Lactate metabolism by human dental plaque and Veillonella under aerobic and anaerobic conditions

Suspensions of human dental plaque, collected with special precautions to protect from exposure to air, metabolized a substantial amount of lactate both under aerobic and anaerobic conditions. Under aerobic conditions, acetate was a main product and a little propionate was produced; anaerobic incubation decreased the acetate production and increased the propionate formation. More lactate was metabolized under aerobic conditions than under anaerobic conditions. Veillonella, a strictly anaerobic microorganism, was considered to contribute largely to this aerobic metabolism of lactate because the resting cells of Veillonella also metabolize lactate actively under aerobic conditions. Activity of NAD-independent lactate dehydrogenases was demonstrated in the cell-free extract of Veillonella and was associated with the efficiency of the cells to metabolize lactate.     

Catabolic pathway for aerobic degradation of lactate by Actinomyces naeslundii

The aerobic metabolism of lactate by oral Actinomyces was studied. Six of 7 strains of Actinomyces naeslundii increased their growth in the presence of lactate under aerobic conditions. Washed cells grown on lactate aerobically degraded lactate and pyruvate to acetate with a concomitant consumption of oxygen. In the presence of catalase, the molar ratios of oxygen consumed to acetate produced were 1 for lactate degradation and 0.5 for pyruvate degradation. The enzymatic activities found in cell extracts revealed that lactate could be converted to pyruvate by NAD-independent lactate dehydrogenase (iLDH) and further to acetyl CoA by pyruvate dehydrogenase (PDH). The acetyl CoA formed could be metabolized into acetate by phosphotrans-acetylase (PTA) and acetate kinase (AK) with the formation of ATP. These results indicate that A. naeslundii metabolizes lactate into acetate by the sequential enzymatic reactions iLDH, PDH, PTA and AK and that hydrogens produced by iLDH and PDH are transferred to oxygen. The activity of lactate degradation and oxygen consumption may modify the environmental conditions of dental plaque.     

Anaerobic and aerobic metabolism of sorbitol in Streptococcus sanguis and Streptococcus mitior

Sorbitol-fermenting strains of Streptococcus sanguis and Streptococcus mitior were grown both anaerobically and in the presence of oxygen in a sorbitol-containing complex medium. Washed-cell suspensions were incubated with an excess of sorbitol, and the production of lactate, formate, ethanol, and acetate was analyzed. Moreover, we determined the lactate dehydrogenase and pyruvate formate-lyase activities in cell-free extracts of anaerobically grown cells. The anaerobically grown cells produced lactate, formate, ethanol, and acetate under anaerobic conditions. When these cells were exposed to air, the amounts of formate, ethanol, and acetate were reduced in comparison with those of the strictly anaerobic cells. Cells grown in the presence of oxygen only produced detectable levels of lactate and acetate. Anaerobically grown cells possessed lactate dehydrogenase and pyruvate formate-lyase activities under strictly anaerobic conditions. The level of pyruvate formate-lyase was dramatically reduced when cells were exposed to air, while the level of lactate dehydrogenase was about the same as that under strictly anaerobic conditions. Thus, the results indicate that S. sanguis and S. mitior both metabolize sorbitol differently under anaerobic and aerobic conditions. This difference may depend on the oxygen-sensitivity of the pyruvate formatelyase of these micro-organisms.     

Metabolism of intracellular polysaccharide in the cells of Streptococcus mutatis under strictly anaerobic conditions

Streptococcus mutans, which had accumulated glycogen-like iodophilic intracellular polysaccharide (IPS), produced large amounts of formate, acetate and ethanol from the IPS by pyruvate formate-lyase (PFL) under strictly anaerobic conditions without exogenous sugar. Under aerobic conditions, the same S. mutans produced exclusively lactate and pyruvate from the IPS because of the inactivation of PFL by oxygen. The total amount of acid produced under anaerobic conditions was larger than that under aerobic conditions. The analysis of intracellular glycolytic intermediates revealed that levels of fructose 1,6-bisphosphate (lactate dehydrogenase (LDH) activator) and glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (PFL inhibitors) were low when IPS was used as a glycolytic substrate, implying that PFL functions more efficiently than LDH in IPS metabolism. These findings suggest that the PFL pathway contributes to the acid production from IPS, and may explain partially why the acids in starved dental plaque are mainly acetate and formate.     

