria monocytogenes CMCC54007 infection by modulating intestinal permeability and immunity.

Enterococcus faecium WEFA23 was previously found effectively against adherence and colonization of Listeria monocytogenes CMCC54007, which might be closely related to its surface layer protein (SLP). In this study, the protective of SLP of E. faecium WEFA23 against infection of L. monocytogenes CMCC54007 was systemically investigated. In vitro assay showed that SLP actively inhibited L. monocytogenes internalization into Caco-2 cell line, with decreasing mRNA level of pro-inflammation cytokines and virulence factors and restoring destroyed intestinal barrier. In vivo assay through excluding SLP of E. faecium WEFA23 by 5 M LiCl represented that SLP increased body weight, reduced mortality and cell counts of L. monocytogenes CMCC54007 in tissues of mice. Further researches showed that SLP protected against L. monocytogenes CMCC54007 infection by modulation of intestinal permeability and immunity, namely, it decreased fluorescein isothiocyanate (FITC)-Dextran in serum, ameliorated destroyed colon structure, and increased number of goblet cells and protein level of TJ protein (Claudin-1, Occludin, and ZO-1) in colon. For immunity, SLP decreased number of CD4+ and CD8+ T cells in liver, mRNA level, and content of pro-inflammatory factors IL-6, IL-1ß, IFN-γ ,TNF-α, and NO, and restored the structure of liver and spleen. Key Points•SLP of E. faecium inhibited L. monocytogenes internalization and colonization•SLP of E. faecium ameliorated host intestinal barrier dysfunction•SLP of E. faecium decreased pro-inflammatory cytokines and cells.

Increased Excess Intracellular Cyclic di-AMP Levels Impair Growth and Virulence of Bacillus anthracis.

Cyclic di-AMP (c-di-AMP) is a recently identified bacterial second messenger that regulates biological processes. In this study, we found that inactivation of two c-di-AMP phosphodiesterases (PDEs), GdpP and PgpH, resulted in accumulation of 3.8-fold higher c-di-AMP levels than in the parental strain Sterne in Bacillus anthracis and inhibited bacterial growth. Moreover, excess c-di-AMP accumulation decreased bacterial toxin expression, increased sensitivity to osmotic stress and detergent, and attenuated virulence in both C57BL/6J and A/J mice. Complementation of the PDE mutant with a plasmid carrying gdpP or pgpH in trans from a Pspac promoter restored bacterial growth, virulence factor expression, and resistance to detergent. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in B. anthracis that is important for host-pathogen interaction.IMPORTANCE Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis Vegetative cells of this species produce anthrax toxin proteins and S-layer components during infection of mammalian hosts. So far, how the expression of these virulence factors is regulated remains largely unknown. Our results suggest that excess elevated c-di-AMP levels inhibit bacterial growth and reduce expression of S-layer components and anthracis toxins as well as reduce virulence in a mouse model of disease. These results indicate that c-di-AMP signaling plays crucial roles in B. anthracis biology and disease.

Lactobacillus rhamnosus GG components, SLP, gDNA and CpG, exert protective effects on mouse macrophages upon lipopolysaccharide challenge.

The aim of this study was to determine whether Lactobacillus rhamnosus GG (LGG) components (surface layer protein, SLP; genomic DNA, gDNA; unmethylated cytosine-phosphate-guanine-containing oligodeoxynucleotide, CpG-ODN), alone or in combination, could affect immunomodulation, and evaluate the signalling mechanism in mouse macrophage RAW264.7 cells challenged with lipopolysaccharide (LPS). LGG components were used to treat cells before LPS stimulation. Cytokine and Toll-like receptor (TLR) expression were assessed using real-time quantitative PCR (RT-qPCR). Mitogen-activated protein kinase (MAPK), extracellular regulated protein kinase (ERK) and nuclear factor-kappa B (NF-κB) signalling pathways were evaluated using immunoblots and immunofluorescence. SLP or SLP + gDNA pre-treatment significantly reduced the LPS-induced mRNA expression of tumour necrosis factor alpha (TNF-α). Pre-treatment with LGG single components (SLP, gDNA or CpG) or their combinations (SLP + gDNA or SLP + CpG) significantly decreased the LPS-induced interleukin-6 (IL-6) mRNA level (P < 0·05). Pre-treatment with SLP or gDNA, alone or in combination, significantly suppressed LPS-induced TLR2 and TLR4 mRNA levels (P < 0·05). SLP pre-treatment also significantly decreased the LPS-induced expression of TLR9 (P < 0·05). Pre-treatment with LGG single components or combinations significantly suppressed the LPS-induced phosphorylation levels of ERK (P > 0·05). In conclusion, pre-incubation with LGG components, singly or in combination, generally inhibited the activation of TLR, MAPK and NF-κB signalling pathways in LPS-stimulated cells, leading to attenuated inflammatory cytokine TNF-α and IL-6 production. These results indicate that nonviable probiotic LGG components exert an anti-inflammation effect on epithelial cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus rhamnosus GG (LGG) is widely used as probiotics. However, its main components are not well known for affecting immunomodulation. This study investigated the effects of pre-treatments with different components such as surface layer protein, genomic DNA and unmethylated cytosine-phosphate-guanine-containing oligodeoxynucleotides, alone or in combination on immunomodulation, and evaluated the signalling mechanism in mouse macrophage RAW264.7 cells challenged with lipopolysaccharide. Pre-incubation with components alone or in combination generally inhibited the activation of Toll-like receptor, mitogen-activated protein kinases, extracellular regulated protein kinases and nuclear factor-kappa B signalling pathways in lipopolysaccharide-stimulated cells, which generally leads to attenuated inflammatory cytokine interleukin-6 and tumour necrosis factor alpha production. These results indicate that nonviable probiotic LGG components exert an anti-inflammation effect on epithelial cells.

SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis.

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3(-/-) mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.

Aggregation of Lactobacillus brevis associated with decrease in pH by glucose fermentation.

Some Lactobacillus brevis strains were found to aggregate upon the addition of glucose, which resulted in glucose fermentation and pH decrease. Surface layer proteins (Slp) that represented the outermost layer of the bacteria decreased under these low pH conditions, probably because of the partial detachment of Slp from the cell surface triggered by the acidic environment. Similar observations of decreased Slp and aggregation were observed under the culture conditions, confirming that L. brevis aggregation was due to the partial Slp detachment under the acidic conditions of glucose fermentation. Such Slp detachment might affect the electrostatic nature of L. brevis cells by initiating the formation of irregular charge across the L. brevis cell surface, thereby leading to aggregation. These observations would be useful for elucidating the aggregation mechanism of lactic acid bacteria, which was considered to be involved in the probiotic effect of the bacteria.

Uromodulin-SlpA binding dictates Lactobacillus acidophilus uptake by intestinal epithelial M cells.

Bacterial access to the gut immune system is a crucial process to promote host immune responses. The probiotic L-92 strain of Lactobacillus acidophilus exerts anti-allergic immunomodulatory effects upon oral administration in mice. Here, we show that microfold cells (M cells) are responsible for L-92 internalization for evoking L-92-mediated immune responses. L-92 specifically bound to uromodulin, a glycosylphosphatidylinositol-anchored protein expressed exclusively on M cells among intestinal epithelial cells. Internalization of L-92 into M cells was significantly reduced in uromodulin-deficient (Umod-/-) mice compared to Umod+/+ mice. Furthermore, the binding of L-92 to uromodulin was significantly decreased after removal of surface layer protein A (SlpA) from the bacteria. Our study thus revealed a crucial role of uromodulin on the M-cell surface for the uptake of SlpA-positive lactic acid bacteria into M cells, possibly leading to subsequent delivery of the bacteria to dendritic cells closely associated with M cells for immunomodulation. Our study also shed light on the possibility that SlpA and uromodulin could be used as vehicle and target, respectively, for efficient mucosal vaccine delivery.

A novel carbohydrate-binding surface layer protein from the hyperthermophilic archaeon Pyrococcus horikoshii.

In Archaea and Bacteria, surface layer (S-layer) proteins form the cell envelope and are involved in cell protection. In the present study, a putative S-layer protein was purified from the crude extract of Pyrococcus horikoshii using affinity chromatography. The S-layer gene was cloned and expressed in Escherichia coli. Isothermal titration calorimetry analyses showed that the S-layer protein bound N-acetylglucosamine and induced agglutination of the gram-positive bacterium Micrococcus lysodeikticus. The protein comprised a 21-mer structure, with a molecular mass of 1,340 kDa, as determined using small-angle X-ray scattering. This protein showed high thermal stability, with a midpoint of thermal denaturation of 79 °C in dynamic light scattering experiments. This is the first description of the carbohydrate-binding archaeal S-layer protein and its characteristics.

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response.

BACKGROUND: Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear. RESULTS: We used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS. CONCLUSIONS: These results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.

Analysis of self-assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus.

