Exopolysaccharide Treatment Research Articles

Effects of elicitors from culture filtrate of Fusarium solani CL105 on flavonoid production of Scutellaria baicalensis calli.

Endophytic fungi can promote secondary metabolite accumulation in medicinal plants. Previously, we observed that the culture filtrate of Fusarium solani CL105 promoted flavonoid production in Scutellaria baicalensis calli. However, the active ingredients and mechanisms associated with this secondary metabolite accumulation remain unclear. This study evaluates the effects of different elicitors from the culture filtrate of F. solani CL105 namely, exopolysaccharide (EPS), exoprotein (EP), and other parts (OP), on the flavonoid production in S. baicalensis calli by HPLC. Subsequently, the underlying mechanism of EPS induced flavonoid production in S. baicalensis calli was revealed by transcriptomics and RT-PCR. The results indicated a significant increase in flavonoid production in S. baicalensis calli following treatment with EPS. Baicalin (1.40 fold), wogonoside (1.91 fold), and wogonin (2.76 fold) were most significantly up-regulated compared with the control. Transcriptome analysis further revealed up-regulation of key enzyme genes (CHS, CHI, FNS, and F6H) involved in flavonoid synthesis after 5 days of EPS treatment. Moreover, the expression of GA2ox and CYP707A-genes involved in gibberellin acid (GA) and abscisic acid biosynthesis (ABA), respectively-were significantly up-regulated. The expression levels of certain transcription factors, including MYB3, MYB8, and MYB13, were also significantly higher than in controls. Our results indicated that EPS was a main active elicitor involved in promoting flavonoid production in S. baicalensis calli. We postulated that EPS might stimulate the expression of MYB3, MYB8, MYB13, GA2ox, and CYP707A, leading to markedly upregulated CHS, CHI, FNS, and F6H expression levels, ultimately promoting flavonoid synthesis. This study provides a novel avenue for large-scale in vitro production of flavonoids in S. baicalensis.

Limosilactobacillus reuteri L26 BiocenolTM and its exopolysaccharide: Their influence onrotavirus-induced immune molecules inenterocyte-like cells.

This study aimed toevaluate the immunomodulatory effect ofthe probiotic Limosilactobacillus reuteri L26 BiocenolTM (L26) and its purified exopolysaccharide (EPS) with respect toantiviral innate immune response. Inour experiment, weused porcine epithelial IPEC-J2 cells asamodel ofthe intestinal barrier inahomologous infection byporcine RotavirusAstrain OSU6 (RVA). The production ofselected molecules ofnon-specific humoral immunity was evaluated atthe mRNA level. The EPS alone significantly increased the level ofinterferon λ3 (IFN-λ3) mRNA inthe non-infected IPEC-J2 cells (P<0.001). Wealso tested whether the treatment ofIPEC-J2 cells byL26 orEPS influences the replication ofRVA byvirus titration and real-time PCR. Wefound that apre-treatment incombination with subsequent continuous stimulation has noinfluence onthe RVA replication. However, both treatments significantly decreased the RVA-induced production ofIFN-λ3 (P<0.05) and the "SOS" cytokine interleukin6 (IL-6; P<0.01), already atthe transcription level. Inaddition, the EPS treatment resulted insignificantly increased IL-10 mRNA inthe RVA-infected cells. Insummary, weassume animmunoregulatory potential ofL.reuteri L26 BiocenolTM and its EPS inthe local intestinal antiviral immune response.

In vitro investigation of the effects of Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 exopolysaccharides on tight junction damage caused by influenza virus infection.

It is a problem that influenza virus infection increases susceptibility to secondary bacterial infection in lungs leading to lethal pneumonia. We previously reported that exopolysaccharides (EPS) derived from Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1) could prevent against influenza virus infection followed by secondary bacterial infection in vitro. Therefore, the present study assessed whether EPS derived OLL1073R-1 protects the alveolar epithelial barrier disfunction caused by influenza virus infection. After A549 cells treated with EPS or without EPS were infected influenza virus A/Puerto Rico/8/34 (IFV) for 12h, the levels of tight junction genes expression and inflammatory genes expression were measured by reverse transcription polymerase chain reaction. As results, EPS treatment could protect against low-titer IFV infection, but not high-titer IFV infection, followed by suppression of the increased expression of inflammatory cytokine gene levels and recovery of the decrease in the expression level of ZO-1 gene that was caused by low-titer IFV infection, leading to an improvement trend in the barrier function. Our findings showed that EPS derived from OLL1073R-1 could inhibit low-titer IFV infection leading to maintenance of the epithelial barrier function through the suppression of inflammatory cytokine genes expression.

