RNA-Based Anti-Inflammatory Effects of Membrane Vesicles Derived from Lactiplantibacillus plantarum.

Bacteria generally release extracellular membrane vesicles (MVs), which are nanoparticles that play important roles in bacterial-bacterial and bacterial-host communication. As probiotics, lactic acid bacteria provide diverse health benefits to their hosts. In this study, we found that the Gram-positive lactic acid bacteria Lactiplantibacillus plantarum subsp. plantarum NBRC 15891 produce high amounts of MVs (LpMVs), and that LpMVs inhibit interleukin (IL)-8 production induced by lipopolysaccharide in intestinal epithelial HT29 cells. Heat- or UV-killed bacterial cells did not exhibit anti-inflammatory effects, and there was no uptake of these bacterial cells; contrarily, LpMVs were taken up into the cytoplasm of HT29 cells. Small RNAs extracted from LpMVs also suppressed IL-8 production in HT29 cells, suggesting that RNAs in the cytoplasm of bacterial cells are encapsulated in the MVs and released from the cells, which may be delivered to HT29 cells to exert their anti-inflammatory effects. In addition, administration of LpMVs to mice with dextran sodium sulfate-induced colitis alleviated colitis-induced weight loss and colon length shortening, indicating that LpMV intake is likely to be effective in preventing or ameliorating colitis.

High productivity of immunostimulatory membrane vesicles of Limosilactobacillus antri using glycine.

Nanosized membrane vesicles (MVs) released by bacteria play important roles in both bacteria-bacteria and bacteria-host interactions. Some gram-positive lactic acid bacteria produce MVs exhibiting immunoregulatory activity in the host. We found that both bacterial cells and MVs of Limosilactobacillus antri JCM 15950, isolated from the human stomach mucosa, enhance immunoglobulin A production by murine Peyer's patch cells. However, the thick cell walls of gram-positive bacteria resulted in low MV production, limiting experiments and applications using MVs. In this study, we evaluated the effects of glycine, which inhibits cell wall synthesis, on the immunostimulatory MV productivity of L. antri. Glycine inhibited bacterial growth while increasing MV production, with 20 g/L glycine increasing MV production approximately 12-fold. Glycine was most effective at increasing MV production when added in the early exponential phase, which indicated that cell division in the presence of glycine increased MV production. Finally, glycine increased MV productivity approximately 16-fold. Furthermore, glycine-induced MVs promoted interleukin-6 production by macrophage-like J774.1 cells, and the immunostimulatory activity was comparable to that of spontaneously produced MVs. Our results indicate that glycine is an effective agent for improving the production of MVs with immunostimulatory activity in gram-positive lactic acid bacteria, which can be applied as mucosal adjuvants and functional foods.

Analysis of the effects of specific growth rate of Lactococcus lactis MG1363 on aerobic metabolism and its application to high-density culture.

Lactic acid bacteria (LAB) are known to produce a large amount of lactate when cultured under non-aerated conditions, which inhibits their growth at high concentrations. Our previous studies have shown that LAB can be cultured without lactate production under aerated conditions at a low specific growth rate. In this study, we investigated the effects of specific growth rate on cell yield and the specific production rates of metabolites in aerated fed-batch cultures of Lactococcus lactis MG1363. The results showed that lactate and acetoin production could be suppressed at specific growth rates below 0.2 h-1, whereas acetate production was the highest at a specific growth rate of 0.2 h-1. When LAB was cultured at a specific growth rate of 0.25 h-1 with the addition of 5 mg/L heme to assist ATP production by respiration, lactate and acetate production was suppressed, and cell concentration reached 19 g-dry-cell/L (5.6 × 10ˆ10 cfu/mL) with a high cell yield of 0.42 ± 0.02 g-dry-cell/g-glucose.

Mechanisms underlying enhanced IgA production in Peyer's patch cells by membrane vesicles derived from Lactobacillus sakei.

We analyzed the mechanisms underlying enhanced IgA production in the cells of Peyer's patch cells via membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC 15893. Depletion of CD11c+ cells from Peyer's patch cells suppressed the enhanced IgA production mediated by membrane vesicles. Meanwhile, the stimulation of bone-marrow-derived dendritic cells with membrane vesicles increased gene expression of inducible nitric oxide synthase, retinaldehyde dehydrogenase 2, and several inflammatory cytokines. The production of nitric oxide and interleukin (IL)-6 by membrane vesicle stimulation was induced via Toll-like receptor 2 on bone marrow-derived dendritic cells. Inhibition of inducible nitric oxide synthase and retinaldehyde dehydrogenase 2, as well as the neutralization of IL-6 in Peyer's patch cells, suppressed the enhanced IgA production by membrane vesicle stimulation. Hence, nitric oxide, retinoic acid, and IL-6 induced by membrane vesicles play crucial roles in the enhanced IgA production elicited by membrane vesicles in Peyer's patch cells.

