Reduction of saliva-promoted adhesion of Streptococcus mutans MT8148 and dental biofilm development by tragacanth gum and yeast-derived phosphomannan

The aim of this study was to investigate materials which reduce saliva-promoted adhesion of Streptococcus mutans onto enamel surfaces, and their potential in preventing dental biofilm development. The effects of hydroxyapatite (HA) surface pretreatment with hydrophilic polysaccharides on saliva-promoted S. mutans adhesion in vitro and de novo dental biofilm deposition in vivo were examined. Saliva-promoted adhesion of S. mutans MT8148 was significantly reduced by pretreatment of the HA surface with tragacanth gum (TG) and yeast-derived phosphoglycans. Extracellular phosphomannan (PM) from Pichia capsulata NRRL Y-1842 and TG reduced biofilm development on lower incisors in plaque-susceptible rats when administered via drinking water at concentrations of 0.5% and 0.01%, respectively. The inhibitory effect of TG on de novo dental biofilm formation was also demonstrated when administered via mouthwash in humans. It is concluded that TG and yeast-derived PM have the potential for use as anti-adherent agents and are effective in reducing de novo dental biofilm formation.     

A high molecular mass cranberry constituent reduces mutans streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite

Previous investigations showed that a high molecular mass, non-dialyzable material (NDM) from cranberries inhibits the adhesion of a number of bacterial species and prevents the co-aggregation of many oral bacterial pairs. In the present study we determined the effect of mouthwash supplemented with NDM on oral hygiene. Following 6 weeks of daily usage of cranberry-containing mouthwash by an experimental group (n = 29), we found that salivary mutans streptococci count as well as the total bacterial count were reduced significantly (ANOVA, P < 0.01) compared with those of the control (n = 30) using placebo mouthwash. No change in the plaque and gingival indices was observed. In vitro, the cranberry constituent inhibited the adhesion of Streptococcus sobrinus to saliva-coated hydroxyapatite. The data suggest that the ability to reduce mutans streptococci counts in vivo is due to the anti-adhesion activity of the cranberry constituent.     

Inhibition of Mg2+-dependent adhesion of polymorphonuclear leukocytes by serum hemopexin: differences in divalent-cation dependency of cell adhesion in the presence and absence of serum

Circulating and nonadherent polymorphonuclear leukocytes (PMNs) become activated to attain adhesive state in an integrin-dependent manner by various stimuli, and perform a variety of microbicidal functions such as phagocytosis and superoxide production. We found that, in the absence of serum, a physiological concentration of hemopexin has a strong inhibitory action on Mg(2+)-dependent adhesion of PMA-activated PMNs to fibrinogen- and serum-coated surfaces. Under these conditions, Ca(2+) had no effect on Mg(2+)-dependent adhesion or the adhesion-inhibitory activity of hemopexin. In contrast, PMNs suspended in serum containing sufficient amounts of hemopexin to inhibit adhesion showed marked adherence, which was inhibited by EGTA. Next, we prepared a small-molecule fraction of serum by ultrafiltration followed by boiling. PMA-activated PMNs was found to adhere in the presence of both hemopexin and the small-molecule fraction, and the adhesion was enhanced by exogenous Ca(2+). EGTA abolished the effect of the small molecule fraction. The data suggest that serum contains adhesion-promoting factor(s) which allows PMNs to adhere despite the presence of hemopexin and that Ca(2+) is required for adhesion-promoting activity. Further study of hemopexin may provide clues for new therapeutic strategies aimed at interfering with PMN adhesion to control inflammation and tissue injury.     

Synthesis of chitooligosaccharide derivative with quaternary ammonium group and its antimicrobial activity against Streptococcus mutans

A derivative of chitooligosaccharide (COS) with quaternary ammonium functionality was synthesized and characterized by means of FT-IR and NMR spectroscopy. Its amtimicrobial activity was evaluated against Streptococcus mutans, which is a principal etiological agent of dental caries in humans. Introduction of quaternary ammonium group to COS has been easily accomplished by coupling of glycidyl trimethylammonium chloride (GTMAC) to COS in aqueous solution without an additional catalyst. The degree of substitution (%), as determined by (1)H NMR, of GTMAC to the COS increased up to 116% at 70 degrees C for 24h. The resulting COS-GTMAC exhibited the growth inhibition of above 80% against S. mutans after 5h, whereas the COS showed the growth inhibition of about 10%. It was found that antimicrobial activity of the COS could be considerably enhanced by the introduction of quaternary ammonium functionality.     

Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.     