Assessment of microbial exopolysaccharides for improving soil fertility and crop yield in semi-arid India

Soil degradation and poor fertility are major concerns in semi-arid regions of India. Microbial exopolysaccharides (EPS) have demonstrated potential in mitigating abiotic and biotic stresses in these areas. However, the studies on the impact of EPS on crop performance and soil fertility under natural field conditions remain limited. To address this knowledge gap, a field experiment was conducted during the rainy season of 2019-20 and 2020-21 at ICAR-NIASM, Baramati, Maharashtra, to examine the effects of microbial EPS on average crop yields of soybean and maize, microbial colonization status in soybean and maize, and soil fertility parameters in basaltic soils of India. Seeds inoculated with Rhizobium or nitrogen fixers and 10 kg ha-1 EPS resulted in a significant increase in soybean and maize yields by 57.7% and 44.8%, respectively, compared to the control. Additionally, the Rhizobium treated seeds with EPS 10 kg ha-1 showed improvements in soil organic carbon, dehydrogenase activity, and nitrogen content by 15.5%, 214.5%, and 70.0%, respectively, in soybean compared to the control. Conversely, the application of microbial EPS did not cause any significant change in soil pH, E.C., or available P, K, Fe, Mn, and Zn contents in both crops. While the presence of EPS within the rhizosphere of soybean actively promoted nodule formation, it also enhanced microbial colonization within the rhizosphere of both soybean and maize. Moreover, the best subset regression analysis revealed that EPS altered the soil parameters for the yield of both crops. The findings of this study suggest that EPS treatment, in combination with Rhizobium or nitrogen fixers, can significantly enhance crop yield, promote nodule formation in soybean, and improve microbial colonization within the rhizosphere of both soybean and maize. These results highlight the potential of microbial EPS as a viable and sustainable solution to address soil degradation and poor fertility in semi-arid regions of India.

Exopolysaccharides from endophytic Glutamicibacter halophytocota KLBMP 5180 functions as bio-stimulants to improve tomato plants growth and salt stress tolerance

Microbial exopolysaccharides (EPSs) can promote plants growth and protect them against various abiotic stresses, but the role of actinobacteria-produced EPSs in plant growth promoting is still less known. Here, we aim to explore the effect of EPSs from an endophyte Glutamicibacter halophytocota KLBMP 5180 on tomato seeds germination and seedlings growth under salt stress. Our study revealed that 2.0 g/L EPSs resulted in increased seed germination rate by 23.5 % and 11.0 %, respectively, under 0 and 200 mM NaCl stress conditions. Further pot experiment demonstrated that EPSs significantly promoted seedlings growth under salt stress, with increased height, root length and fibrous roots number. Plant physiological traits revealed that EPSs increased chlorophyll content, enhanced the activity of antioxidant enzymes, soluble sugar, and K+ concentration in seedlings; malondialdehyde and Na+ contents were reduced. Additionally, auxin, abscisic acid, jasmonic acid, and salicylic acid were accumulated significantly in seedlings after EPSs treatment. Furthermore, we identified 1233 differentially expressed genes, and they were significantly enriched in phytohormone signal transmission, phenylpropanoid biosynthesis, and protein processing in endogenous reticulum pathways, etc. Our results suggest that KLBMP 5180-produced EPSs effectively ameliorated NaCl stress in tomato plants by triggering complex regulation mechanism, and showed application potentiality in agriculture.