The formation of stable and functional surface layers (S-layers) via self-assembly of surface-layer proteins on the cell surface is a dynamic and complex process. S-layers facilitate a number of important biological functions, e.g., providing protection and mediating selective exchange of molecules and thereby functioning as molecular sieves. Furthermore, S-layers selectively bind several metal ions including uranium, palladium, gold, and europium, some of them with high affinity. Most current research on surface layers focuses on investigating crystalline arrays of protein subunits in Archaea and bacteria. In this work, several complementary analytical techniques and methods have been applied to examine structure-function relationships and dynamics for assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus: (1) The secondary structure of the S-layer protein was analyzed by circular dichroism spectroscopy; (2) Small-angle X-ray scattering was applied to gain insights into the three-dimensional structure in solution; (3) The interaction with bivalent cations was followed by differential scanning calorimetry; (4) The dynamics and time-dependent assembly of S-layers were followed by applying dynamic light scattering; (5) The two-dimensional structure of the paracrystalline S-layer lattice was examined by atomic force microscopy. The data obtained provide essential structural insights into the mechanism of S-layer self-assembly, particularly with respect to binding of bivalent cations, i.e., Mg2+ and Ca2+. Furthermore, the results obtained highlight potential applications of S-layers in the fields of micromaterials and nanobiotechnology by providing engineered or individual symmetric thin protein layers, e.g., for protective, antimicrobial, or otherwise functionalized surfaces.

Purification of Lactobacillus acidophilus surface-layer protein and its immunomodulatory effects on RAW264.7 cells.

BACKGROUND: Surface-layer proteins (SLP) have been found in the outermost layer of the cell wall in many types of lactobacillus are considered to be an important factor with respect to intestinal immunity. RESULTS: The present study compared the effects of SLP extracted by different concentrations of LiCl and carbamide, and subsequently identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism and differential scanning calorimetry. Furthermore, RAW 264.7 cells were used to evaluate the immunomodulatory effects of SLP. SLP were derived from Lactobacillus acidophilus CICC6074 with a molecular weight of 46 kDa, and consisted of 16.9% α-helix, 42.3% ß-sheet, 20.8% ß-turns and 22.5% random coils. SLP promoted NO secretion and higher quantities of NO were produced as the SLP concentrations increased. SLP concentrations over 50 µg mL-1 significantly decreased the amount of tumor necrosis factor-α secreted by RAW264.7 cells. CONCLUSION: SLP can trigger immunomodulatory effects in RAW 264.7 cells. This provides crucial information that will enable the further use of L. acidophilus in food, medicine and other products. © 2017 Society of Chemical Industry.

Influence of biofilm surface layer protein A (BslA) on the gel structure of myofibril protein from chicken breast.

BACKGROUND: Different techniques have been applied to alter myofibril protein (MP) structure, which further promotes protein-protein interactions and influencing the MP gelling characteristics. Influence of BslA from natto food (protein concentration, 30 mg mL-1 ; at 0.001, 0.005, 0.01, 0.05 and 0.1 g kg-1 ) on the characteristics of MP gel of chicken breast was investigated. RESULTS: Results show that cooking loss significantly (P < 0.05) decreased with increased percentage of BslA. Hardness of MP gel did not significantly change at 0.01 g kg-1 BslA. Differential scanning calorimetry disclosed that MP was modified by the addition of BslA. Moreover, BslA produced a high value of storage modulus (G') and low value of phase angle (tan δ) during heating, especially at 0.01 g kg-1 . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis proved the formation of higher-molecular-weight polymers by developing non-disulfide covalent bonds between MP at 0.01 g kg-1 BslA. Surface hydrophobicity of the MP gel was decreased with increased percentage of BslA. Scanning electron microscopy confirmed the increasing number of uniform cavities of MP gel with the increased percentage of BslA. CONCLUSION: Addition of 0.01 g kg-1 BslA significantly improved the water holding capacity and rheological properties of MP by developing non-disulfide covalent bonds. © 2017 Society of Chemical Industry.

Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani.

Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5' splice site located 8 nt upstream of the usual 5' GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1-EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5' splice site is shown to be affected by structures in addition to IBS1-EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3' exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression.

Truncation Derivatives of the S-Layer Protein of Sporosarcina ureae ATCC 13881 (SslA): Towards Elucidation of the Protein Domain Responsible for Self-Assembly.