Effect of acetate on sorbitol fermentation by oral lactobacilli

The rate of acid production and end-products from sorbitol were measured under anaerobic conditions in washed-cell suspensions of oral strains of Lactobacillus casei subsp, casei and Lactobacillus casei subsp, rhamnosus . The enzymatic activities were assayed in cell extracts of these strains. The cells fermented sorbitol to lactate, formate, ethanol and acetate under anaerobic conditions. Exposure of the cells to air (oxygen) led to inactivation of pyruvate formatelyase and inhibition of anaerobic sorbitol fermentation. In the presence of acetate, air-exposed cells fermented sorbitol with a concomitant consumption of acetate and production of ethanol and lactate. Acetate also enhanced acid production from sorbitol in cells kept under anaerobic conditions and resulted in formation of lactate and ethanol. Cell extracts of all the strains had NADH-coupled acetate-reducing activity, which consisted of sequential reactions of acetate kinase, phosphotrans-acetylase, acylating aldehyde dehydrogenase and alcohol dehydrogenase. These findings indicate that oral lactobacilli can utilize acetate as an electron acceptor for maintaining their intracellular redox balance during anaerobic sorbitol fermentation in the absence of pyruvate formate-lyase activity.     

Production and degradation of formate by Veillonella dispar ATCC 17745

Under strictly anaerobic conditions, the resting cells of V. dispar ATCC 17745 produced formate as well as acetate and propionate from pyruvate or from lactate. Pyruvate formate-lyase activity was found when the activity was assayed under strictly anaerobic conditions. Under aerobic conditions, however, the resting cells did not produce formate from pyruvate or from lactate, though the cells actively metabolized pyruvate or lactate (mainly to acetate). This was ascribed to pyruvate formate-lyase activity being easily lost when the cell-free extract was exposed to the air. A part of the produced formate was further degraded to CO2 by the resting cells.     

Glucose metabolism by Prevotella intermedia and Prevotella nigrescens

Glucose metabolism by Prevotella intermedia and Prevotella nigrescens were investigated. Glucose increased the anaerobic growth of these bacteria and promoted the accumulation of intracellular polysaccharide. The polysaccharide was confirmed to be glycogen-like glucan by the absorption spectrum of iodinepolysaccharide complex and the sugar composition. The washed cells consumed glucose anaerobically and converted a part of glucose into the metabolic end-products acetate, formate and succinate. The rest of glucose was confirmed to be accumulated as intracellular polysaccharide. The cells grown in the presence of glucose produced acetate, formate and succinate without exogenous glucose along with the consumption of intracellular polysaccharide. The metabolism of glucose and intracellular polysaccharide required bicarbonate. Prevotella cells had hexokinase and a set of the usual enzymes of the Embden-Meyerhof-Parnas pathway except that phosphofructokinase was pyrophosphate-dependent. A series of enzymes, including phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarase and fumarate reductase, was found for succinate formation. Another series of enzymes, pyruvate oxidoreductase, pyruvate formate-lyase, phosphotransacetylase and acetate kinase was found for acetate and formate formation. Glucose 1,6-bisphosphate-dependent phosphoglucomutase and fructose 1,6-bisphosphate-activated UDP-glucose pyrophosphorylase were detected for glycogen synthesis, while glycogen phosphorylase was for glycogen degradation. The capacity of intracellular polysaccharide formation in addition to glucose fermentation could be advantageous for survival in the supragingival area as well as in the subgingival area.     

Oxygen and the sugar metabolism in oral streptococci.

