Effect of chlorhexidine on molecular weight distribution of fructans produced by fructosyltransferase in solution and immobilized on surface

The effect of chlorhexidine (CHX), a potent antibacterial agent, was tested on the molecular weight distribution (MWD) of fructans synthesized by cell-free fructosyltransferase (FTF) in solution in comparison to FTF immobilized onto hydroxyapatite (HA). Size-exclusion chromatography (SEC) analysis has shown that cell-free FTF, both in solution and immobilized on HA, produces both low MW (1.9-2.2 kDa) and high MW (913-1047 kDa) fructans. CHX at a concentration of 0.02% altered the MWD of the fructans by reducing the polydispersity ratio and changing the MWD of the fructans synthesized both by immobilized FTF and by FTF in solution. These changes of the fructans in the presence of CHX adds a new prospective to the anticaries effect of CHX in addition to its antibacterial properties.     

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.     

Immobilization of fructosyltransferase by chitosan and alginate for efficient production of fructooligosaccharides

The effective system of reusing mycelial fructosyltransferase (FTase) immobilized with two polymers, chitosan and alginate were evaluated for continuous production of fructooligosaccharides (FOS). The alginate beads were successfully developed by maintaining spherical conformation of using 0.3% (w/v) sodium alginate with 0.1% (w/v) of CaCl₂ solution for highest transfructosylating activity. The characteristics of free and immobilized FTase were investigated and results showed that optimum pH and temperature of FTase activity were altered by immobilized materials. A successive production of FOS by FTase entrapped alginate beads was observed at an average of 62.96% (w/w) up to 7 days without much losing its activity. The data revealed by HPLC analysis culminate 67.75% (w/w) of FOS formation by FTase entrapped alginate beads and 42.79% (w/w) by chitosan beads in 36 h of enzyme substrate reaction.     

RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression in Streptococcus mutans

Glucosyltransferases (Gtfs) and fructosyltransferase (Ftf), and the exopolysaccharides they produce, facilitate bacterial adherence and biofilm formation, and enhance the virulence of Streptococcus mutans. In this study, we used continuous chemostat cultures and reporter gene fusions to study the expression of ftf and gtfBC in response to carbohydrate availability and pH, and to asses the role of a protein similar to catabolite control protein A (CcpA), RegM, in regulation of these genes. Expression of ftf was efficient at pH 7.0 and 6.0, but was repressed at pH 5.0 under glucose-excess conditions. At pH 7.0, ftf expression was 5-fold lower under glucose-limiting conditions than in cells growing with an excess of glucose. Expression of gtfBC was also sensitive, albeit to a lesser extent, to pH and glucose availability. Inactivation of regM resulted in decreases of as much as 10-fold in both ftf and gtfBC expression, depending on growth conditions. These findings reinforce the importance of pH and carbohydrate availability for expression of two primary virulence attributes of S. mutans and reveal a critical role for RegM in regulation of expression of both gtfBC and ftf.     

Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii

The binding of salivary amylase to Streptococcus gordonii has previously been shown to involve a 20-kDa amylase-binding protein (AbpA). S. gordonii also releases an 82-kDa protein into the supernatant that binds amylase. To study this 82-kDa component, proteins were precipitated from bacterial culture supernatants by the addition of acetone or purified amylase. Precipitated proteins were separated by SDS-PAGE and transferred to a sequencing membrane. The P2 kDa band was then sequenced, yielding a 25 N-terminal amino acid sequence, CGFIFGRQLTADGSTMFGPTEDYP. Primers derived from this sequence were used in an inverse PCR strategy to clone the full-length gene from S. gordonii chromosomal DNA. An open reading frame of 1959 bp was noted that encoded a 652 amino acid protein having a predicted molecular mass of 80 kDa. The first 24 amino acid residues were consistent with a hydrophobic signal peptide, followed by a 25 amino acid N-terminal sequence that shared identity (24 of 25 residues) with the amino acid sequence of purified AbpB. The abpB gene from strains of S. gordonii was interrupted by allelic exchange with a 420-bp fragment of the abpB gene linked to an erythromycin cassette. The 82-kDa protein was not detected in supernatants from these mutants. These abpB mutants retained the ability to bind soluble amylase. Thus, AbpA, but not AbpB, appears sufficient to be the major receptor for amylase binding to the streptococcal surface. The role of AbpB in bacterial colonization remains to be elucidated.     

