Effects of pH, potassium, magnesium, and bacterial growth phase on lysozyme inhibition of glucose fermentation by Streptococcus mutans 10449.

The effects of physiological (saliva and plaque fluid) concentrations of potassium and magnesium and growth phase on lysozyme inhibition of glucose fermentation by S. mutans 10449 were investigated. Glucose fermentations were carried out in a pH-stat at pH 7.0 or 5.5. Cells were at least two times more sensitive to lysozyme in the early-to-middle exponential phase compared with the stationary phase. S. sobrinus 6715 exhibited three-fold greater lysozyme resistance than S. rattus BHT or S. mutans 10449. The concentration of potassium which reduced lysozyme inhibition of S. mutans 10449 fermentation by 50% was 0.2 and 10 mmol/L for stationary and exponential phase cells, respectively. Corresponding values for magnesium were < or = 0.01 and 0.50 mmol/L. Potassium and magnesium exhibited little pH dependence in their reduction of lysozyme inhibition of fermentation by exponential- or stationary-phase S. mutans 10449. The results suggest that: (i) lysozyme interaction with stationary-phase cells involves more non-inhibitory modes than with exponential-phase cells, and (ii) lysozyme may be more effective as an antibacterial agent in saliva than in plaque fluid.

Streptoccus mutans dextransucrase: purification, properties, and requirement for primer dextran.

We attempted to purify dextransucrase from S mutans strain 6715 to investigate its properties and determine if multiple species of the enzyme existed. It was concluded that the properties of this enzyme such as the pH (5.5), temperature (37 C) optimum, and Km for sucrose (3 mM) are very similar to those reported for S sanguis, S bovis, S mutans strain OMZ-176 isozymes, S mutans strain GS-5, and the single dextransucrase purified from S mutans strain HS-6. The IEF enzyme preparation consisted of two enzyme species, possibly differing in their ability to synthesize different dextran linkages. The minor enzyme activity demonstrated a strict primer dependency. Similarly, primer dependency has been reported for dextransucrases from S mutans, S sanguis, and L mesenteroides. S mutans strain 6715 dextransucrase also showed both the insertion and stepwise mechanisms for dextran synthesis. Sucrose was the sole glucose donor, whereas dextran was a specific, highly efficient glucose acceptor. The complex primer kinetics are not fully understood at this time and require further investigation. Without linkage analysis of the products of our enzymes, we can only postulate that each enzyme has a different function in the synthesis of interresidue and interchain alpha1-3 and alpha1-6 bonds. Insoluble dextran synthesis may involve a special enzyme mechanism characteristic of S mutans. This synthesis would require both enzymes, possibly in some aggregated form, with one enzyme synthesizing endogenous primer dextran. This endogeneous primer or some cell wall polysaccharide could stimulate both enzymes to rapidly synthesize heterogeneously linked insoluble dextran.

Localized sinus inflammation in a rabbit sinusitis model induced by Bacteroides fragilis is accompanied by rigorous immune responses.

We evaluated inflammatory and immune responses against Bacteroides fragilis in a rabbit sinusitis model. Bacteroides was inoculated into the left maxillary sinus, and inflammatory (histology, cell number/cytology, lactose dehydrogenase, and apoptosis) and immune responses in the sinus, airway, and peripheral blood (PB) were determined for up to 4 weeks. In the inflamed sinus, the lactose dehydrogenase level was markedly elevated, with neutrophilic infiltration, severe tissue inflammation, and increased apoptosis. Low-grade tissue inflammation was present in the contralateral and sham-operated sinuses, but other parameters remained unchanged, and so did those in the airway and PB in the inoculated rabbits. Serum IgG antibody levels increased rapidly, were highest at 3 weeks, and began to decline at 4 weeks. Cellular immune responses (proliferation and interferon-gamma mRNA expression) against Bacteroides were detected in the PB of all inoculated rabbits. Vigorous immune responses against Bacteroides may have localized but failed to terminate inflammation in the sinus, indicating importance of microenvironmental factors.

Whole saliva proteases: development of methods for determination of origins.

1) Recognition and characterization of protease activities present in saliva by PAGE examination of substrate protein cleavage patterns appears possible. 2) BSA proteolysis by whole saliva is not due to activities present in the major gland secretions and may be due, in part, to the oral microflora.

Adherence of Streptococcus sanguis to hydroxyapatite coated with lysozyme and lysozyme-supplemented saliva.