Promotion of Growth of Alfalfa by Erwinia persicina Cp2 Exopolysaccharides under NaCl Stress

Soil salinity poses a significant challenge to plant growth and agricultural productivity. Research demonstrated the potential of exopolysaccharides (EPS) to enhance plant growth and improve resistance to abiotic stress. Nevertheless, the precise regulatory mechanism by which EPS mediates salt tolerance in alfalfa (Medicago sativa) remains largely unexplored. To investigate the protective effects of EPS from Erwinia persicina Cp2 in enhancing salt stress tolerance in alfalfa, a hydroponic experiment was conducted on the ‘Aohan’ cultivar of alfalfa, and changes in germination rate, biomass, chlorophyll content, electrolyte leakage (EL), hydrogen peroxide (H2O2), malondialdehyde (MDA), proline (Pro), soluble sugar (SS), soluble protein (SP), and activities of superoxide dismutase (SOD) and peroxidase (POD) were analyzed with and without Cp2 EPS under 75 mmol/L NaCl conditions. The results show that the exposure of alfalfa to salt conditions seriously inhibited its germination and growth. However, it is interesting that the application of Cp2 EPS greatly alleviated the damage of salt stress on alfalfa and promoted the germination of alfalfa as well as the root length, seedling length, fresh weight, and dry weight of the plants. In addition, the increases in MDA, H2O2 content, and EL rate caused by salt stress were inhibited after Cp2 EPS treatment, while chlorophyll, Pro, SP, and SS contents as well as SOD and CAT activities were increased. Therefore, Cp2 EPS can significantly alleviate the toxic effect of 75 mmol/L NaCl stress on alfalfa, and comprehensive analysis shows that 1.5 g/L Cp2 EPS had the best alleviating effect on alfalfa at this salt stress concentration. This study lays a practical and theoretical foundation for the development of biogenic agents used to alleviate the growth of alfalfa in saline alkali soil.

Exopolysaccharide from Lacticaseibacillus rhamnosus induces IgA production in airways and alleviates allergic airway inflammation in mouse model.

The currently observed high prevalence of allergic diseases has been associated with changes in microbial exposure in industrialized countries. Defined bacterial components represent a new strategy for modulating the allergic immune response. We show that intranasal administration of exopolysaccharide (EPS) isolated from Lacticaseibacillus (L.) rhamnosus LOCK900 induces TGF-β1, IgA, and regulatory FoxP3+ T-cells in the lungs of naïve mice. Using the ovalbumin mouse model, we demonstrate that intranasal administration of EPS downregulates the development of allergic airway inflammation and the Th2 cytokine response in sensitized individuals. At the same time, EPS treatment of sensitized mice, similar to EPS-induced responses in naïve mice, significantly increased the level of total, OVA-specific, and also bacteria-specific IgA in bronchoalveolar lavage and the number of IgA-producing B-cells in the lung tissue of these mice. Thus, EPS derived from L. rhamnosus LOCK900 can be considered a safe candidate for preventing the development of allergic symptoms in the lungs of sensitized individuals upon exposure to an allergen.

Probiotic-derived exopolysaccharide interferes with T H2 priming and inhibits allergic inflammation

Abstract Allergic asthma is an increasing burden in industrialized countries, where treatments are limited to targeting symptoms and end-stage effectors rather than root causes. In recent years, the use of prophylactic, inhaled probiotic cocktails has shown promise in preventing allergic asthma. However, the mechanisms by which such probiotics prevent allergic symptoms and the exact bacterial product(s) responsible for probiotic activity remain to be elucidated. In this study, we focused on exopolysaccharide (EPS) from Bacillus subtilis, a ubiquitous soil bacterium that is commonly found as a dietary supplement. We found that in a mouse model of allergic airway disease, EPS treatment, prior to allergic sensitization and challenge, limits lung mucus hypersecretion and eosinophilia. Furthermore, EPS treatment inhibited accumulation of T H2, but not T H1 and T H17 subsets. To determine whether EPS was affecting T H2 cell priming by dendritic cells (DCs), we compared the composition and phenotype of DCs in the lung and mediastinal lymph nodes (medLN) after EPS treatment. Surprisingly, pre-treatment with EPS induced DC maturation as measured by an increase in costimulatory molecules and a decrease in the ability to uptake allergen and migrate to the medLN. DC maturation by EPS was TLR4-dependent, as TLR4 KO DCs did not respond to EPS. Together, our data suggest that EPS inhibits allergic sensitization by inducing DC maturation prior to allergen exposure, thereby leading to decreased T H2 priming and allergic inflammation. Thus, we provide a potential mechanism by which inhalation of a single probiotic-derived molecule can prevent the development of allergic inflammation. Supported by grants from NIH (3R01 AI125644-05S1, 1F31 HL156659-01A1)

