Interactions of Streptococcus mutans fimbria-associated surface proteins with salivary components.

Streptococcus mutans has been implicated as the major causative agent of human dental caries. S. mutans binds to saliva-coated tooth surfaces, and previous studies suggested that fimbriae may play a role in the initial bacterial adherence to salivary components. The objectives of this study were to establish the ability of an S. mutans fimbria preparation to bind to saliva-coated surfaces and determine the specific salivary components that facilitate binding with fimbriae. Enzyme-linked immunosorbent assay (ELISA) established that the S. mutans fimbria preparation bound to components of whole saliva. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot techniques were used to separate components of whole saliva and determine fimbria binding. SDS-PAGE separated 15 major protein bands from saliva samples, and Western blot analysis indicated significant binding of the S. mutans fimbria preparation to a 52-kDa salivary protein. The major fimbria-binding salivary protein was isolated by preparative electrophoresis. The ability of the S. mutans fimbria preparation to bind to the purified salivary protein was confirmed by Western blot analysis and ELISA. Incubation of the purified salivary protein with the S. mutans fimbria preparation significantly neutralized binding of the salivary protein-fimbria complex to saliva-coated surfaces. The salivary protein, whole saliva, and commercial amylase reacted similarly with antiamylase antibody in immunoblots. A purified 65-kDa fimbrial protein was demonstrated to bind to both saliva and amylase. These data indicated that the S. mutans fimbria preparation and a purified fimbrial protein bound to whole-saliva-coated surfaces and that amylase is the major salivary component involved in the binding.

Differential activation of human neutrophils by Streptococcus mutans isolates from root surface lesions and caries-free and caries-active subjects.

Phagocytosis of bacterial pathogens is an important defense mechanism and may contribute to regulating Streptococcus mutans-induced dental caries, particularly at root surfaces. This study was undertaken to examine and compare differences in polymorphonuclear leukocyte or neutrophil activation by clinical isolates of S. mutans collected from the saliva of caries-free or caries-active individuals with S. mutans isolates from root surface lesions. S. mutans clinical isolates (5 caries-free, 5 caries-active, 5 root caries isolates and a laboratory strain) were incubated with neutrophils in the presence of normal human serum and the luminol dependent chemiluminescence was measured for 1 h at 37 degrees C. Results indicated that the caries active and laboratory strains activated neutrophils equally. The mean integration stimulated by caries-free strains, however, displayed a 25-30% enhanced neutrophil activation over the caries-active and laboratory strains. In contrast, neutrophil activation by root caries strains of S. mutans was 45-50% lower than all other S. mutans strains, possibly suggesting a natural selection for S. mutans strains that can evade neutrophil recognition and subsequent phagocytosis. Stimulation of neutrophils with the cell wall and membrane surface component preparations indicated that extracts from all four groups activated neutrophils significantly. Again, caries-free preparations activated neutrophils significantly more than caries active, laboratory strain and root caries isolates. This selection may become more important on root surfaces due to increased exposure to crevicular fluid and neutrophils. The data provide evidence for the presence or onset of mechanisms or biological alterations in S. mutans developed to circumvent neutrophil recognition and/or phagocytosis, thus increasing S. mutans survival and colonization on tooth surfaces, resulting in an enhanced risk of dental caries, particularly at root surfaces.

Antigenic characterization of fimbria preparations from Streptococcus mutans isolates from caries-free and caries-susceptible subjects.