Adhesion mechanisms of Bifidobacterium animalis subsp. lactis JCM 10602 to dietary fiber.

Adherence of probiotics to dietary fibers present in the intestinal tract may affect adhesion to intestinal epithelial cells. The properties of the adhesion of bifidobacteria to mucin or epithelial cells have been well studied; however, adhesion of bifidobacteria to dietary fiber has not been investigated. The adhesion ratio of six Bifidobacterium strains to cellulose and chitin was examined; among the strains, Bifidobacterium animalis subsp. lactis JCM 10602 showed high adherence to both cellulose and chitin, and two strains showed high adherence to only chitin. The ratios of adhesion of B. animalis to cellulose and chitin were positively and negatively correlated with ionic strength, respectively. These data suggest that hydrophobic and electrostatic interactions are involved in the adhesion to cellulose and chitin, respectively. The adhesion ratios of the cells in the late logarithmic phase to cellulose and chitin decreased by approximately 40% and 70% of the cells in the early logarithmic phase, respectively. Furthermore, the adhesion ratio to cellulose decreased with increasing bile concentration regardless of the culture phase of the cells. On the other hand, the adhesion ratio to chitin of cells in the early logarithmic phase decreased with increasing bile concentration; however, that of cells in the late logarithmic phase increased slightly, suggesting that adhesins differ depending on the culture phase. Our results indicated the importance of considering adhesion to both dietary fibers and the intestinal mucosa when using bifidobacteria as probiotics.

Suppression of lactate production by aerobic fed-batch cultures of Lactococcus lactis.

Aerobic fed-batch cultures were studied as a means of suppressing the production of lactate, which inhibits the growth of lactic acid bacteria (LAB). LAB produce lactate via lactate dehydrogenase (LDH), regenerating nicotinamide adenine dinucleotide (NAD+) consumed during glycolysis. Therefore, we focused on NADH oxidase (NOX), employing oxygen as an electron acceptor, as an alternative pathway to LDH for NAD+ regeneration. To avoid glucose repression of NOX and NAD+ consumption by glycolysis exceeding NAD+ regeneration by NOX, glucose was fed gradually. When Lactococcus lactis MG 1363 was aerobically fed at a specific growth rate of 0.2 h-1, the amount of lactate produced per amount of grown cell was reduced to 12% of that in anaerobic batch cultures. Metabolic flux analysis revealed that in addition to NAD+ regeneration by NOX, ATP acquisition by production of acetate and NAD+ regeneration by production of acetoin and 2,3-butanediol contributed to suppression of lactate production.

Suppression of lactate production of Lactobacillus reuteri JCM1112 by co-feeding glycerol with glucose.

Lactate produced by lactic acid bacteria inhibits their growth. To suppress lactate production, it is necessary to regenerate NAD+ consumed by glycolysis with alternative pathways other than lactate dehydrogenase. In a heterofermentative lactic acid bacterium, Lactobacillus reuteri JCM1112, suppression of lactate production by regenerating NAD+ when producing 1,3-propanediol from glycerol was investigated. The bacterium produced lactate with a yield of 4.7 ± 0.8 g·g-cell-1 in a batch culture using glucose as the sole carbon source. When glycerol was added to glucose at a molar ratio (rGly/Glc) of three in the batch culture, the bacterium produced 1,3-propanediol at 1.6 ± 0.7 g·g-cell-1·h-1 and the lactate yield decreased to 3.6 ± 0.5 g·g-cell-1. When glycerol was co-fed with glucose exponentially to give a target specific growth rate of 0.1 h-1 (rGly/Glc = 3), the lactate yield decreased to 1.5 ± 0.2 g·g-cell-1. The lactate production when glycerol was added together with glucose was reduced to one-third of that observed in the batch culture using glucose as a carbon source.

Suppression of lactate production by using sucrose as a carbon source in lactic acid bacteria.

Lactic acid bacteria (LAB) grow by producing lactate from sugar. However, the accumulation of lactate inhibits their growth. Here, the lactate productivity per cell in a semi-solid medium prepared with a chlorella powder in several LAB strains was much lower than that in the conventional MRS medium. Furthermore, the lactate production was suppressed not only in semi-solid medium, but also in chlorella liquid medium. The lactate productivity by Lactococcus lactis subsp. lactis NBRC 12007 in the chlorella liquid medium and MRS medium was 3.0 and 6.9 g-lactate·g-cell-1, respectively. The productivity of lactate in the chlorella liquid medium decreased to 44% of that in MRS medium. Gas chromatography/mass spectrometry (GC/MS) analysis of the culture supernatants revealed that the utilization of sucrose in the chlorella powder led to the suppression of lactate production. Comparison of the metabolites extracted from the cells indicated that the two ATP generating pathways, the arginine deiminase pathway and the decarboxylation reaction of glutamate and GABA, which are usually repressed by glucose, are activated in chlorella medium. It was considered that these pathways which do not require NAD+ for generation of ATP are not repressed when sucrose is used as a carbon source. Thus, the utilization of these pathways results in the suppression of the lactate production.

Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source.

We report a method for suppression of lactate production by lactic acid bacteria (LAB) in culture. LAB produce lactate to regenerate NAD+ that is consumed during glycolysis. Glucose suppresses NAD+ regeneration pathways other than lactate dehydrogenase and non-glycolytic ATP production pathways. Therefore, the carbon source was changed to sucrose, and fed-batch culture was performed to limit the glycolytic flux and thus suppress lactate production. As a result, lactate productivity (i.e., the amount of lactate produced per amount of grown cell) in the sucrose/fed-batch culture was decreased compared to that in glucose/batch culture, in all five LAB strains examined. The productivity level decreased to 24% and 46% in Lactobacillus reuteri JCM 1112 and Lactococcus lactis JCM 7638, respectively. Metabolic flux analysis of Lactobacillus reuteri JCM 1112 revealed increased contributions of the mannitol production pathway to NAD+ regeneration and the arginine deiminase pathway to ATP production in the sucrose/fed-batch culture.

IgA-enhancing effects of membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC15893.

Immunoglobulin (Ig) A in the mucus of the intestinal tract plays an important role in preventing the invasion of pathogenic microorganisms and regulating the composition of the gut microbiota. Several strains of probiotic lactic acid bacteria (LAB) are known to promote intestinal IgA production. Bacteria are also known to naturally release spherical membrane vesicles (MVs) that are involved in various biological functions such as quorum sensing, pathogenesis, and host immunomodulation. However, the production of MVs by LAB and their effects on host immunity remain poorly understood. In this study, we investigated the MV production by Lactobacillus sakei subsp. sakei NBRC15893 isolated from kimoto, the traditional seed mash used for brewing sake. MVs were separated from the culture broth of L. sakei NBRC15893 through filtration and density gradient ultracentrifugation and were observed by transmission electron microscopy. The MVs showed a spherical morphology, with a diameter of 30-400 nm, and contained proteins and nucleic acids. In addition, both the LAB cells and purified MVs promoted IgA production by murine Peyer's patch cells. This MV- and cell-induced IgA production was suppressed by neutralization of Toll-like receptor (TLR) 2, which recognizes cell wall components of gram-positive bacteria, using an anti-TLR2 antibody. Collectively, our results indicate that MVs released from L. sakei NBRC15893 enhance IgA production by activating host TLR2 signaling through its cell wall components. Thus, it is important to consider novel interactions between gut microbiota and hosts via MVs, and MVs derived from probiotic bacteria could have promising applications as safe adjuvants.

Analysis of gene expression profiles of Lactobacillus paracasei induced by direct contact with Saccharomyces cerevisiae through recognition of yeast mannan.

Co-culture of lactic acid bacteria (LAB) and yeast induces specific responses that are not observed in pure culture. Gene expression profiles of Lactobacillus paracasei ATCC 334 co-cultured with Saccharomyces cerevisiae IFO 0216 were analyzed by DNA microarray, and the responses induced by direct contact with the yeast cells were investigated. Coating the LAB cells with recombinant DnaK, which acts as an adhesive protein between LAB and yeast cells, enhanced the ratio of adhesion of the LAB cells to the yeast cells. The signals induced by direct contact were clarified by removal of the LAB cells unbound to the yeast cells. The genes induced by direct contact with heat-inactivated yeast cells were very similar to both those induced by the intact yeast cells and those induced by a soluble mannan. The top 20 genes upregulated by direct contact with the heat-inactivated yeast cells mainly encoded proteins related to exopolysaccharide synthesis, modification of surface proteins, and transport systems. In the case of the most upregulated gene, LSEI_0669, encoding a protein that has a region homologous to polyprenyl glycosylphosphotransferase, the expression level was upregulated 7.6-, 11.0-, and 8.8-fold by the heat-inactivated yeast cells, the intact yeast cells, and the soluble mannan, respectively, whereas it was only upregulated 1.8-fold when the non-adherent LAB cells were not removed before RNA extraction. Our results indicated that the LAB responded to direct contact with the yeast cells through recognition of mannan on the surface of the yeast.

Simple chronic colitis model using hypopigmented mice with a Hermansky-Pudlak syndrome 5 gene mutation.