The adherence of [3H]thymidine-labeled Streptococcus sanguis strains to bare hydroxyapatite and to hydroxyapatite coated with a range of concentrations of lysozyme, poly-L-lysine, poly-L-glutamic acid, whole saliva supernatant, and combinations of some of the above was studied. Adherence of several strains of S. sanguis to bare hydroxyapatite and saliva-coated hydroxyapatite was compared. Saliva present as a pellicle on the hydroxyapatite inhibited adherence of some strains (903, M-5, 73X11) and stimulated that of others (S35, B-4, 66X49). Strains 903 and S35 were chosen for further study. Adherence of both strains was stimulated up to fivefold by the presence of adsorbed lysozyme or poly-L-lysine on the hydroxyapatite, whereas poly-L-glutamic acid inhibited adherence (80 to 95%). Adherence of strain S35 to hydroxyapatite coated with combinations of saliva and (i) lysozyme, (ii) poly-L-lysine, or (iii) poly-L-glutamic acid was unaffected compared with adherence to hydroxyapatite coated with saliva alone. In contrast, adherence of strain 903 to hydroxyapatite coated with combinations of saliva and either lysozyme or poly-L-lysine was inhibited up to ca. 90% compared with hydroxyapatite coated with saliva alone. Strain 903 was also unaffected by combinations of poly-L-glutamic acid and saliva on the hydroxyapatite. Adherent cells of both strains were completely (greater than 90%) eluted with high-ionic-strength buffer from either bare hydroxyapatite or hydroxyapatite coated with lysozyme alone. Adherent cells of strain S35 were only poorly eluted (25%) from hydroxyapatite coated with either saliva alone or saliva and lysozyme. Strain 903 elution from hydroxyapatite coated with either saliva alone or saliva and lysozyme was essentially complete. These observations were taken to indicate that the two test strains adhered to saliva-coated hydroxyapatite by different mechanisms. Protein-coated hydroxyapatite was shown not to be saturated under the conditions described here. Examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the variously supplemented salivary pellicles formed on the hydroxyapatite demonstrated that major changes in salivary protein composition did not occur when lysozyme, poly-L-lysine, or poly-L-glutamic acid was used to supplement saliva. Lysozyme-dependent aggregation of strain 903 was shown not to occur under the conditions of our experiments. We suggest that the basis for stimulation of adherence to hydroxyapatite coated only with lysozyme is an increase in the cationic surface area available for electrostatic adherence of the microorganisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of human saliva on glucose uptake by Streptococcus mutans and other oral microorganisms.

We examined the effects of human whole salivary supernatant and parotid fluid on glucose uptake by Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, Actinomyces viscosus, Staphylococcus aureus, and Escherichia coli. The following three effects of saliva were observed: (i) inhibition of glucose uptake (S. mutans, S. sanguis), (ii) promotion of a transient, rapid (0 to 30 s) burst of glucose uptake (S. mutans, S. sanguis), and (iii) enhancement of glucose uptake (S. mitis, A. viscosus, S. aureus, E. coli). We observed no differences between the effects of whole salivary supernatant and the effects of parotid fluid. Heat treatment (80 degrees C, 10 min) of saliva or the addition of dithiothreitol abolished inhibition of glucose uptake. Supplementation of saliva with H(2)O(2) potentiated inhibition of glucose uptake. S. mitis and A. viscosus, which were stimulated by saliva alone, were inhibited by H(2)O(2)-supplemented saliva; 50% inhibition of glucose uptake by S. mutans and S. mitis required ca. 10 muM H(2)O(2) in 50% (vol/vol) saliva. Loss of the inhibitory action of saliva occurred at about 5% (vol/vol) saliva. Supplementation of saliva dilutions with SCN(-) and H(2)O(2) extended the inhibitory activity to solutions containing ca. 0.2% (vol/vol) saliva. We suggest that the salivary lactoperoxidase-SCN(-)-H(2)O(2) system is responsible for the inhibitory activity of saliva reported here. Furthermore, we concluded that lactoperoxidase and SCN(-) are present in saliva specimens in concentrations that exceed minimal inhibitory levels by factors of ca. 500 and 10 to 20, respectively. The resistance of A. viscosus, S. aureus, and E. coli to the inhibitory potential of saliva alone was probably due to the production of catalase by these organisms. The resistance of S. mitis may have been due to special effects of saliva on H(2)O(2) accumulation by this organism compared with S. mutans and S. sanguis. The basis of saliva-dependent enhancement of glucose uptake and the basis of promotion of a transient, rapid burst of glucose uptake are unknown. The role of the salivary lactoperoxidase-SCN(-)-H(2)O(2) system in the oral microbial ecosystem is discussed.

Simple filter paper procedure for estimation of glucose uptake via group translocation by whole-cell suspensions of bacteria.

A simple and rapid filter paper technique is described for processing samples from glucose uptake studies with whole cells of gram-positive or gram-negative bacteria that transport glucose via group translocation. The procedure yields results equivalent to those obtained with a conventional membrane filtration method and requires no special filtration equipment or source of vacuum.

Effect of endogenous phosphoenolpyruvate potential on fluoride inhibition of glucose uptake by Streptococcus mutans.

The fluoride sensitivity of glucose uptake by whole cell suspensions of Streptococcus mutans was studied. Preincubation of the organism with up to 1 mM glucose markedly reduced the fluoride sensitivity of subsequent glucose uptake at pH 7.0 and 5.5. Glucose preincubation was shown to result in the establishment of a stable pool of three-carbon glycolytic intermediates. On the basis of inhibition studies and thin-layer chromatography of cell extracts, we suggest that 3- and 2-phosphoglycerate are the principal constituents of the pool. Increased concentrations of glucose used in preincubation mixtures was associated with increased pool sizes of the glycolytic intermediates and increased fluoride resistance. Transport of 2-deoxy-D-glucose by permeabilized cells was inhibited by fluoride when 2-phoshoglycerate served as the energy source. Increased concentrations of 2-phosphoglycerate were shown to overcome the fluoride inhibition of transport. The data suggest that establishment of a stable pool of glycolytic intermediates that includes 2-phosphoglycerate (or its progenitors) may contribute significantly to the apparent refractoriness of plaque microbes to fluoride in vivo.