Extracellular Polysaccharides Matrix—An Often Forgotten Virulence Factor in Oral Biofilm Research

Oral diseases related to dental biofilms continue to afflict the majority of the world＇s population. Among them, dental caries continues to be the single most prevalent and costly oral infectious disease （Marsh, 2003; Dye et al., 2007）. Dental caries results from the interaction of specific bacteria with constituents of the diet within a dental biofilm known as plaque （Bowen, 2002）. Sucrose is considered to be the ＂arch criminal＂ from the dietary aspect because it serves as a substrate for synthesis of extracellular （EPS） and intracellular （IPS） polysaccharides in dental biofilm and is also fermentable （Bowen, 2002）.

Inhibitory Effect of Extracellular Polysaccharide EPS-II from Pseudoalteromonas on Candida adhesion to Cornea in vitro

Objective Fungal keratitis（FK） is a vision-threatening infection,whose treatment requires more effective and safer anti-fungal agent exploitation urgently.With this aim,we focused on the effect of an extracellular polysaccharide on fungal adhesion to human corneal epithelial cells.Methods We performed the cytotoxicity assays of the extracellular polysaccharide EPS-II from an antarctic bacterium Pseudoaltermonas and evaluated its inhibitory effect on Candida albicans cells＇ adherence to human corneal epithelial cells（HCECs）.Results EPS-II,which displayed minor cytotoxicity but also promoted proliferation of HCECs,could inhibit the adherence of yeast cells to HCECs in a dose-dependent manner.EPS-II could also suppress the subsequent PI3K/AKT signaling pathway,and thereby decrease the expression of early inflammatory cytokines.Conclusions ExtracellularpolysaccharideEPS-IIwassuggestedasanewnaturalagentforattenuatingFK.

Production and characterization of an extracellular polysaccharide of antarctic marine bacteria Pseudoalteromonas sp. S-15-13

Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseudoalteromonas sp. S - 15 - 13 were investigated, and the EPS was separated and purified for characterization analysis. The results showed that the optimal conditions for the EPS production were culture period, 56 h; growth temperature, 8 ℃ ; carbon source, 1.0% glucose; NaCI concentration, 3.0% ; pH 6.0 - 7.0. The EPS was purified by cold ethanol precipitation, proteins removal, ion exchange chromatography and gel chromatography technology. The molecular mass of EPS - H was 62 kDa as determined by the high performance gel permeation chromatography. Its sugar composition was a homopolymer of marmose analyzed by gas chromatograph spectroscopy. After repeated freezing and thawing of the bacteria hiomass in the presence of EPS, the bacterial growth was much higher than that observed after freezing in the absence of EPS and the difference augmented with the increase of freeze-thaw cycles. It is hypothesized that the adaptation of Pseudoalteromonas sp. S- 15 - 13 to the antarctic marine conditions, characterized by low temperature, high NaCl concentration and repeated freeze-thaw cycles, might be related to the EPS production ability.

Isolation, chemical characteristics and immunity activity of an extracellular polysaccharide EPS I isolated from Antarctic bacterium Pseudoalteromonas sp.S-15-13

A new extracelluar polysaccharide （EPS） was isolated and purified from Antarctic bacterium S-15-13, identified as Pseudoalteromonas sp. After being separated and purified by DEAE-Sephadex A-50 ionexchange and Sephadex G-100 gel chromatography, two mains fractions （EPS I and EPS Ⅱ ） were ob-tained. EPS I was composed of mannose, glucose and galactose with a molecular weight of 23kDa and EPS Ⅱ was composed of mannose only with a molecular weight of 62kDa. The effect of the polysaccharide EPS I on the cellular immune response of mice was investigated. Results demonstrated that EPS I could markedly facilitate lymphocyte proliferation, and might be a strong immunomodulator.