Streptococci have several ways of adapting themselves to the constantly changing environment of the human oral cavity. This paper discusses the adaptation of sugar metabolism to variations in oxygen levels. In all streptococci the Embden-Meyerhof pathway of glycolysis works under aerobic as well as anaerobic conditions, but pyruvate is converted into different metabolic end products depending on the oxygen levels. Under anaerobic conditions all streptococci form formate, acetate, and ethanol by a pyruvate formate-lyase pathway. If sugar is in excess, they also form lactate using a lactate dehydrogenase. Under aerobic conditions pyruvate formate-lyase is inactivated. This enzyme is then replaced by a pyruvate oxidase in some streptococci and by a pyruvate dehydrogenase in others. The characteristics of these enzymes help streptococci like S. sanguis, S. oralis, S. gordonii, and S. mitis to compete successfully with other bacteria in those sites of the oral cavity that are freely exposed to saliva, while mutans streptococci have to colonize anaerobic sites such as those in-between the teeth and in the occlusal fissures of the teeth.     

The role of the succinate pathway in sorbitol fermentation by oral Actinomyces viscosus and Actinomyces naeslundii

The sorbitol fermentation by Actinomyces viscosus and Actinomyces naeslundii was studied with washed sorbitol-grown cells. The fermentation was followed by titration of acids produced at pH 7.0 under anaerobic conditions. Metabolic end-products and intracellular levels of NAD, NADH and glycolytic intermediates during the fermentation were also analyzed. Cell extracts were examined for certain enzyme activities. Bicarbonate was required for acid production from sorbitol and from a mixture of glucose and sorbitol. Malate and fumarate could also support the acid production of A. viscosus. The main end-products were succinate and lactate but not ethanol. Cell extracts showed no activities of alcohol and aldehyde dehydrogenases, but they had activities of malate dehydrogenase and fumarate reductase. In the absence of bicarbonate, malate or fumarate, the intracellular NADH/NAD ratio increased and the levels of 3- and 2-phospho-glycerate and phosphoenolpyruvate decreased. The results indicate that oral sorbitol-fermenting actinomyces lack the ethanol pathway that can contribute to NADH oxidation. To maintain intracellular redox balance during anaerobic sorbitol fermentation, these bacteria can oxidize surplus NADH through a succinate pathway.     

Biochemical mechanisms of enhanced inhibition of fluoride on the anaerobic sugar metabolism by Streptococcus sanguis

The effect of fluoride on acid production by Streptococcus sanguis ATCC 10556 was compared under anaerobic and aerobic conditions. The rate of acid production under constant-pH and pH-free-fall conditions was determined during glucose metabolism by resting cells. Anaerobic glycolysis was inhibited more strongly by fluoride than was aerobic glycolysis. Intracellular levels of 3-phosphoglyceric, 2-phosphoglyceric, and phosphoenolpyruvic acids were lower under anaerobic conditions than under aerobic conditions. Thus, S. sanguis had a low phosphoenolpyruvate (PEP) potential under anaerobic conditions. This low PEP potential was suggested to account for the more effective fluoride inhibition of enolase and, consequently, the reduced transport of sugar by the PEP-dependent phosphotransferase system of this micro-organism.     

Acid production by streptococci growing at low pH in a chemostat under anaerobic conditions

Streptococcus mutans and other oral streptococci were grown in continuous culture under strictly anaerobic conditions. When the cultural pH was kept at 7.0, the main acid products were formate and acetate, as reported previously. However, more lactate was produced at pH values of 5.5 or 6.0, with a concomitant decrease in formate and acetate production. This change in fermentation products could partly be ascribed to a change in intracellular pH and difference in the pH optima between pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH). At extracellular pH values of 7.0 and 5.5, the intracellular pH values of S. mutans NCIB 11723 were 7.5 and 6.6, respectively. The pH optima of PFL and LDH were 7.8 and 5.5-6.3, respectively. The cells had also a larger amount of LDH during growth at pH 5.5 than at pH 7.0.     

