Greg R. Germaine

Rapid Filter Paper Assay for the Dextransucrase Activity from Streptococcus mutans

A convenient, sensitive, and reliable assay for the conversion of radiolabeled sucrose to alcohol-insoluble dextran by the Streptococcus mutans dextransucrase has been developed.

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-γ 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.

Streptococcus 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.

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 ≤ 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.

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.

Role of gal repressor depletion in λdg transduction escape synthesis

Transduction escape synthesis of galactokinase in Escherichia coli has been studied with the following results. (1) Escape synthesis of host-directed galactokinase is observed upon infection of a λ-sensitive E. coli with phage λdgA-J, containing a deletion of the galactokinase region. (2) When a λ-lysogen of a galactokinase-negative E. coli is infected with purified λdg, escape synthesis of galactokinase is observed. The differential rate of enzyme synthesis is shown to be dependent upon the multiplicity of infecting λdg. The data describe a hyperbolic curve without added inducer and a strictly linear relationship is found with inducer added. (3) Simultaneous infection of the λ-lysogen with both λdg and λdgA-J reveals an enhanced escape synthesis of λdg-directed galactokinase, while infection with λ together with λdg did not enhance escape synthesis. (4) gal repressor concentration and an apparent repressor-operon binding constant are estimated from these data by applying a simple model relating the rate of enzyme synthesis to the numbers of operons and repressors per cell. Approximate values of 17 gal repressors per cell and a dissociation constant, KG = 6 × 10−9m, for the gal repressor-operon system are obtained. The data support the hypothesis that the event of prime importance in transduction escape synthesis is depletion of cellular repressor.