氧对口腔链球菌产生过氧化氢的影响

目的观察环境中氧含量对口腔链球菌过氧化氢产生速率的影响。方法采用ABTS-HRP微量板法测定在不同氧含量条件下口腔链球菌过氧化氢产生的速率。结果口腔链球菌在严格厌氧条件下过氧化氢产生速率为9.29nmol/(min×109细胞);当氧含量增高,口腔链球菌过氧化氢合成速率加快;在不同氧含量的环境中口腔链球菌产生过氧化氢的速率差异存在显著性:有氧振荡培养>有氧静置培养>厌氧(P<0.05)。结论环境中氧含量是影响口腔链球菌过氧化氢产生速率的重要因素。     

Colonization and vertical transmission of Streptococcus mutans in Turkish children

The aim of the study was to establish the colonization of Streptococcus mutans and to determine the possibility of intra-familial transmission in a group of Turkish children and their parents. A total of 56 children participated in the study together with their parents (20 fathers and 49 mothers). Saliva samples were collected from the individuals and cultivated on S. mutans selective TYCSB agar. The typical isolates of S. mutans were identified by using classical microbiological methods, as well as molecular typing of S. mutans clones which was performed by using AP PCR with OPA5 primer for the detection of transmission. The vertical transmission of salivary S. mutans was detected among 14 mother-father-child, 35 mother-child (one twins) and 6 father-child combinations. The homologies of strain types were recorded as 24% and 16.6% for mother-child and father-child combinations, respectively. A significant positive correlation (p<0.001) was found between the infected children and their parents with high S. mutans counts.     

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.     

The effect of oxygen on the growth and acid production of Streptococcus mutans and Streptococcus sanguis

Abstract Pure cultures of Streptococcus mutans NCTC 10499 and Streptococcus sanguis ATCC10556 were grown in a glucose-limited chemostat under varying concentrations of oxygen in the gas phase. Both streptococci consumed large amounts of oxygen by the partial oxidation of sugars, thus maintaining an anaerobic environment. With increasing oxygen concentrations the degradation products from glucose become more oxidized. Ethanol gradually disappeared from the culture fluid while the acetate concentration increased. In the case of S. sanguis , the products became even more oxidized at higher oxygen concentrations, and carbon dioxide was formed instead of formate. Sudden increase in the oxygen concentration in the gas phase caused elevated oxygen tensions in the cultures, which led to a decrease in the growth rate of the streptococci.     

Cloning and characterization of an alpha-enolase of the oral pathogen Streptococcus mutans that binds human plasminogen

Streptococcus mutans is the etiologic agent of dental caries and is a causative agent of infective endocarditis. While the mechanisms by which S. mutans cells colonize heart tissue is not clear, it is thought that bacterial binding to extracellular matrix and blood conponents is crucial in the development of endocarditis. Previously, we have demonstrated that S. mutans cells have the capacity to bind and activate plasminogen to plasmin. Here we report the first cloning and characterization of an α-enolase of S. mutans that binds plasminogen. The functional identity of the purified recombinant α-enolase protein was confirmed by its ability to catalyze the conversion of 2-phosphoglycerate to phosphoenolpyruvate. The protein exhibited a K_m of 9.5 mM and a V_max of 31.0 mM/min/mg. The α-enolase protein was localized in the cytoplasmic, cell wall and extracellular fractions of S. mutans. Binding studies using an immunoblot analysis revealed that human plasminogen binds to the enolase enzyme of S. mutans. These findings identify S. mutans α-enolase as a binding molecule used by this oral pathogen to interact with the blood component, plasminogen. Further studies of this interaction may be critical to understand the pathogenesis of endocarditis caused by S. mutans.     

Dpr蛋白在寡发酵链球菌抗过氧化氢中的作用

摘要：【目的】寡发酵链球菌（Streptococcus oligofermentans）是从无龋人的口腔中分离到的一株链球菌,好氧条件下产生、同时也耐受高浓度（4.4 mmol/L）的过氧化氢。本研究探讨dpr基因对寡发酵链球菌抗过氧化氢的贡献。【方法】克隆和表达寡发酵链球菌dpr基因,分析Dpr蛋白的功能;构建寡发酵链球菌的dpr基因突变株,比较野生株和突变株对不同浓度过氧化氢的耐受程度;并将寡发酵链球菌dpr基因克隆到对过氧化氢耐受力低的变形链球菌中,分析其对变形链球菌过氧化氢耐受能力的影响。【结果】     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Electrophoretic studies of extracellular glucosyltransferases and fructosyltransferases from seventeen strains of Streptococcus mutans

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60.Abbreviations GTase glucosyltransferase - FTase fructosyltransferase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PAG-IEF polyacrylamide gel-isoelectric focussing - PAS periodic acid-Schiff     

Crystal structure of prephenate dehydrogenase from Streptococcus mutans

Prephenate dehydrogenase (PDH) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. This enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. The crystal structure of PDH from Streptococcus mutans in a complex with NAD⁺ shows that the enzyme exists as a homo-dimer, each monomer consisting of two domains, a modified nucleotide binding N-terminal domain and a helical prephenate C-terminal binding domain. The latter is the dimerization domain. A structural comparison of PDHs from mesophilic S. mutans and thermophilic Aquifex aeolicus showed differences in the long loop between β6 and β7, which may be a reason for the high K_m values of PDH from Streptococcus mutans.     