A Polysaccharide-Degrading Marine Bacterium Flammeovirga sp.MY04 and Its Extracellular Agarase System

Bacteria of the genus Flammeovirga can digest complex polysaccharides (CPs), but no details have been reported regarding the CP depolymerases of these bacteria. MY04, an agarolytic marine bacterium isolated from coastal sediments, has been identified as a new member of the genus Flammeovirga. The MY04 strain is able to utilize multiple CPs as a sole carbon source and grows well on agarose, mannan, or xylan. This strain produces high concentrations of extracellular proteins (490 mg L-1 ± 18.2 mg L-1 liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose. These proteins have an activity of 310 U mg-1 ± 9.6 U mg-1 proteins. The extracellular agarase system (EAS) in the crude extracellular enzymes contains at least four agarose depolymerases, which are with molecular masses of approximately 30-70 kDa. The EAS is stable at a wide range of pH values (6.0-11.0), temperatures (0-50℃), and sodium chloride (NaCl) concentrations (0- 0.9 mol L-1). Two major degradation products generated from agarose by the EAS are identified to be neoagarotetraose and neoagarohexaose, suggesting that β-agarases are the major constituents of the MY04 EAS. These results suggest that the Flammeovirga strain MY04 and its polysac-charide-degradation system hold great promise in industrial applications.

Metabolic activity of Streptococcus mutans biofilms and gene expression during exposure to xylitol and sucrose

The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media： Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy（CLSM）-based analyses of the microbial vitality, respiratory activity（5-cyano-2,3-ditolyl tetrazolium chloride, CTC） and production of extracellular polysaccharides（EPS） were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters： culture growth, vitality, CTC activity and EPS production. However,xylitol exposure caused a difference in gene expression compared to the control. Gtf C was upregulated only in the presence of xylitol.Under xylitol exposure, gtf B was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three.Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms.

Effect of fluid motion on colony formation in Microcystis aeruginosa

Microcystis aeruginosa, generally occurring in large colonies under natural conditions, mainly exists as single cells in laboratory cultures. The mechanisms involved in colony formation in Microcystis aeruginosa and their roles in algal blooms remain unknown. In this study, based on previous research findings that fluid motion may stimulate the colony formation in green algae, cul^are experiments were conducted under axenic conditions in a circular water chamber where the flow rate, temperature, light, and nutrients were controlled. The number of cells of Microcystis aeruginosa, the number of cells per colony, and the colonial characteristics in various growth phases were observed and measured. The results indicated that the colony formation in Microcystis aeruginosa, which was not observed under stagnant conditions, was evident when there was fluid motion, with the number of cells per largest colony reaching 120 and the proportion of the number of cells in colonial form to the total number of cells and the mean number of cells per colony reaching their peak values at a flow rate of 35 crn/s. Based on the analysis of colony formation process, fluid motion stimulates the colony formation in Microcystis aeruginosa in the lag growth phase, while flushes and disaggregates the colonies in the exponential growth phase. The stimulation effect in the lag growth phase may be attributable to the involvement of fluid motion in a series of physiological processes, including the uptake of trace elements and the synthesis and secretion of polysaccharides. In addition, the experimental groups exhibiting typical colonial characteristics in the lag growth phase were found to have higher cell biomass in the later phase.

An Inner Membrane Protein(Imp) of Xanthomonas oryzae pv. oryzicola Functions in Carbon Acquisition, EPS Production, Bacterial Motility and Virulence in Rice

Xanthomonas oryzae pv. oryzicola （Xoc） causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein （Imp）, showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R？imp was constructed in the wild-type RS105. The R？imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide （EPS） production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

Effects of nutrient profiles and light regime on the production of exopolysaccharide by Porphyridium cruentum

In order to increase the yield of polysaccharides from Porphyridium cruentum and the cell biomass, we investigated several important cultural conditions such as nutrient-salts, light irradiance as well as growth conditions in the flat plate photobioreacturs （FPPBR） with different light-path. We have found that the optimal （OCM II ） was a superior medium for extracellular polysaccharide production than the f/2 and Koch medium, and the polysaccharide production with OCM II was increased by 1.79 times and 1.62 times compared with that of f/2 and Koch medium; the yields of polysaccharides were also improved by optimizing the light intensity and irradiation length Furthermore, we significantly enhanced the yields of both the biomass and extracellular polysaccharidc using a small light-path photobioreactor, and about 2.1 times increase of polysaccharide production was observed in FPPBR-30 than in FPPBR-100. Overall, the process for polysaccharides production was improved and this would facilitate further studies on such polysaccharidcs such as their antiviral and antitumor activities.