Amelioration of graft versus host disease in humanized mice by exopolysaccharide from a commensal bacterium

Abstract Graft versus host disease (GVHD) is the major and often lethal complication of allogenic bone marrow transplantation. Prophylactic regimens for GVHD are given as standard pre-transplantation therapy; however, up to 60% of these patients develop GVHD, and require additional immunosuppressive intervention. We identified a probiotic exopolysaccharide (EPS) that induces inhibitory macrophages and tolerogenic dendritic cells (DCs) that inhibit T cell proliferation. We tested if EPS-treated human DCs, derived in vitrofrom CD34 +cord blood cells, would inhibit allogeneic T cell proliferation in a mixed lymphocyte reaction (MLR) and found that they did, demonstrating that EPS can induce tolerogenic DCs in vitro.We translated these studies in vivousing a humanized mouse model of aGvHD and demonstrated that EPS-treated human DCs administered at the time of transfer of human peripheral blood mononuclear cells (hPBMCs) significantly increased survival of these mice compared to mice given PBS-treated DCs, or no DCs. Several subsets of DCs have been described, e.g., cDCs, moDCs and plasmacytoid DCs, each with different phenotypes and functions. Using labeled EPS, we showed that the tolerogenic CD34-derived DCs generated by EPS treatment are conventional DCs (cDCs). We found that EPS binds to two cDC populations, conventional DC1 (cDC1) and conventional DC2 (cDC2), but only cDC1 were inhibitory in our MLR cultures. Our data indicate that EPS-DCs, potentially cDC1, can be used as a cell-based therapy to mitigate aGvHD in humans. NIH 1R41AI155281-01

Antioxidant activity and cytotoxicity of exopolysaccharide from mushroom Hericium coralloides in submerged fermentation.

Mushrooms of the genus Hericium spp. represent a series of delicious edible mushrooms with medicinal value. Here, for the first time, the species native to Iran, the mushroom Hericium coralloides, was collected in Mazandaran province, identified, and registered with the NCBI under accession number MW136052. The production of exopolysaccharides (EPS) in submerged culture was optimized using the response surface method. Among the physicochemical and culture medium conditions tested, rotation speed and concentration of maltose and peptone of soybean significantly affected the production of EPS. The proposed model predicts maximum EPS production (0.13 g/L) at 50 g/L maltose, 3 g/L soy peptone, and 1 g/L yeast extract, pH = 6.5, 200 rpm, inoculum at 5% v/v, and 22 °C. The molecular weight of the EPS chains was 413 and 1578 Da. EPS has antioxidant action (EC50 = 6.59 mg/mL) and cytotoxic activity against cancer cells. The viability of AGS and MKN-45 cancer cell lines declined to 20 and 30% after 48 h of the EPS treatment. H. coralloides EPS could be considered a natural dietary anti-cancer supplement. Further studies are necessary to understand the mechanism of the H. coralloides EPS activity on the cell cycle of cancer cells and to prove its action in vivo.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13399-022-03386-0.

Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway

BackgroundBone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms.MethodshDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria.ResultsThe calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture.ConclusionsThe stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.