群体感应(QS)在牙菌斑形成中的研究进展

形成牙菌斑的生物膜中存在大量微生物,各种微生物通过高丝氨酸内酯(AHL)或寡肽等不同的信号分子产生群体感应(QS),形成的群体感应使各种微生物建立了区系平衡,对龋齿及牙周炎等口腔疾病的治疗产生严重影响。因组成生物膜的各种微生物对抗生素的敏感性和耐受性有显著差异,因此在治疗中增加了对宿主免疫的相应要求。为进一步探讨QS系统在牙菌斑形成中的作用特点,本文就牙菌斑的形成与抗药机制、群体感应系统及其信号分子、群体感应系统的抑制因子和研究展望进行综述。     

Production of fructosyltransferase by <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. ATCC 20524 in batch and two-step batch cultures

A comparison of fructosyltransferase (EC 2.4.1.9) production by Aureobasidium sp. ATCC 20524 in batch and two step batch cultures was investigated in a 1-l stirred tank reactor using a sucrose supply of 200 g/l. Results showed that the innovative cultivation in two step of Aureobasidium sp. produced more fructosyltransferase (FFase) than the single batch culture at the same sucrose concentration with a maximal enzyme production of 523 U/ml, which was 80.5% higher than the one obtained in the batch culture. The production of fructooligosaccharides (FOSs) was also analyzed; their concentration reached a maximum value of 160 g/l the first day in the two-step culture and 127 g/l in the single-batch mode. The use of the two-step batch culture with Aureobasidium sp. ATCC 20524 in allowing the microorganism to grow up prior to the induction of sucrose (second step), proved to be a powerful method for producing fructosyltransferase and FOSs.     

细菌生物膜与龋齿相关性的研究进展

牙菌斑是由多种微生物在牙面上沉积,有机基质互相集聚、交联而形成的生物膜结构,生物膜中微生物相互依存、相互竞争,构成了复杂的微生态关系。牙菌斑生物膜的形成是导致龋齿重要过程。本文综述了细菌生物膜与龋齿发生的关系,以期为龋齿的预防与治疗提供新的思路。     

Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes

Bacterial fructosyltransferase enzymes belonging to glycoside hydrolase family 68 (GH68) are not known to require a metal cofactor. Here, we show that Ca2+ ions play an important structural role in the Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes. Analysis of the Bacillus subtilis Lev 3D structure [Meng, G. and Futterer, K. (2003) Nat. Struct. Biol. 10, 935-941] has provided evidence for the presence of a bound metal ion, most likely Ca2+. Characterization of site-directed mutants in the putative Ca2+ ion-binding sites of Lb. reuteri Lev and Inu revealed that the Inu Asp520 and Lev Asp500 residues play an important role in Ca2+ binding. Sequence alignments of family GH68 proteins showed that this Ca2+ ion-binding site is (largely) present only in proteins of Gram-positive origin.     

益生菌防治龋病的研究进展

随着人们生活水平日益提高,口腔健康越来越受到关注。近年来,益生菌对口腔菌群和宿主健康的调节作用已成为广泛研究的话题。已有证据表明,益生菌可以竞争性地抑制口腔中致病细菌的黏附和定植,分泌抗菌物质以减少病原菌。本文综述了相关益生菌对变形链球菌这一公认的龋齿病原菌的作用,包括:(1)生物表面活性剂、细菌素、胞外多糖等益生菌代谢产物对变形链球菌牙菌生物膜的抑制作用;(2)刺激宿主免疫反应,调节口腔菌群组成;(3)下调或阻断变形链球菌细胞中与黏附在牙齿表面形成生物膜相关的基因表达。     

Inhibitory effect of zingiber officinale towards Streptococcus mutans virulence and caries development: in vitro and in vivo studies

Background

Streptococcus mutans is known as a key causative agent of dental caries. It metabolizes dietary carbohydrate to produce acids which reduce the environmental pH leading to tooth demineralization. The ability of this bacterium to tolerate acids coupled with acid production, allows its effective colonization in the oral cavity leading to the establishment of highly cariogenic plaque. For this reason, S. mutans is the only bacterium found in significantly higher numbers than other bacteria in the dental plaque. The aim of this study was to evaluate the effect of crude extract and methanolic fraction of Z. officinale against S. mutans virulence properties.