Antibiofilm activity of 3,3'-diindolylmethane on Staphylococcus aureus and its disinfection on common food-contact surfaces

This study explored the antibiofilm efficacy of 3,3’-diindolylmethane(DIM)on Staphylococcus aureus and its disinfection on common food-contact surfaces.The minimum biofilm inhibitory concentration(MBIC)of DIM on S.aureus was 62.5μmol/L,while it did not impede the bacterial growth evaluated by growth curve and XTT reduction assay.DIM in the concentration range of 31.2-62.5μmol/L demonstrated a dose-dependent antibiofilm activity to S.aureus,as confirmed by light microscopic(LM),confocal laser scanning microscopic(CLSM),and scanning electron microscopic(SEM)analyses.At DIM of62.5μmol/L,the biomass of S.aureus biofilm was significantly reduced by 97%and its average thickness by 58%(P<0.05).DIM of 62.5μmol/L inhibited the bacterial initial adhesion and proliferation,as well as cell motility;the release of extracellular DNA(eDNA)and extracellular polysaccharide(EPS)were reduced by 75%and 69%,respectively.DIM exhibited a strong inhibition to S.aureus biofilm formation on common food-contact surfaces,including 304 stainless steel,glass,and polyvinyl chloride(PVC)but not disperse the mature biofilm.Overall,our investigation identified DIM as a promising antibiofilm agent and its suitability to prevent the biofilm formation of S.aureus on common food-contact surfaces utilized during food processing.

Screening of Toxin Mutant of Dickeya zeae and Its Biological Characters

[Objective]This study aimed to screen toxin mutant of Dickeya zeae(Erwinia chrysanthemi pv.zeae)and investigate its biological characters.[Method]We obtained a toxin mutant strain D.zeae Ech7-3-42 by using acridine orange as a mutagenic agent and compared their biological characteristics and virulence between the toxin mutant and wild strain.[Result]There was no significant difference in pectin lyase,protease,cellulase and the production of extracellular polysaccharide and lipopolysaccharide,but significant difference in toxin biological activities and virulence.Ech7-3-42 mutant did not produce toxin,as well as the loss of virulence on rice and HR on tobacco,but did not lose the ability to soft rot on potato.Mutant strain Ech7-3-42 can infect rice root and then enriched in the root neck and stalk,but it could not cause rice foot rot.Dickeya zeae(wild and mutant strain)could be detected by PCR in the root neck and below the 1-2 cm long stem area,but could not be detected in the leaves.[Conclusion]We believed that toxin may be one of the important factors for D.zeae virulence on rice.

Synthesis of L-Hexopyranosyl Fluorides Enabled by Radical Decarboxylative Fluorination: Assembly of a Pentasaccharide Repeating Unit Corresponding to Extracellular Polysaccharide S-88

L-Hexoses are key components of many biologically relevant natural products and pharmaceuticals.As rare sugars,L-hexoses are not readily obtained from natural sources.Access to L-hexose building blocks from commercially available and inexpensive D-sugars is highly desirable from the viewpoints of organic synthesis and drug discovery.As demonstrated by the convenient preparation of L-glucosyl,L-galactosyl,and L-mannosyl fluorides from readily availableβ-D-C-glucosyl,β-D-C-mannosyl,andβ-D-C-galactosyl derivatives,we describe a novel and efficient approach to the demanding L-glycosyl fluorides.

Highly active probiotic hydrogels matrixed on bacterial EPS accelerate wound healing via maintaining stable skin microbiota and reducing inflammation