Studies on the carbohydrate metabolism of cariogenic Streptococcus mutans strain PK-1

Under anaerobic condition the resting cells of cariogenic streptococcus strain PK-1 metabolized glucose via the Embden-Meyerhof pathway and about 2 moles of lactate were formed for each mole of glucose consumed. Under aerobic condition the greater part of the glucose was also metabolized by the way of the Embden-Meyerhof pathway to pyruvate, but the pyruvate was converted not only to lactate but to acetoin, CO₂ and acetate. It is noteworthy that under aerobic condition -lactate formation did not decrease remarkably, though -lactate formation was decreased by half. Under aerobic condition a part of the glucose consumed was considered to be metabolized via the hexose monophosphate shunt but none of the glucose was observed to be metabolized via the citric acid cycle or the Entner-Doudoroff pathway.

Evidence was obtained that sucrose phosphorylase was induced in sucrose-grown cells.

Fructose-1; 6-diphosphate activated the -lactate dehydrogenase of this organism, but did not activate -lactate formation. However, the effect was less than that on the lactate dehydrogenase of some other streptococci.     

The time-course of acid excretion, levels of fluorescence dependent on cellular nicotinamide adenine nucleotide and glycolytic intermediates of Streptococcus mutans cells exposed and not exposed to air in the presence of glucose and sorbitol

The aim of this study was to examine glucose and sorbitol metabolism in Streptococcus mutans cells exposed and not exposed to air at the coexistence of these compounds by measuring acid excretion, levels of fluorescence dependent on cellular NADH and glycolytic intermediates. An aliquot of bacterial cells grown under strictly anaerobic conditions (anaerobic cells) was exposed temporarily to air (aerobic cells). When glucose was added to the anaerobic cells metabolizing sorbitol, the acid excretion was increased. The level of NADH decreased initially and then increased to the higher plateau level than that during glucose metabolism. The aerobic cells neither metabolized sorbitol nor contained glycolytic intermediates. However, 2 min after glucose was added in the presence of sorbitol, the acid excretion was started slowly and the intermediates appeared. The level of NADH was decreased at first and then increased. These results suggested that the anaerobic S. mutans cells metabolized glucose and sorbitol simultaneously, and that in the presence of sorbitol the aerobic cells could start to metabolize glucose 2 min after glucose was added, as the intermediates (phosphoenopyruvate potential) for the glucose transport were accumulated.     

The time‐course of acid excretion,levels of fluorescence dependent on cellular nicotinamide adenine nucleotide and glycolytic intermediates of Streptococcus mutans cells exposed and not exposed to air in the presence of glucose and sorbitol

The aim of this study was to examine glucose and sorbitol metabolism in Streptococcus mutans cells exposed and not exposed to air at the coexistence of these compounds by measuring acid excretion, levels of fluorescence dependent on cellular NADH and glycolytic intermediates. An aliquot of bacterial cells grown under strictly anaerobic conditions (anaerobic cells) was exposed temporarily to air (aerobic cells). When glucose was added to the anaerobic cells metabolizing sorbitol, the acid excretion was increased. The level of NADH decreased initially and then increased to the higher plateau level than that during glucose metabolism. The aerobic cells neither metabolized sorbitol nor contained glycolytic intermediates. However, 2 min after glucose was added in the presence of sorbitol, the acid excretion was started slowly and the intermediates appeared. The level of NADH was decreased at first and then increased. These results suggested that the anaerobic S. mutans cells metabolized glucose and sorbitol simultaneously, and that in the presence of sorbitol the aerobic cells could start to metabolize glucose 2 min after glucose was added, as the intermediates (phosphoenopyruvate potential) for the glucose transport were accumulated.     

Effect of pH on acid production from sorbitol in washed cell suspensions of oral bacteria

The acid production from sorbitol and glucose was studied under anaerobic conditions in resting cell suspensions of bacteria from the predominant sorbitol-fermenting human dental plaque flora, belonging to the genera Streptococcus, Lactobacillus and Actinomyces. The acid production activity of the bacterial cells was followed by titration with alkali, at environmental pH 7.0, 6.0 and 5.0 after addition of carbohydrate solution. The metabolic end products formed in the suspensions were analyzed thereafter by isotachophoretic and enzymatic methods. The results showed that sorbitol was fermented at a slower rate than glucose. Lowering the environmental pH decreased the acid production activity from the two carbohydrates. Compared with glucose, the catabolism of sorbitol was affected to greater extent by the pH conditions. The total amount of acids formed from sorbitol was considerably less than from glucose. Lactic acid, which was the major end product in glucose-challenged suspensions, was produced only in low concentrations from sorbitol by all strains tested. The ratio strong (formic + lactic)/weak acids was moreover lower for sorbitol than for glucose. The present results further illustrate some of the mechanisms behind the low cariogenic potential of this sugar substitute.     