Results

We investigated in vitro and in vivo activity of crude extract and methanolic fraction at sub- MIC levels against cariogenic properties of S. mutans. We found that these extracts strongly inhibited a variety of virulence properties which are critical for its pathogenesis. The biofilm formation in S. mutans was found to be reduced during critical growth phases. Furthermore, the glucan synthesis and adherence was also found to be inhibited. Nevertheless, the insoluble glucan synthesis and sucrose dependent adherence were apparently more reduced as compared to soluble glucan synthesis and sucrose- independent adherence. Biofilm architecture inspected with the help of confocal and scanning electron microscopy, showed dispersion of cells in the treated group as compared to the control. The Quantitative Real Time PCR (qRT-PCR) data had shown the down regulation of the virulence genes, which is believed to be one of the major reasons responsible for the observed reduction in the virulence properties. The incredible reduction of caries development was found in treated group of rats as compared to the untreated group which further validate our in vitro data.

Conclusion

The whole study concludes a prospective role of crude extract and methanolic fraction of Z. officinale in targeting complete array of cariogenic properties of S. mutans, thus reducing its pathogenesis. Hence, it may be strongly proposed as a putative anti- cariogenic agent.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0320-5) contains supplementary material, which is available to authorized users.     

Effects of mutating putative two-component systems on biofilm formation by Streptococcus mutans UA159

Streptococcus mutans has at least six pairs of open reading frames that are homologous to bacterial two-component regulatory systems. Putative response regulators from five out of six of these pairs were successfully mutated by insertion of a kanamycin resistance marker and the effects of inactivation of the genes on the ability of the cells to form biofilms in an in vitro model were assessed. Disruption of the response regulators of four systems had no effect on biofilm formation, whereas disruption of one response regulator caused a substantial decrease in biofilm formation as compared to the wild-type S. mutans.     

Biodegradation of acid blue-15, a textile dye,by an up-flow immobilized cell bioreactor

Acid blue-15, a complex and resonance-stabilized triphenylmethane (TPM) textile dye, resistant to transformation, was decolorized/degraded in an up-flow immobilized cell bioreactor. A consortium comprised of isolates belonging to Bacillus sp., Alcaligenes sp. and Aeromonas sp. formed a multispecies biofilm on refractory brick pieces used as support material. The TPM dye was degraded to simple metabolic intermediates in the bioreactor with 94% decolorization at a flow rate of 4 ml h^–1.     

Enrichment and characterization of histones by two-dimensional hydroxyapatite/reversed-phase liquid chromatography-mass spectrometry

Here we report a novel two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) method that combines offline hydroxyapatite (HA) chromatography with online reversed-phase liquid chromatography-mass spectrometry (HA/RP LC-MS). The 2D LC-MS method was used to enrich and characterize histones and their posttranslational modifications. The 2D HA/RP LC-MS approach separates histones based on their relative binding affinity to DNA and relative hydrophobicity. HA/RP separations showed improvement in the number of histone isoforms detected as compared with one-dimensional RP LC-MS of acid-extracted histones. The improved histone fractionation resulted in better detection of lower abundant histone variants as well as their posttranslationally modified isoforms. Histones eluted from the HA/RP in the following order: H1, H2A/H2B heterodimers followed by H3/H4 heterotetramers, as predicted from their spatial organization in nucleosomes for binding affinity to DNA. Comparison between HA-purified and acid-extracted histones revealed similar histone profiles with the exception that the HA fractions showed a greater number of H1 isoforms. Two elution conditions were also examined: batch elution and salt gradient elution. Although both elution techniques were able to fractionate the histones sufficiently, the salt gradient approach has the most potential for purification of selected histone isoforms.     