Exopolysaccharide from Lactobacillus rhamnosusZFM231 alleviates DSS‐induced colitis in mice by regulating gut microbiota

The exopolysaccharides (EPS) produced by Lactobacillus and other probiotics are associated with many benefits, such as immune regulation, antioxidant properties, antitumor effect, and regulation of intestinal microbe homeostasis. In the present study, the modulatory effect of EPS produced by Lactobacillus rhamnosus ZFM231 on the intestinal flora of mice with inflammatory bowel disease induced by dextran sulfate solution was investigated. Results indicated that weight loss, colonic length, the disease activity index score and colonic tissue damage in mice were significantly improved by EPS treatment. Compared with the model group, in the EPS-treated group, the diversity of and the composition of gut microbiota at both phylum and genus levels were found to recover to the levels of normal group, indicating the effective modulation on gut microbiota by EPS; short-chain fatty acids, including acetic acid, propionic acid and butyric acid produced by intestinal microbial metabolism, increased significantly; the level of anti-inflammatory factor transforning growth factor-β significantly increased and the level of pro-inflammatory factor tumor necrosis factor-α significantly decreased in the colonic cells of EPS-treated mice. It is clear that EPS produced by L. rhamnosus ZFM231 could find application in functional foods with the property of anti-ulcerative colitis. The experimental results provide new insights into the probiotic effect of EPS. © 2022 Society of Chemical Industry.

Exopolysaccharides from Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 prevent influenza virus infection and attenuate secondary bacterial infection risk.

The present study assessed the inhibitory action of exopolysaccharides (EPS) produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 against influenza virus infection followed by secondary bacterial infection. We found that the presence of 200 or 400 μg ml-1 of EPS significantly protected against influenza virus infection in a dose-dependent manner when A549 cells were treated with EPS before infection but not after it. The expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1), an adhesion molecule for bacteria adherence, on A549 cells was significantly enhanced during influenza virus infection compared with viral-non-infected A549 cells. However, this upregulated CEACAM-1 expression was significantly decreased by EPS treatment before viral infection in association with the reduction in the virus titre in A549 cells. In a bacterial adhesion assay using Staphylococcus aureus, the bacterial adherence to viral-infected A549 cells was significantly greater than that to viral-non-infected A549 cells, and the increased bacterial adherence induced by influenza virus infection tended to be decreased by EPS treatment before the infection. Our findings show that EPS treatment before viral infection can inhibit influenza virus infection and alleviate secondary bacterial infection through decreased CEACAM-1 expression.

Abstract P5-12-10: A commensal’s exopolysaccharide inhibits breast cancer proliferation in vitro but promotes tumor growth in vivo

Abstract Background: Recent decades have started to realize the importance of the microbiome in cancer initiation, progression and response to therapy. Recent findings suggest that both the local and distal microbiome may influence tumor growth. Although many studies focused on the gut microbiome, the breast itself also possesses a unique microbiome which differs between healthy and breast cancer patients, and between tumor subtypes. However, it is poorly understood whether these bacteria in the gut or the breast microenvironment may interact with breast cancer cells and if that interaction alters tumor growth. Bacteria produce metabolites and other signaling molecules to alter the host’s response. We have identified and purified an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis, which was shown to act directly on the host’s innate immune cells to protect against various inflammatory diseases. This study aims to investigate if, and how the bacterial product (EPS) alters proliferation and tumor growth of breast cancer cells.Methods: Cell proliferation of bulk cells was conducted on PBS or EPS treated breast cancer cells (T47D, MDA-MB-468, MDA-MB-453, HCC1428, BT549, MDA-MB-231, ZR-75-30, and MCF-7) using trypan blue exclusion and the XTT assay. Flow cytometry was used to assess apoptosis with Annexin V, and cell cycle progression with propidium iodide. Cancer stem cell survival was assessed using the mammosphere forming assay. CRISPR/Cas9-mediated knockout of the Toll-like-receptor 4 (TLR4) was used to assess the requirement of TLR4 signaling on EPS-mediated phenotypes. RNA-sequencing was performed on both EPS-sensitive and resistant breast cancer cell lines after treatment with PBS or EPS for 20 hours. T47D xenografts studies were performed in vivo using EPS pretreatment and continual treatment via i.p. injection.Results: EPS inhibited proliferation of some breast cancer cell lines (T47D, MDA-MB-468, MDA-MB-453, HCC1428) in a concentration dependent manner while having little effect on others (BT549, MDA-MB-231, ZR-75-30, MCF7). EPS acted via distinct mechanisms depending on the cell line, increasing apoptosis in MDA-MB-468 cells while inducing G0/G1 cell cycle arrest in T47D cells. Although EPS directly inhibited bulk cell proliferation, the cancer stem cell population of T47D cells was increased by EPS. EPS is known to require TLR4 signaling to modulate immune cells. CRISPR/Cas9 knockout of TLR4 demonstrated that it was not required for EPS-mediated inhibition of T47D breast cancer cells, suggesting a novel mechanism of action in this cell line. RNA-sequence analysis showed that EPS altered interferon signaling and cell cycle progression pathways. Interferon signaling activates the STAT family of transcription factors, and western blots confirmed that 3h of EPS treatment induced STAT1 phosphorylation and p21 accumulation, indicating that EPS activated this pathway. Contrary to in vitro proliferation data, in vivo results showed that EPS-treated cells formed significantly larger tumors compared to PBS controls (273mg vs 59mg). The slope of tumor growth was also significantly faster in the EPS-treated group. Conclusions: These findings demonstrated the multifacet of EPS’s effect on breast cancer cells. EPS inhibits the proliferation of certain breast cancer cells in vitro, possibly via activation of STAT1 and p21 signaling, but also increases survival of cancer stem cells and promotes more aggressive tumor growth in vivo. This highlights the complexity of the influence of commensal bacteria on breast cancer growth, which can be highly context-dependent. Deeper understanding of this complex interplay between the host-microbe-tumor will be vital to the development of novel bacterial strategies in the future. Citation Format: Mai Rachel Nguyen, Katherine L Knight, Clodia Osipo. A commensal’s exopolysaccharide inhibits breast cancer proliferation in vitro but promotes tumor growth in vivo [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-10.