Alterations of energy metabolism and glutathione levels of HL-60 cells induced by methacrylates present in composite resins

OBJECTIVES: Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and bisphenol A glycerolate (1 glycerol/phenol) dimethacrylate (Bis-GMA) are largely present in auto- or photopolymerizable composite resins. Since the polymerization reaction is never complete, these molecules are released into the oral cavity tissues and biological fluids where they could cause local adverse effects. The aim of this work was to verify the hypothesis that the biological effects of HEMA, TEGDMA and Bis-GMA - at a non-cytotoxic concentration - depend on the interaction with mitochondria and exert consequent alterations of energy metabolism, GSH levels and the related pathways in human promyelocytic cell line (HL-60). METHODS: The biological effects of methacrylic monomers were determined by analyzing the following parameters: GSH concentration, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activity, oxygen and glucose consumption and lactate production along with cell differentiation and proliferation. RESULTS: All monomers induced both cellular differentiation and decrease in oxygen consumption. Cells treated with TEGDMA and Bis-GMA showed a significant enhancement of glucose consumption and lactate production. TEGDMA and HEMA induced GSH depletion stimulating G6PDH and GR activity. CONCLUSIONS: All the monomers under study affect the metabolism of HL-60 cells and show differentiating activity. Since alterations in cellular metabolism occurred at compound concentrations well below cytotoxic levels, the changes in energy metabolism and glutathione redox balance could be considered as potential mechanisms for inducing clinical and sub-clinical adverse effects and thus providing useful parameters when testing biocompatibility of dental materials.     

L-serine enhances the anaerobic lactate metabolism of Veillonella dispar ATCC 17745

Under anaerobic conditions, the rate of metabolism of lactate by starved resting cells of Veillonella dispar ATCC 17745 was very low. Because pyruvate was metabolized well by the starved cells, oxidation of lactate to pyruvate, which is the first step of the lactate metabolism, must have been limited in the cells. In the starved cells, the levels of the metabolic intermediates, oxalacetate or fumarate, of which reductions to malate or to succinate could be coupled with lactate oxidation to pyruvate and initiate lactate metabolism, were quite low, suggesting that these had been reduced during the starvation steps under strictly anaerobic conditions. Thus, the starved cells were unable to start the anaerobic lactate metabolism because of shortage of such reducible substrates. L-serine greatly enhanced anaerobic lactate metabolism of the starved cells. This enhancement may have been due to metabolism of L-serine itself and conversion to oxalacetate and fumarate, which made it possible to begin lactate oxidation.     

Stimulatory effect of bicarbonate on the glycolysis of Actinomyces viscosus and its biochemical mechanism

The effects of bicarbonate on acid production by 4 human strains of Actinomyces viscosus were estimated under anaerobic conditions. The rate of acid production was accelerated by bicarbonate 3-4 times as much as that without bicarbonate. The analyses of intracellular glycolytic intermediates, NAD and NADH revealed a decrease in NADH:NAD ratio and an increase in the level of 3-phosphoglycerate in the cells when bicarbonate was present. Furthermore, when bicarbonate was available, malate dehydrogenase and fumarate reductase in the succinate pathway were expected to function as NADH-oxidizing enzymes in addition to lactate dehydrogenase. These observations indicate the efficient regeneration of NAD in the presence of bicarbonate. Thus, the stimulation of A. viscosus glycolysis by bicarbonate was thought to stem from the activation of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) by the decrease in the level of NADH, because NADH was a strong inhibitor of G3PDH in this microorganism.     