Kinetic characterization of P-type membrane ATPase from Streptococcus mutans

The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F₁F_o-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K_0.5=0.27 mM) and low (K_0.5=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg²⁺ saturating condition (high affinity site, K_0.5=0.10 mM, and low affinity site, K_0.5=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.     

An in vitro synergetic evaluation of the use of nisin and sodium fluoride or chlorhexidine against Streptococcus mutans

The objective of this study is to investigate the synergetic action between nisin and sodium fluoride or chlorhexidine against Streptococcus mutans, a primary cariogenic pathogen. In the antibacterial assay, a synergetic effect on S. mutans was found between nisin and sodium fluoride, but there was no interaction between nisin and chlorhexidine by the checkerboard, the fractional inhibitory concentration (FIC) and the fractional bactericidal concentration (FBC) tests. S. mutans survival rates showed a significant decline after treatment with a combination of nisin and sodium fluoride in a time-kill study. Scanning electron microscopy showed that the damage to S. mutans with the combined nisin and sodium fluoride treatment was the most severe among all of the different single and combined antimicrobial treatments. Furthermore, in the antibiofilm test, nisin in combination with sodium fluoride produced a stronger bactericidal effect on a S. mutans biofilm for 4 h and 16 h compared with sodium fluoride alone by confocal laser scanning microscopy. Nisin in combination with sodium fluoride exerted a high bactericidal effect on S. mutans and thereby has the potential to be used as an effective drug combination to prevent dental caries.     

Inhibition of proton-translocating ATPases of Streptococcus mutans and Lactobacillus casei by fluoride and aluminum

One of the major effects of fluoride on oral bacteria is a reduction in acid tolerance, and presumably also in cariogenicity. The reduction appears to involve transport of protons across the cell membrane by the weak acid HF to dissipate the pH gradient, and also direct inhibition of the F₁F₀, proton-translocating ATPases of the organisms, especially for Streptococcus mutans. This direct inhibition by fluoride was found to be dependent on aluminum. The dependence on aluminum was indicated by the protection against fluoride inhibition afforded by the Al-chelator deferoxamine and by loss of protection after addition of umolar levels of Al³⁺, which were not inhibitory for the enzyme in the absence of fluoride. The F₁ form of the enzyme dissociated from the cell membrane previously had been found to be resistant to fluoride in comparison with the F₁F₀ membrane-associated form. However, this difference appeared to depend on less aluminum in the F₁ preparation in that the sensitivity of the F₁ enzyme to fluoride could be increased by addition of umolar levels of Al³⁺. The effects of Al on fluoride inhibition were apparent when enzyme activity was assayed in terms of phosphate release from ATP or with an ATP-regenerating system containing phosphoenolpyruvate, pyruvate kinase, NADH and lactic dehydrogenase. Also, Be²⁺ but not other metal cations, e.g. Co²⁺, Fe²⁺, Fe³⁺, Mn², Sn²⁺, and Zn²⁺, served to sensitize the enzyme to fluoride inhibition. The differences in sensitivities of enzymes isolated from various oral bacteria found previously appeared also to be related to differences in levels of Al. Even the fluoride-resistant enzyme of isolated membranes of Lactobacillus casei ATCC 4646 could be rendered fluoride-sensitive through addition of Al³⁺. Thus, the F₁F₀ ATPases of oral bacteria were similar to E₁E₂ ATPases of eukaryotes in being inhibited by Al-F complexes, and the inhibition presumably involved formation of ADP-Al-F _inf3 ^sup- complexes during catalysis at the active sites of the enzymes.     

Correlation of oxygen utilization and hydrogen peroxide accumulation with oxygen induced enzymes in Lactobacillus plantarum cultures

Two strains of Lactobacillus plantarum accumulated H₂O₂ when grown aerobically in a complex glucose based medium. The H₂O₂ accumulation did not occur immediately on exposure of the culture to O₂ but was delayed for a time which, in the case of one strain, was dependent on the amount of inoculum used to seed the culture. The accumulation was always preceded by an increase in the rate of O₂ utilization by the cultures. The latter coincided approximately with an increase in specific activity of NADH oxidase, pyruvate oxidase and NADH peroxidase. H₂O₂ was not a product of NADH oxidase in vitro but was formed in substantial quantities from O₂ during oxidation of pyruvate. The three enzymes were induced by O₂ and H₂O₂; the induction of NADH oxidase responded to lower levels of O₂ (but not of H₂O₂) than the pyruvate oxidase or the NADH peroxidase.Abbreviations MRSG Mann, Rogosa and Sharpe medium (1960) with glucose as fermentation source - TPP thiamin pyrophosphate     