Antimicrobial and antibiofilm activity of pleurocidin against cariogenic microorganisms

Dental caries is a common oral bacterial infectious disease of global concern. Prevention and treatment of caries requires control of the dental plaque formed by pathogens such as Streptococcus mutans and Streptococcus sobrinus. Pleurocidin, produced by Pleuronectes americanus, is an antimicrobial peptide that exerts broad-spectrum activity against pathogenic bacteria and fungi. Moreover, pleurocidin shows less hemolysis and is less toxic than other natural peptides. In the present study, we investigated whether pleurocidin is an effective antibiotic peptide against common cariogenic microorganisms and performed a preliminary study of the antimicrobial mechanism. We assayed minimal inhibitory concentration (MIC), minimal bactericide concentration (MBC) and bactericidal kinetics and performed a spot-on-lawn assay. The BioFlux system was used to generate bacterial biofilms under controllable flow. Fluorescence microscopy and confocal laser scanning microscopy (CLSM) were used to analyze and observe biofilms. Scanning electron microscopy was used to observe the bacterial membrane. MIC and MBC results showed that pleurocidin had different antimicrobial activities against the tested oral strains. Although components of saliva could affect antimicrobial activity, pleurocidin dissolved in saliva still showed antimicrobial effects against oral microorganisms. Furthermore, pleurocidin showed a favorable killing effect against BioFlux flow biofilms in vitro. Our findings suggest that pleurocidin has the potential to kill dental biofilms and prevent dental caries.     

Effects of oxygen on the growth and metabolism of Actinomyces viscosus

Abstract Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h⁻¹) with exposure to variable amounts of oxygen. The Y_glucose increased from 62.5 g · mol⁻¹ under anaerobic conditions to 149 g · mol⁻¹ under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [¹⁴C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.     

Identification of archaeal rDNA from subgingival dental plaque by PCR amplification and sequence analysis

A PCR assay for the amplification of small subunit ribosomal DNA (SSU rDNA) of Euryarchaea was developed and used to detect archaeal rDNA in 37 (77%) out of 48 pooled subgingival plaque samples from 48 patients suffering from periodontal disease. One major group of cloned periodontal sequences was identical to Methanobrevibacter oralis and a second minor group to Methanobrevibacter smithii. These two groups and a third novel group were found to be more than 98% similar to each other over an 0.65-kb segment of the 16S rRNA gene sequenced. M. oralis was found to be the predominant archaeon in the subgingival dental plaque. Phylogenetic analysis of partial SSU rDNA sequences revealed evidence for a distinct cluster for human and animal Methanobrevibacter sp. within the Methanobacteriaceae family.     

Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor.

Matriptase is an epithelial-derived, integral membrane, trypsin-like serine protease. We have shown previously that matriptase exists both in complexed and noncomplexed forms. We now show that the complexed matriptase is an activated, two-chain form, which is inhibited in an acid-sensitive, reversible manner through binding to its cognate, Kunitz-type inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Conversely, the majority of the noncomplexed matriptase is a single-chain zymogen, which lacks binding affinity to HAI-1, suggesting that matriptase, similar to most other serine proteases, is activated by proteolytic cleavage at a canonical activation motif. We have now generated mAbs specific for the conformational changes associated with the proteolytic activation of matriptase. Using these mAbs, which specifically recognize the two-chain form of matriptase, we demonstrate that matriptase is transiently activated on 184A1N4 human mammary epithelial cell surfaces following their exposure to serum. The ability of serum to activate matriptase is highly conserved across reptilian, avian, and mammalian species. This serum-dependent activation of matriptase on epithelial cell surfaces is followed by ectodomain shedding of both matriptase and its Kunitz-type inhibitor.     

Effects of fibrin on the integration hydroxyapatite coating implants: experimental study in a rabbit model

The purpose of this study was to investigate the effects of the addition of fibrin (SAF) to titanium alloy implants coated with hydroxyapatite (HAP) on osteogenesis in rabbits. A titanium (Ti) alloy implant was inserted into the femoral neck of twenty-four adult rabbits. Six rabbits were included on each of the following groups: Ti control, HAP-coated Ti module, HAP-coated Ti module with added fibrin glue and Ti module also with added fibrin glue. After seven weeks, bone growth was examined radiographically and by histo-morphometry. The SAF/HAP mixture did caused to a significant increase in bone growth compared to the other groups. The addition of fibrin did not result in an increase in new-bone growth and increase the formation of fibrous tissue in contact with the implant. We concluded that SAF did not demonstrate osteoinductive properties.