The cell surface of Sporosarcina ureae ATCC 13881 is covered by an S-layer (SslA) consisting of identical protein subunits that assemble into lattices exhibiting square symmetry. In this work the self-assembly properties of the recombinant SslA were characterised with an emphasis on the identification of protein regions responsible for self-assembly. To this end, recombinant mature SslA (aa 31-1097) and three SslA truncation derivatives (one N-terminal, one C-terminal and one CN-terminal) were produced in a heterologous expression system, isolated, purified and their properties analysed by in vitro recrystallisation experiments on a functionalised silicon wafer. As a result, recombinant mature SslA self-assembled into crystalline monolayers with lattices resembling the one of the wild-type SslA. The study identifies the central protein domain consisting of amino acids 341-925 self-sufficient for self-assembly. Neither the first 341 amino acids nor the last 172 amino acids of the protein sequence are required to self-assemble into lattices.

Targeting surface-layer proteins with single-domain antibodies: a potential therapeutic approach against Clostridium difficile-associated disease.

Clostridium difficile is a leading cause of death from gastrointestinal infections in North America. Antibiotic therapy is effective, but the high incidence of relapse and the rise in hypervirulent strains warrant the search for novel treatments. Surface layer proteins (SLPs) cover the entire C. difficile bacterial surface, are composed of high-molecular-weight (HMW) and low-molecular-weight (LMW) subunits, and mediate adherence to host cells. Passive and active immunization against SLPs has enhanced hamster survival, suggesting that antibody-mediated neutralization may be an effective therapeutic strategy. Here, we isolated a panel of SLP-specific single-domain antibodies (VHHs) using an immune llama phage display library and SLPs isolated from C. difficile hypervirulent strain QCD-32g58 (027 ribotype) as a target antigen. Binding studies revealed a number of VHHs that bound QCD-32g58 SLPs with high affinity (K D = 3-6 nM) and targeted epitopes located on the LMW subunit of the SLP. The VHHs demonstrated melting temperatures as high as 75 °C, and a few were resistant to the gastrointestinal protease pepsin at physiologically relevant concentrations. In addition, we demonstrated the binding specificity of the VHHs to the major C. difficile ribotypes by whole cell ELISA, where all VHHs were found to bind 001 and 027 ribotypes, and a subset of antibodies were found to be broadly cross-reactive in binding cells representative of 012, 017, 023, and 078 ribotypes. Finally, we showed that several of the VHHs inhibited C. difficile QCD-32g58 motility in vitro. Targeting SLPs with VHHs may be a viable therapeutic approach against C. difficile-associated disease.

Lactobacillus reuteri glyceraldehyde-3-phosphate dehydrogenase functions in adhesion to intestinal epithelial cells.

This study was aimed to identify key surface proteins mediating the adhesion of lactobacilli to intestinal epithelial cells. By using Caco-2 and IPEC-J2 cells labeled with sulfo-NHS-biotin in the western blotting, a protein band of an approximately 37 kDa was detected on the surface layer of Lactobacillus reuteri strains ZJ616, ZJ617, ZJ621, and ZJ623 and Lactobacillus rhamnosus GG. Mass spectrometry analysis using the adhesion-related protein from L. reuteri ZJ617 showed that it was 100% homologous to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. reuteri JCM 1112 (GenBank: YP_001841377). The ability of L. reuteri ZJ617 to adhere to epithelial cells decreased significantly by treatment with LiCl or by blocking with an anti-GAPDH antibody, in comparison with the untreated strain (p < 0.05). Immunoelectron microscopic and immunofluorescence analyses confirmed that GAPDH is located on the surface layer of L. reuteri ZJ617. The results indicated that the GAPDH protein of L. reuteri ZJ617 acts as an adhesion component that plays an important role in binding to the intestinal epithelial cells.

Xylan-mediated aggregation of Lactobacillus brevis and its relationship with the surface properties and mucin-mediated aggregation of the bacteria.

Some Lactobacillus brevis strains were found to aggregate upon the addition of xylan after screening for lactic acid bacteria that interact with plant materials. The S-layer proteins of cell surface varied among the strains. The strains that displayed xylan-mediated aggregation retained its ability even after the removal of S-layer proteins. L. brevis had negative zeta potentials. A correlation between the strength of aggregation and zeta potential was not observed. However, partial removal of S-layer proteins resulted in decreases in the electric potential and aggregation ability of some strains. Therefore, xylan-mediated aggregation of L. brevis was considered to be caused by an electrostatic effect between the cells and xylan. L. brevis also aggregated in the presence of mucin, and the strengths of aggregation among the strains were similar to that induced by xylan. Thus, xylan- and mucin-mediated L. brevis aggregation was supposed to be caused by a similar mechanism.