An exopolysaccharide from Bacillus subtilis alleviates airway inflammatory responses via the NF-κB and STAT6 pathways in asthmatic mice.

Bacillus subtilis is an intestinal probiotic for immune homeostasis and its exopolysaccharide (EPS) is known to possess anti-inflammatory and antioxidant properties. The underlying mechanisms are not yet fully understood. In the present study, we investigated the effects of the EPS (50, 100, 200 mg/kg) on airway inflammation in asthmatic mice. Our results showed that EPS treatment of asthmatic mice significantly alleviated pathological damage in the lungs, remarkably decreased the counts of total inflammatory cells including lymphocytes, and eosinophils in the bronchoalveolar lavage fluid (BALF) and reduced indexes of oxidative damage. Moreover, the expression of type II T-helper cell (Th2) cytokines (interleukin- (IL)4 and -5) subsequent to EPS treatment was found to be dramatically down-regulated in a concentration-dependent manner. Additionally, the EPS treatment reduced JAK1, STAT6 and nuclear factor-κB (NF-κB) expression in the lungs of asthmatic mice. Taken together, these results suggest that the EPS from B. subtilis alleviates asthmatic airway inflammation, which involves the reduction in reactive oxygen species (ROS) and the down-regulation of the STAT6 and NF-κB inflammatory pathways, which can further reduce Th2 cytokine expression and eosinophilic inflammation. Thus, our findings provide a potential mechanism through which the EPS mitigates asthma, suggesting that the EPS could be a potential source of an anti-asthmatic drug.

Gut-Liver Axis reveals the protective effect of exopolysaccharides isolated from Sporidiobolus pararoseus on alcohol-induced liver injury

• A study about Exopolysaccharides (EPS) from S. pararoseus against ALD. • The effects of EPS on ALD in oxidative stress, inflammation, and lipid accumulation. • The effects of EPS on ALD based on liver-gut axis. • The mechanism analysis of EPS to protect intestinal barrier function. Chronic and excessive alcohol consumption can lead to the development of alcoholic liver disease (ALD), which is characterized by oxidative damage, inflammation, and impaired intestinal barrier function. To evaluate the effect of exopolysaccharides (EPS) from Sporidiobolus pararoseus on ALD, alcohol was used in this study to cause liver damage in mice. Then, the antioxidant and anti-inflammation activities and protective effect of EPS on the intestinal barrier function were further explored in an ALD model. The results show that the histopathological change and liver injury were significantly improved in the EPS group. EPS treatment significantly increased the levels of superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) (P < 0.05), and weak reduced the elevation of malondialdehyde (MDA) induced by ALD. Several inflammatory markers, including tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β were reduced significantly after the intervention by EPS (P < 0.01). In addition, EPS also inhibited the injury of the small intestine and regulated the intestinal barrier function. A mechanism study shows that EPS restrained oxidative stress and protected intestinal barrier function by activating Nrf2/HO-1/PPAR-γ signaling pathway and upregulating the expression of ZO-1 and occludin, indicating that EPS showed protective activity against ALD through the gut-hepatic axis. Therefore, EPS has potential as a protective agent against ALD.