Skin microbiota plays an important role in wound healing,but skin injuries are highly susceptible to wound infections,leading to disruption of the skin microbiota.However,conventional antibacterial hydrogels eliminate both probiotics and pathogenic bacteria,disrupting the balance of the skin microbiota.Therefore,it is important to develop a wound dressing that can fend off foreign pathogenic bacteria while preserving skin microbiota stability.Inspired by live bacteria therapy,we designed a probiotic hydrogel(HAEPS@L.sei gel)with high viability for promoting wound healing.Lactobacillus paracasei TYM202 encapsulated in the hydrogel has the activity of promoting wound healing,and the hydrogel matrix EPS-M76 has the prebiotic activity that promotes the proliferation and metabolism of Lactobacillus paracasei TYM202.During the wound healing process,HAEPS@L.sei gel releases lactic acid and acetic acid to resist the growth of pathogenic bacteria while maintaining Firmicutes and Proteobacteria balance at the phylum level,thus preserving skin microbiota stability.Our results showed that live probiotic hydrogels reduce the incidence of inflammation during wound healing while promoting angiogenesis and increasing collagen deposition.This study provides new ideas for developing wound dressings predicated on live bacterial hydrogels.

Feruloylated Arabinoxylans Are Oxidatively Cross- Linked by Extracellular Maize Peroxidase but Not by Horseradish Peroxidase

Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross- linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H2O2/peroxidase, O2/laccase, or reactive oxygen species acting non-enzymically, To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Crosslinking was suppressed by iodide, an H2O2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H2O2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [^3H]arabinoxylans in vitro only if H2O2 was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases （assayed on o-dianisidine） were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylancross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [^3H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.

Extracellular Polysaccharide from Rhizopus nigricans Inhibits Hepatocellular Carcinoma via miR-494-3p/TRIM36 Axis and Cyclin E Ubiquitination

Background and Aims:This study was designed to uncov-er the mechanism for extracellular polysaccharide(EPS1-1)-mediated effects on hepatocellular carcinoma(HCC)devel-opment.Methods:HCC cells were treated with EPS1-1,miR-494-3p mimic,sh-TRIM36,and pcDNA3.1-TRIM36.The levels of miR-494-3p and TRIM36 were measured in nor-mal hepatocytes,THLE-2,and HepG2 and HuH7HCC cell lines,along with the protein expression of cyclin D/E and p21.The proliferation,cell cycle,and apoptosis of HCC cells were assayed.The interactions between miR-494-3p and TRIM36,and between TRIM36 and cyclin E were assessed.Finally,the expression and localization of TRIM36 and cyclin E were monitored,and tumor apoptosis was detected,in tumor xenograft model.Results:EPS1-1 suppressed HCC cell proliferation and cyclin D/E expression and promoted apoptosis and p21 expression.miR-494-3p was upregulated and TRIM36 was downregulated in HCC cells.Transfection with miR-494-3p mimic or sh-TRIM36 facilitated HCC cell proliferation and the expression of cyclin D/E protein but they inhibited apoptosis and p21 expression in the pres-ence of EPS1-1.Overexpression of TRIM36 further con-solidated EPS1-1-mediated inhibition of HCC proliferation,cyclin D/E,and the promotion of apoptosis and p21 expres-sion.Those effects were reversed by miR-494-3p overex-pression.TRIM36 was a target gene of miR-494-3p,and TRIM36 induced cyclin E ubiquitination.EPS1-1 suppressed cyclin E expression,promoted TRIM36 expression and tu-mor apoptosis,all of which were abrogated by increasing the expression of miR-494-3p in vivo.Conclusions:EPS1-1 protected against HCC by limiting its proliferation and sur-vival through the miR-494-3p/TRIM36 axis and by inducing cyclin E ubiquitination.

Screening of probiotics with efficient α-glucosidase inhibitory ability and study on the structure and function of its extracellular polysaccharide