Binding of Streptococcus mutans to extracellular matrix molecules and fibrinogen

We have determined the ability of Streptococcus mutans cells to bind to extracellular matrix (ECM) molecules and fibrinogen. S. mutans cells were found to bind fibronectin, laminin, collagen type I, and fibrinogen. An isogenic S. mutans strain with a defect in the expression of the major surface protein of S. mutans, antigen I/II, possessed a reduced ability to bind fibronectin, collagen, and fibrinogen but not laminin, suggesting that antigen I/II contributes during pathological processes to the interaction of S. mutans cells with fibronectin, collagen type I, and fibrinogen.     

Protective immunity in rats by intranasal immunization with Streptococcus mutans glucan-binding protein D encapsulated into chitosan-coated poly(lactic-co-glycolic acid) microspheres

Recombinant Streptococcus mutans glucan-binding protein D (rGbpD) was incorporated into poly(lactic-co-glycolic acid) (PLGA) microspheres which then were surface-coated with chitosan. The microspheres, with a mean diameter of ca. 1.8 μm, were intranasally administered in rats. There were elevated salivary immunoglobulin A and serum immunoglobulin G antibody responses to rGbpD, as well as lower molar caries scores in immunized animals as compared to sham immunized ones. The chitosan-coated PLGA microspheres are thus potentially useful for antigen delivery in dental caries vaccination.     

Growth of mixed cultures of Actinomyces viscosus and Streptococcus mutans under dual limitation of glucose and oxygen

Abstract Streptococcus mutans and Actinomyces viscosus are among the dominant species in human dental plaque. In their natural environment, carbohydrate- and oxygen-limited conditions are likely to occur frequently. Therfore, mixed cultures of the 2 species were studied under dual limitation of glucose and oxygen. Over a wide range of oxygen-supply rates, coexistence of A. viscosus and S. mutans was observed, within this range A. viscosus increased almost linearly with oxygen supply. A mathematical model based on Monod-type type kinetics and accounting for uncompetitive inhibition of growth by oxygen was developed to simulate these mixed cultures. The model predicted coexistence over a fairly large range of aeration rates. This finding, in combination with the results of the chemostat experiments, led to the conclusion that coexistence of the two species     

Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans

Streptococcus mutans, a gram-positive immobile bacterium, is an oral pathogen considered to be the principal etiologic agent of dental caries. Although some researches suggest that trace metals, including iron, can be associated with dental caries, the function of salivary iron and lactoferrin in the human oral cavity remains unclear. The data reported in this study indicates that iron-deprived saliva (Fe3+ < 0.1 microM) increases S. mutans aggregation and biofilm formation in the fluid and adherent phases as compared with saliva (Fe3+ from 0.1 to 1 microM), while iron-loaded saliva (Fe3+ > 1 microM) inhibits both phenomena. Our findings are consistent with the hypothesis that S. mutans aggregation and biofilm formation are negatively iron-modulated as confirmed by the different effect of bovine lactoferrin (bLf), added to saliva at physiological concentration (20 microg/ml) in the apo- or iron-saturated form. Even if saliva itself induces bacterial aggregation, iron binding capability of apo-bLf is responsible for the noticeable increase of bacterial aggregation and biofilm development in the fluid and adherent phases. On the contrary, iron-saturated bLf decreases aggregation and biofilm development by supplying iron to S. mutans. Therefore, the iron-withholding capability of apo-Lf or native Lf is an important signal to which S. mutans counteracts by leaving the planktonic state and entering into a new lifestyle, biofilm, to colonize and persist in the human oral cavity. In addition, another function of bLf, unrelated to its iron binding capability, is responsible for the inhibition of the adhesion of S. mutans free, aggregated or biofilm on abiotic surfaces. Both these activities of lactoferrin, related and unrelated to the iron binding capability, could have a key role in protecting the human oral cavity from S. mutans pathogenicity.     

Superoxide,hydrogen peroxide,and the respiratory burst of fungally infected plant cells

The principal route of oxygen utilization in the respiratory burst of fungally infected plants was determined from stoichiometries of the uptake and electronic reduction of oxygen in cotton cells exposed to Aspergillus favus walls. Using 2,2-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) and epinephrine as redox reagents to manipulate oxygen transitions, we found that oxygen consumption doubled when superoxide disproportionation was abolished and was abolished when disproportionation doubled. Of four possible pathways for oxygen consumption, only monovalent reduction of molecular oxygen to superoxide was consistent with this inversely proportional relationship. According to the observed rate of oxygen consumption in this pathway and in the absence of competition to disproportionation of superoxide, infected cells are capable of generating intracellular concentrations of 1 M hydrogen peroxide in 13 min.