环境氧条件影响寡发酵链球菌抑制变形链球菌作用的研究

目的 探讨外界环境氧气条件的改变对寡发酵链球菌(Streptococcus oligofermentans,So)与变形链球菌(Streptococcus mutans,Sm)之间抑制作用的影响,以及对So产过氧化氢能力的影响,以期探讨这两种细菌在不同口腔环境条件下的共存模式.方法 通过二氧化碳培养法建立细菌培养的有氧和厌氧环境;运用平板培养法观察So与Sm之间的抑制作用;运用4-氨基安替吡啉-辣根过氧化物酶法测定有氧和厌氧环境下So对数生长期各时间段过氧化氢的产量和So生长周期过氧化氢初始产生速率.结果 同时接种So和Sm,厌氧环境下未见So抑制Sm,有氧环境下So轻度抑制Sm,受抑制区面积约为菌膜面积的1/5;先接种So再接种Sm,两种环境下均可见So抑制Sm,厌氧环境下受抑制区面积约为菌膜面积的1/5,有氧环境下约为4/5;先接种Sm再接种So,两种环境下均未见So生长;有氧环境下So对数生长期各时间段过氧化氢产量明显高于厌氧环境(P<0.05);厌氧环境下So生长周期过氧化氢初始产生速率为每分钟(11.84±3.97) μmol/L,仅为有氧环境时[(24.13±4.46) μmol/L]的49%(P<0.05).结论 环境氧条件影响So对Sm的抑制作用,有氧环境下抑制作用更强;有氧环境下So产过氧化氢的能力强于厌氧环境.

Abstract：

Objective To investigate the effect of environmental oxygen on the inhibition between Streptococcus oligofermentans(So) and Streptococcus mutans(Sm) and the producibilities of hydrogen peroxide by So. Methods The aerobic and anaerobic environment was established by the carbon dioxide cultivation. The inhibition between So and Sm was observed by plating method. The production and synthesis rates of hydrogen peroxide by So were determined in both aerobic and anaerobic environment by 4-ATTP-horseradish peroxidase method at A510. Results When both Sm and So were inoculated at the same time, Sm was not inhibited under the anaerobic environment, vice versa. Sm was slightly inhibited by So under the aerobic environment, the inhibition area was 1/5 of all bacterial membrane. When So was cultivated first and then Sm applied, So could inhibite Sm growth under both anaerobic and aerobic conditions. The inhibition area was 1/5 of bacterial membrane under the anaerobic environment, and 4/5 under the aerobic environment. When Sm was cultivated first and then So applied, So was unable to proliferate under both conditions. During the logarithmic phase, the production of H2O2 by So under the aerobic environment was higher than under the anaerobic environment (P<0.05).The initial synthesis rate of H2O2 by So during growth cycle under the anaerobic condition was (11.84±3.97) μmol/L per minute, which was only 49% of that under the aerobic environment[(24.13±4.46) μmol/L per minute].Conclusions The oxygen has the effect on the inhibition between So and Sm, and the inhibition in the aerobic environment is much stronger than in the anaerobic environment. The synthesis ability of hydrogen peroxide by So under the aerobic environment is higher than under the anaerobic environment.     

Lactate dehydrogenase isoenzymes of the human periodontal ligament

The isoenzymes of lactate dehydrogenase (E. C. 1. 1. 1. 27.) from the periodontal ligament on the root surface of extracted erupted permanent human teeth have been separated and quantitated by means of cellulose acetate membrane electrophoresis.
LDH-3 and LDH-4 were the predominating isoenzymes. and smaller amounts of LDH-2 and LDH-5 were also present. Only traces of LDH-1 could be seen. The results indicate the presence of both aerobic and anaerobic metabolic pathways in the human periodontal ligament.
These pathways bring about an efficient, economical energy production under normal circumstances, and make the survival of this tissue possible under hypoxic conditions, when the blood supply is briefly interrupted. The results further show that determinations of the survival time of the periodontal ligament on a tooth outside its alveolus have to be made by activity measurements of both aerobic and anaerobic enzymes.