Pigmentation in mammals is important for protection of skin and eyes from ultraviolet radiation. Dysregulation of pigmentation is often associated with other conditions that are not directly linked to pigmentation. Here, we isolated spontaneously occurring hypopigmented mice that occasionally experienced severe diarrhea during lactation. Treatment of these mice with dextran sulfate sodium salt, a conventional method to induce acute colitis, caused chronic diarrhea with granulomatous colitis. Gene mapping and sequencing revealed that the mice had a nonsense mutation in the Hermansky-Pudlak syndrome (Hps)5 gene. As some HPS patients can develop granulomatous colitis, the simple induction of chronic colitis in spontaneously mutated Hps5-deficient mice may become an invaluable model for exploring treatment options in patients with HPS as well as other patients with inflammatory bowel disease.

Optimal production of a fusion protein consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in fed-batch culture of Pichia pastoris.

BACKGROUND/AIM: The aim of the present study was to establish the strategy for producing a single-chain variable fragment (scFv) antibody fused with interleulin-2 (IL2) by Pichia pastoris and to optimize production during fed-batch cultivation in a 5-l fermenter. MATERIALS AND METHODS: We constructed a fusion sequence consisting of an scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (designated MK-1 antigen) and human interleulin-2 (IL-2) gene, ligated the sequences to expression vector pPICZα-A and separately transformed the constructs into Pichia pastoris strains GS115 and KM71H. RESULTS: The highest concentration of secreted fusion protein, 738 ± 44 mg/l, was obtained after a 60-h induction. To investigate the specific binding activity of the partially purified fusion protein, we used an enzyme-linked immunosorbent assay and antigen from a whole-cell lysate. Student's t-test showed that the specific binding activity of the partially-purified fusion protein to the lysate of Chinese hamster ovary cell lines expressing the MK-1 antigen was significantly higher than that of the lysate of CHO cell lines that do not express MK-1. CONCLUSIONS: The method described here permits the production of substantial amounts of the fusion protein for conducting functional studies on the biological role of these fusion proteins.

Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-ß-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

Strategy for preventing bacterial contamination by adding exogenous ethanol in solid-state semi-continuous bioethanol production.

A strategy for preventing reduction of ethanol yield in fermentation caused by bacterial contamination was developed. In solid-state ethanol fermentation in which saccharification, fermentation and ethanol recovery are performed simultaneously, the addition of exogenous ethanol to the fermentation mixture at the start of fermentation at 50 g kg(-1) prevented contamination, and the ethanol yield reached 0.50 g g(-1).

Ethanol production from biomass by repetitive solid-state fed-batch fermentation with continuous recovery of ethanol.

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 g l(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

Lactic acid bacteria display on the cell surface cytosolic proteins that recognize yeast mannan.

Fluorescent-labeled invertase, a hyperglycosylated mannoprotein from Saccharomyces cerevisiae, was found to bind to Lactococcus lactis IL1403 at acidic pH. Proteins on the cell wall of the bacterium affinity-purified using invertase as a ligand were identified to be heat shock proteins such as DnaK and GroEL and glycolytic enzymes such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. DnaK bound to both the bacterium and yeast at pH 4 and aggregated them at above 0.1 mg/ml, whereas no significant difference between the circular dichroism spectra of DnaK at pH 4 and 7 was observed. These results indicate that the cytosolic proteins, including DnaK displayed on the cell wall, cause the lactic acid bacterium to adhere to the yeast.

Enhanced antibody production following intermediate addition based on flux analysis in mammalian cell continuous culture.

Generally, mammalian cells utilize glucose and glutamine as primary energy sources. To investigate the effect of energy sources on metabolic fluxes and antibody production, glucose- or glutamine-limited serum-free continuous culture of hybridoma 3A21 cells, which produce anti-ribonuclease A antibody, was carried out. The cell volume and dry cell weight were evaluated under various steady-state conditions. The specific consumption and production rates were evaluated on the basis of dry cell weight. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate causes the specific ATP and antibody production rates to decrease. The fluxes between malate and pyruvate also decreased with the increase in specific growth rate. To increase the ATP production rate under steady-state conditions by the enhancement of fluxes between malate and pyruvate, the reduced metabolic fluxes were increased by an intermediate (pyruvate, malate, and citrate) addition. As a result, higher specific ATP and antibody production rates were achieved following the intermediate addition at a constant dilution rate.

Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag).

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

Effect of CO2 concentration on the growth and exopolysaccharide production of Bifidobacterium longum cultivated under anaerobic conditions.

For substantial cell growth and exopolysaccharide (EPS) production of Bifidobacterium longum JBL05, anaerobic conditions (dissolved oxygen concentrations below 0.05 ppm) and the supplement of CO2 (> or = 20%) were required. Under these conditions, B. longum JBL05 produced EPS amounts of up to 2.9 g/l.