Effect of carbohydrates on fructosyltransferase expression and distribution in Streptococcus mutans GS-5 biofilms

Streptococcus mutans produces a fructosyltransferase (FTF) enzyme, which synthesizes fructan polymers from sucrose. Fructans contribute to the virulence of the biofilm by acting as binding sites for S. mutans adhesion and as extracellular nutrition reservoir for the oral bacteria. Antibodies raised against a recombinant S. mutans FTF were used to test the effect of glucose, fructose, and sucrose on FTF expression in S. mutans GS-5 biofilms. Biofilms formed in the presence of fructose and glucose showed a higher ratio of FTF compared to biofilms formed in the presence of sucrose. Confocal laser scanning microscopy images of S. mutans biofilms indicated a carbohydrate-dependent FTF distribution. The layer adjacent to the surface and those at the liquid interface displayed high amounts cell-free FTF with limited amount of bacteria while the in-between layers demonstrated both cell-free FTF and cells expressing cell-surface FTF. Biofilm of S. mutans grown on hydroxyapatite surfaces expressed several FTF bands with molecular masses of 160, 125, 120, 100, and 50 kDa, as detected by using FTF specific antibodies. The results show that FTF expression and distribution in S. mutans GS-5 biofilms is carbohydrate regulated.     

Purification and Characterization of a Surface Protein from Lactobacillus fermentum 104R That Binds to Porcine Small Intestinal Mucus and Gastric Mucin

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.     

Protein-mediated adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium

The mechanism of adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium was investigated. Adhesion inhibition tests involving chelators, monosaccharides, periodate and concanavalin A and the use of bacteria grown in the presence of tunicamycin failed to clarify the adhesive mechanism. Washed bacterial cells had reduced adhesive capacity, except in the presence of spent broth culture supernatant fraction or cell washings. Spent culture supernatant fractions of erythrosine-supplemented broth did not enhance adhesion of washed cells. The adhesion-promoting factor(s) in the spent broth culture supernatant fractions and cell washings bound to both bacterial and epithelial cell surfaces, but did not promote adhesion of two other Lactobacillus strains which were not of mouse origin, thereby indicating host specificity for the adhesion-promoting activity. Chemical characteristics of the adhesion-promoting factor were determined by pretreatment of the dialysis retentate of spent broth culture supernatant fractions with proteolytic enzymes, concanavalin A-Sepharose or periodate before the adhesion assay. The adhesin was non-dialysable, pronase-sensitive, heat sensitive at 100 degrees C, had no affinity for concanavalin A-Sepharose and contained no carbohydrate groups active in the adhesion process. The protein profiles of dialysis retentates of spent broth culture supernatant fractions after bacterial growth in the absence and presence of erythrosine were determined by 2-dimensional SDS-PAGE. Gel filtration by HPLC was used for purification of an adhesion-promoting fraction. The host-specific adhesion of L. fermentum strain 737 was mediated by a protein, with an Mr of 12-13000, that was not detectable in cells grown in the presence of erythrosine. A model for the mode of binding of the adhesin to host epithelia and bacterial surfaces is proposed.     

The role of fructans on dental biofilm formation by Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus

Dental plaque biofilm plays a pivotal role in the progression of dental diseases. Polysaccharides are of great importance in the ecology of the dental biofilm. We studied the effect of fructans, glucans and a mixture of both fructans and glucans, synthesized in situ by immobilized fructosyltransferase or glucosyltransferase, on the adhesion of Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus to hydroxyapatite beads coated with human saliva (sHA). The adhesion of A. viscosus to sHA was found to be fructan-dependent. Adhesion of both S. sobrinus and S. mutans was found to be mediated mainly by glucans, while the adhesion of S. gordonii was found to be both glucan- and fructan-dependent. Treatment with fructanase prior to A. viscosus adhesion resulted in a significant reduction in adhesion to sHA, while adhesion of S. sobrinus, S. mutans and S. gordonii was slightly influenced by fructanase treatment. Treatment with fructanase after adhesion of S. gordonii to sHA resulted in a significant reduction in their adhesion to sHA. Our results show that fructans may play a role in the adhesion and colonization of several cariogenic bacteria to sHA, thus contributing to the formation of dental plaque biofilm.     