Amelioration of Graft-versus-Host Disease by Exopolysaccharide from a Commensal Bacterium.

Acute graft-versus-host disease (aGvHD) is a severe, often lethal, complication of hematopoietic stem cell transplantation, and although prophylactic regimens are given as standard pretransplantation therapy, up to 60% of these patients develop aGvHD, and require additional immunosuppressive intervention. We treated mice with a purified probiotic molecule, exopolysaccharide (EPS) from Bacillus subtilis, shortly before and after induction of aGvHD and found that, whereas only 10% of control mice survived to day 80, 70% of EPS-treated mice survived to 80 d. EPS treatment of donor-only mice resulted in ∼60% survival. Using a biosensor mouse model to assess inflammation in live mice during aGvHD, we found that EPS prevented the activation of alloreactive donor T cells. In vitro, EPS did not affect T cells directly but, instead, induced bone marrow-derived dendritic cells (BMDCs) that displayed characteristics of inhibitory dendritic cells (DCs). Development of these BMDCs required TLR4 signaling through both MyD88 and TRIF pathways. Using BMDCs derived from IDO knockout mice, we showed that T cell inhibition by EPS-treated BMDCs was mediated through the suppressive effects of IDO. These studies describe a bacterial molecule that modulates immune responses by inducing inhibitory DCs in a TLR4-dependent manner, and these cells have the capacity to inhibit T cell activation through IDO. We suggest that EPS or EPS-treated DCs can serve as novel agents for preventing aGvHD.

Antioxidant and Immunomodulatory Properties of Partially purified Exopolysaccharide from Lactobacillus Casei Isolated from Chinese Northeast Sauerkraut

ABSTRACT Background Exopolysaccharides (EPS) from Lactobacillus spp. have been found to have biological activities. Our previous work demonstrated the antibiofilm activity of EPS from Lactobacillus casei NA-2 (L.casei NA-2) isolated from northeast Chinese sauerkraut (Suan Cai). The present study has focussed on the antioxidant and immunomodulatory activities of the EPS in vitro. Methods Antioxidant properties of the EPS were evaluated by the radical-scavenging activities in vitro. The immunomodulatory effects of EPS were assayed by measuring nitric oxide (NO), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and reactive oxygen species (ROS) in RAW 264.7 macrophages, and the mechanism was investigated through NF-κB and JNK. Result EPS contains 88% total sugar, with the molecular weights (Mw) of 1.3 × 106 Da, 6.4 × 105 Da, 2.0 × 105 Da, and 1.4 × 104 Da. EPS showed antioxidant activity by scavenging hydroxyl radicals (42% at 1.2 mg/mL), superoxide radicals (76% at 100 µg/mL), and DPPH (80% at 10 mg/mL); and did not affect the proliferation of unstimulated or lipopolysaccharide (LPS)-induced RAW 264.7 cells at the concentrations ranging from 31.25 to 500 µg/mL. Results showed EPS promoted the production of ROS and TNF-α involved in NF-κB p65 and JNK signaling pathways in unstimulated RAW 264.7 cells. On the other hand, the levels of NO and iNOS were reduced after EPS treatment in LPS-induced RAW 264.7 cells. Conclusion Our results showed the protective effect against oxidative damage and potential immunomodulatory and anti-inflammatory properties of EPS from Lactobacillus casei NA-2.