Inhibition ofα-glucosidase activity is an important strategy in lowering the concentration of blood sugar.In this paper,using domestic and foreign characteristic food-derived substances as the sources of lactobacillus,the performance of them were evaluated by measuring the strains’α-glucosidase inhibitory ability.Finally,the cell-free extracellular supernatants(CFS)of Lactobacillus rhamnosus LB1lac10 was determined to have the highest α-glucosidase inhibition ability.Based on the Nanopore third-generation sequencing technology platform,the genome of LB1lac10 was sequenced and functional gene annotation was performed.After that,the biological activity and structural composition of the exopolysaccharide produced by L.rhamnosus LB1lac10 were studied.The purified exopolysaccharide EPS1-1 also showed efficientα-glucosidase inhibitory ability.The structure and conformation characteristics of EPS1-1 were further analyzed.The EPS1-1 from L.rhamnosus LB1lac10 had a molecular weight of 88,650 Da,and it was mainly composed of mannose,glucuronic acid,glucose,xylose,galactose,and arabinose.From the FT-IR and NMR analyses,EPS1-1 had functional groups of a typical polysaccharide structure and contained two types of glycosidic bonds with α-configuration pyranose.The main glycosidic bond corresponded to→4)-α-D-Glcp-(1→,which might be an important reason why EPS1-1 could inhibit α-glycosidase.Thermodynamic studies showed that EPS1-1 had high heat resistance to meet the needs of food processing.The results suggested that L.rhamnosus LB1lac10 could be used as a potential probiotic to lower blood sugar,and the EPS1-1 has the potential to serve as a naturalα-glycosidase inhibitor to regulate the concentration of blood glucose.

Structure of an Extracellular Polysaccharide from a Strain of Lactic Acid Bacteria

A new extracellular polysaccharide (EPS-I) isolated and purified from Z_ 222 ,a strain of Lactic acid bacteria has been investigated. Sugar composition analysis,methylation analysis and 1H NMR and 13 C NMR spectroscopy reveal that the EPS-I is composed of a pentasaccharide repeating unit. The sequence of sugar residue was determined by using two-dimensional NMR spectroscopy,including heteronuclear multiple-bond correlation (HMBC) and nuclear overhauser effect spectroscopy (NOESY).

The production-influencing factors of extracellular polysaccharide(EPS)from a strain of lactic acid bacteria and EPS extraction

The influencing factors of extracellular polysac-charide(EPS)produced from a strain of lactic acid bacteria(LAB L15)were studied by using the phenol-H2SO4 method.It was demonstrated that the strain produced EPS at the most amount when it was incubated for 40-48 h and when the pH value was 4 under 30℃.Glucose was the most suitable carbon source for LAB-producing EPS.The rough EPS was obtained from L15 culture after centrifugation,dialysis,deprotein,decoloration,and ethanol-precipitation.The sample was at least composed of two polysaccharides that were completely different in molecular weight and the amount.The purified EPS was passed through the SephadexG-200 column and it showed that it was a sample purified by thin layer chromatography.

Nitrate removal and extracellular polymeric substances of autohydrogenotrophic bacteria under various pH and hydrogen flow rates

In recent years there has been an increasing interest in the use of autohydrogenotrophic bacteria to treat nitrate from wastewater. However, our knowledge about the characteristics of extracellular polymeric substances（EPS） releasing by these activities is not yet very advanced. This study aimed to investigate the change in EPS compositions under various p H values and hydrogen flow rates, taking into consideration nitrogen removal. Results showed that p H 7.5 and a hydrogen flow rate of 90 m L/min were the optimal operating conditions, resulting in 100% nitrogen removal after 6 hr of operation. Soluble and bound polysaccharides decreased, while bound proteins increased with increasing p H. Polysaccharides increased with increasing hydrogen flow rate. No significant change of bound proteins was observed at various hydrogen flow rates.

Purification and molecular weight distribution of a key exopolysaccharide component of Bacillus megaterium TF10

Extracellular polymeric substances（EPS） are organic metabolic compounds excreted by microorganisms. They largely impact microbial aggregate structures and functions.Extracellular polysaccharides（EP） in EPS are responsible for the formation of microbial aggregates. In this work, we successfully separated and characterized EP from EPS of the bacterium Bacillus megaterium TF10. Extraction of EP from EPS was optimized using Sevag＇s reagent. Chemical characteristics, functional groups, and molecular weight（MW） distribution of EP were compared with the harvested EPS and soluble microbial products（SMP）. We found that the polymers of lower MW and free proteins were successfully removed by Sevag＇s reagent. The higher MW components of EPS were predominantly polysaccharides,while the polymers of lower MW tended to secrete to the supernatant and were described as SMP. A part of the proteins in the EP was polysaccharide-bonded. Our results can be further used in elucidating the complex flocculation mechanisms in which EP play a major role.