大蒜素抗变异链球菌的致龋效果

目的 研究大蒜素对口腔变异链球菌生长及其菌斑生物膜粘附的抑制作用。方法 二倍稀释法梯度稀释测最小抑菌浓度（minimum inhibitory concentration,MIC）,将MIC以上2个梯度浓度对应的培养物涂布于BHI培养基上进行次代培养获得最低杀菌浓度（minimum bactericidal concentration,MBC）;酶标仪测A值观察不同浓度大蒜素抑菌效应;抑制产酸试验观察抑制细菌产酸效应;结晶紫法研究亚抑菌浓度提取物对变异链球菌粘附能力及生物膜总量的影响;采用激光共聚焦荧光显微镜（laser scanning confocal microscopy,LSCM）观察常态牙菌斑生物膜生长过程中及药物处理后牙菌斑生物膜中死菌和活菌的构成,研究其对牙菌斑生物膜结构和活性的影响。结果 抑菌试验中,得到大蒜素MIC为12.8 mg/L,MBC为25.8 mg/L。MIC及亚抑菌浓度抑菌试验显示均有一定的抑菌性,抑制率为2.17%~67.12%,并且抑菌性与浓度梯度成正相关。产酸试验显示24 h内大蒜素明显抑制细菌产酸（P<0.01）,细菌粘附试验结果显示大蒜素在MIC时生物膜的生成速度最慢,生物膜的总量最低（P<0.01）。共聚焦荧光显微镜可见大蒜素组随药物浓度增加,菌斑生物膜较薄,绿色的活菌及团块明显减少,抑制生物膜的生长。结论 大蒜素对变异链球菌生长、产酸与粘附有一定抑制作用。     

黄连素对变异链球菌抑制作用的体外研究

目的分析黄连素对体外变异链球菌生长、产酸、粘附的影响,探讨其防龋作用。方法采用微量肉汤稀释法进行最小抑菌浓度(MIC)的测定;然后通过试管粘附法测定不同浓度药液对变异链球菌粘附作用的影响;最后计算不同浓度药液作用24h后pH值的变化。结果黄连素对变异链球菌的MIC为1.25mg/mL,MBC为5.00mg/mL。实验组对变异链球菌的粘附及产酸的抑制作用随着药物浓度的增加而增强,与阴性对照组相比差异有统计学意义(P0.05)。结论黄连素可抑制体外变异链球菌的生长、产酸及粘附。     

Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities

Tumor cell adhesion to the extracellular matrix is an important consideration in tumor metastasis. Recent results show that multiple adhesion-promoting domains for melanoma cells can be purified from proteolytic digests of fibronectin [McCarthy, J. B., Hagen, S. T., & Furcht, L. T. (1986) J. Cell Biol. 102, 179-188]. Monoclonal antibodies were generated against a tryptic/catheptic 33K heparin binding fragment of fibronectin derived from the carboxyl terminal of the A chain. This region contains a tumor cell adhesion-promoting domain(s). The amino-terminal sequence was determined for this fragment, as well as a tryptic 31K fragment which is located to the carboxyl-terminal side of the 33K heparin binding fragment in A chains of fibronectin. The partial sequence data demonstrate that arginyl-glycyl-aspartyl-serine (RGDS) or the related arginyl-glutamyl-aspartyl-valine (REDV) is not present in the 33K heparin binding fragment, confirming earlier results which demonstrated that cells adhere to this fragment by an RGDS-independent mechanism. Two monoclonal antibodies, termed AHB-1 and AHB-2, recognized epitopes common to heparin binding fragments derived from the carboxyl terminus of both the A and B chains of fibronectin. Monoclonal antibody AHB-2 inhibited melanoma adhesion to the 33K heparin binding fragment of fibronectin in a concentration-dependent manner, whereas monoclonal antibody AHB-1 had no effect on adhesion to this fragment. Neither monoclonal antibody inhibited adhesion to intact fibronectin. However, monoclonal AHB-2 potentiated the inhibitory effect of suboptimal levels of exogenous RGDS on cell adhesion to intact fibronectin. AHB-2 recognized an epitope common to both the A- and B-chain carboxyl-terminal heparin binding region of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake, transport and regulation of JBP485 by PEPT1 in vitro and in vivo

Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide with anti-hepatitis activity that has been chemically synthesized. Previous experiments in rats showed that JBP485 was well absorbed by the intestine after oral administration. The human peptide transporter (PEPT1) is expressed in the intestine and recognizes compounds such as dipeptides and tripeptides. The purposes of this study were to determine if JBP485 acted as a substrate for intestinal PEPT1, and to investigate the characteristics of JBP485 uptake and transepithelial transport by PEPT1. The uptake of JBP485 was pH dependent in human intestinal epithelial cells Caco-2. And JBP485 uptake was also significantly inhibited by glycylsarcosine (Gly-Sar, a typical substrate for PEPT1 transporters), JBP923 (a derivative of JBP485), and cephalexin (CEX, a β-lactam antibiotic and a known substrate of PEPT1) in Caco-2 cells. The rate of apical-to-basolateral transepithelial transport of JBP485 was 1.84 times higher than that for basolateral-to-apical transport. JBP485 transport was obviously inhibited by Gly-Sar, JBP923 and CEX in Caco-2 cells. The uptake of JBP485 was increased by verapamil but not by cyclosporin A (CsA) and inhibited by the presence of Zn²⁺ or the toxic metabolite of ethanol, acetaldehyde (AcH) in Caco-2 cells. The in vivo uptake of JBP485 was increased by verapamil and decreased by ethanol in vivo, which was consisted with the in vitro study. PEPT1 mRNA levels were enhanced after exposure of the cells to JBP485 for 24 h, compared to control. In conclusion, JBP485 was actively transported by the intestinal oligopeptide transporter PEPT1. This mechanism is likely to contribute to the rapid absorption of JBP485 by the gastrointestinal tract after oral administration.     

Effects of Lactobacillus reuteri PTA 5289 and L. paracasei DSMZ16671 on the Adhesion and Biofilm Formation of Streptococcus mutans

Probiotics have decreased the counts of salivary mutans streptococci (MS) in clinical studies. The aim of this study was to compare the effects of Lactobacillus reuteri PTA 5289 and L. paracasei DSMZ16671 on the adhesion of a reference strain and a clinical isolate of Streptococcus mutans and on the counts of MS in a biofilm. The adhesion of S. mutans Ingbritt and the clinical isolate S. mutans 2366 to a smooth glass surface and saliva-coated hydroxyapatite (SHA) were studied in the presence of and without the lactobacilli. A three-species biofilm formed on saliva-coated hydroxyapatite discs was used in the biofilm experiments. The lactobacilli did not affect adhesion to the glass surface but interfered with binding to SHA. No effects of the lactobacilli were detected on the MS levels in the three-species biofilms. The results of the SHA binding experiments best reflected the results of the existing clinical studies.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Enhanced adhesion of Streptococcus mutans to hydroxyapatite after exposure to saliva

Streptococcus mutans cells form robust biofilms on human teeth and are strongly related to caries incidents. Hence, understanding the adhesion of S. mutans in the human oral cavity is of major interest for preventive dentistry. In this study, we report on atomic force microscopy–based single‐cell force spectroscopy measurements of S. mutans cells to hydroxyapatite surfaces. We observe for almost all measurements a significant difference in adhesion strength for S. mutans as well as for Staphylococcus carnosus cells. However, the increase in adhesion strength after saliva exposure is much higher for S. mutans cells compared to S. carnosus cells. Our results demonstrate that S. mutans cells are well adapted to their natural environment, the oral cavity. This ability promotes the biofilm‐forming capability of that species and hence the production of caries‐provoking acids. In consequence, understanding the fundamentals of this mechanism may pave a way towards more effective caries‐reducing techniques.     

Diet-induced elevation of circulating HSP70 may trigger cell adhesion and promote the development of atherosclerosis in rats

Although accumulating evidence indicates that heat shock protein 70 (HSP70) could be secreted into plasma and its levels have been found to have an ambiguous association with atherosclerosis, our knowledge for the exact role of circulating HSP70 in the development of atherosclerosis is still limited. In the present study, we report an adhesion-promoting effect of exogenous HSP70 and evaluate the potential involvement of elevated circulating HSP70 in the development of atherosclerosis. Time-dependent elevation of plasma HSP70 was found in diet-induced atherosclerotic rats, whose effect was investigated through further in vitro experiments. In rat aortic endothelial cell (RAEC) cultures, exogenous HSP70 incubation neither produced cell injuries by itself nor had protective effects on cell injuries caused by Ox-LDL or homocysteine. However, exogenous HSP70 administration could lead to a higher adhesion rate between rat peripheral blood monocytes (PBMCs) and RAECs. This adhesion-promoting effect appeared only when PBMCs, rather than RAECs, were pretreated with HSP70 incubation. PBMCs in an HSP70 environment released more IL-6 to supernatant, which subsequently up-regulated the expression of ICAM-1 in RAECs. These results indicate that the diet-induced elevation of circulating HSP70 could trigger cell adhesion with the help of IL-6 as a mediator, which provides a novel possible mechanism for understanding the role of circulating HSP70 in the pathogenesis of atherosclerosis.