Abstract 6101: Exopolysaccharide secreted by Bacillus subtilis alters breast cancer cell growth and stemness

Abstract Recently, multiple studies have shown that a unique microbiome is associated with the breast, with distinct differences in microbial compositions between healthy and patients with benign versus malignant breast cancer tissue. It is well known that bacteria can produce metabolites and other signaling molecules to alter the host's response. However, little is known about whether bacteria in the breast microenvironment interact with breast cancer cells and if that interaction changes cancer progression. We aim to investigate if, and how commensal bacteria and/or their bacterial products alter growth and metastasis of breast cancer cells. Upon initial analysis of sequencing data deposited by Heiken et al, we identified the presence of the gut commensal bacterium Bacillus subtilis in some breast tissues of patients with breast cancer. Previous studies have shown that exopolysaccharide (EPS) secreted by B. subtilis can induce tolerance in the myeloid compartment to protect the host from inflammatory diseases such as diarrhea, sepsis, and graft-versus-host-disease. We first examined the effect of EPS on breast cancer proliferation by performing in vitro proliferation assays on a diverse panel of breast cancer cell lines, including hormone receptor positive and negative lines. Results showed that EPS suppressed proliferation of multiple breast cancer cell lines (T47D, MDA-MB-468, MDA-MB-453, HCC1428) in a concentration dependent manner while other cell lines were unaffected (BT549, MDA-MB-231, ZR-75-30, MCF7). Annexin V apoptosis assay and cell cycle staining indicated that EPS acted via distinct mechanisms depending on the cell line, increasing apoptosis in MDA-MB-468 cells while inducing G0/G1 cell cycle arrest in T47D cells. In addition to proliferation by bulk tumor cells, the survival of breast cancer stem cells emerges as a significant challenge to breast cancer therapies as these stem-like cells are thought to repopulate the tumor and lead to recurrence. We tested if EPS treatment alters cancer stem cell survival using the mammosphere forming assay, a 3D culture system which specifically selects for cells with stem cell characteristics. Our preliminary mammosphere results show that pretreatment with EPS increased cancer stem cell survival in T47D and MCF7 cells. Overall, we found that EPS secreted by B. subtilis can directly suppress the proliferation of some breast cancer cell lines but enhance cancer stem cell survival. Thus, effects of B. subtilis on breast cancer cells are potentially multifaceted. Future work will focus on assessing the effect of EPS on breast tumor growth in vivo and elucidating the signaling mechanism by which EPS inhibits bulk cancer cells but stimulates cancer stem cells. Elucidating this mechanism would further our understanding of how local breast microbes may influence breast cancer growth and outcome. Citation Format: Mai Rachel Nguyen, Clodia Osipo, Katherine L. Knight. Exopolysaccharide secreted by Bacillus subtilis alters breast cancer cell growth and stemness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6101.

Characterisation and bioactivities of an exopolysaccharide from an Antarctic bacterium Shewanella frigidimarina W32–2

The continent of Antarctica breeds numerous special extremophiles that have adapted to the extreme variation of environment with a wide range of novel biodiversity. A cold-adapted bacterium Shewanella frigidimarina W32–2, isolated from Antarctic sediments, was identified to be a promising candidate of exopolysaccharides (EPS) production. In the present study, the physiochemistry and bioactivities of the EPS were investigated to promote its application in aquaculture. Physiological characterisation of this strain was firstly performed by means of combined API 20NE and API ZYM tests. A biochemical analysis revealed that the EPS contained a majority of carbohydrates (56.34 ± 1.29%), uronic acids (8.23 ± 0.55%) and high content of sulphate groups (35.43 ± 1.84%), which were also confirmed by the FTIR spectrum showing various functional groups with potential antioxidant activities. The bioactive properties of the EPS in vitro and in vivo were both evaluated. The effect of the EPS on the non-specific immunity of red tilapia via serum injection exhibited 100% of survival rate. Improvement of activities in different serum enzymes, including AKP, ACP, SOD, CAT and LZM, was observed generally at the early stage of EPS treatment (except LZM at later stage). This study explores novel EPS from an extremophile exhibiting powerful bioactivity with immense potential in aquaculture sustainability.