The adhesion of pathogenic bacteria to the host surface is an essential step in the development of numerous infections, including dental caries. Attachment of Streptococcus mutans, the main etiological agent of human dental caries, to the tooth surface may be mediated by glucan synthesized by glucosyltransferase (GTF) and by cell surface proteins, such as P1, which bind to salivary receptors. Fimbriae on the surfaces of many microorganisms are known to function in bacterial adhesion. Previous studies in this laboratory have initially characterized the fibrillar surface of S. mutans. The purpose of this investigation was the comparison of the antigenic properties of fimbria preparations of S. mutans isolates from five caries-resistant (CR) and six caries-susceptible (CS) subjects. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of S. mutans fimbrial preparations revealed five major protein bands at 200, 175, 157, 86, and 66 kDa in preparations from CR and CS subjects. Immunoblot analysis indicated the presence of the same major bands recognized by anti-S. mutans fimbria antisera. Furthermore, the 175- and 157-kDa bands were recognized by antibodies to P1 and GTF, respectively. Immunoblot analysis with antisera to the fimbria preparation, to P1, or to GTF indicated that the levels of fimbria-reactive components and P1 and GTF antigens were higher in S. mutans fimbria preparations from CS subjects than in those from CR individuals. For example, four of six fimbria preparations from CS patients had demonstrable P1, and all had GTF. In contrast, only two of five CR fimbrial preparations exhibited P1 and GTF. Enzyme-linked immunosorbent assay demonstrated similar results for levels of GTF antigen in the fimbrial preparations from CR and CS subjects. The results suggest that differences between the compositions of S. mutans fimbriae in CR and CS individuals may play an important role in the virulence of this microorganism in dental caries.

Evaluación de los niveles de la adhesina P1 en fimbrias de Streptococcus mutans aisladas de pacientes susceptibles y resistentes a la caries dental / Evaluation of P1 adhesin levels in Streptococcus mutans fimbriae isolated from caries susceptible and caries resistant patients

El objetivo de este trabajo fue la determinación de los niveles de P1 en fimbrias aisladas de S. mutans, provenientes de pacientes susceptibles (CS) y resistentes (CR) a la caries dental. El análisis electroforético de las preparaciones demostró la existencia de 5 bandas mayores migrando a 200, 175, 157, 86 y 66 kDa, tanto en los CR como en los CS. La inmunotransferencia indicó la presencia de las mismas 5 bandas, que fueron reconocidas por el antisuero antifimbria S. mutans. Adicionalmente, la banda de 175 kDa fue reconocida por el anticuerpo anti-P1. El análisis de la inmunotransferencia demostró que los niveles de componentes reactivos con las fimbrias y P1 fueron mayores en los sujetos CS que en los CR. Estos resultados sugieren que las diferencias existentes en la composición de las fimbrias de S. mutans de CS y CR pueden jugar un papel importante en la virulencia de este microorganismo en la caries dental.

Influence of heat inactivation of human serum on the opsonization of Streptococcus mutans.

Phagocytosis of bacteria, such as Streptococcus mutans, is important to host defense. One mechanism by which phagocytosis can be enhanced is by antibody or complement-mediated opsonization of bacteria. Many studies utilize opsonization of bacteria to enhance a cellular response, but little information has been found examining methodology or validity of the opsonization process following the denaturization of the serum. Human serum was inactivated by heat in order to disrupt the classical and alternative pathways of the complement cascade. S. mutans isolated from human subjects were opsonized with heat-inactivated human serum before exposing them to viable neutrophils in vitro. Luminol-dependent chemiluminescence (CL) was used to measure neutrophil activation. Human serum used to opsonize the bacteria was denatured by incubation at 57 degrees C for intervals of 30 and 60 min to inactivate complement. The results from the opsonization data indicated that there was significantly increased CL with 60-min inactivation of the serum (34% increase in mean integration mV.min; p < or = 0.05) over the nonopsonized control. This indicated a successful opsonization of the bacteria. In addition, the data demonstrate that the inactivation of serum requires a minimum of 60 min at 57 degrees C to disrupt the complement cascade, while 30- and 15-min inactivations produced no significant increase in CL activity over the control. Standard sandwich ELISA assays, detecting complement binding to S. mutans, confirmed successful heat inactivation of serum showing a significant decrease (p < or = 0.001) in complement binding to S. mutans after 30 min, but could not explain the increased CL response after 60-min heat deactivation of the serum.

Effect of exercise on milk immunoglobulin A.

The major immunoglobulin (Ig) in human secretions is IgA. The immune properties of breast milk are well documented; however, the immunological influence of maximal exercise has not been established. The objective of this study was to investigate the role that exercise has on breast milk IgA and IgA subclasses. Breast milk was collected from 17 lactating women (4.6 +/- 2.3 months postpartum) before and after randomized exercise and control periods. The exercise treatment was a maximal graded treadmill test (VO2max = 30.3 +/- 5.7 mL x min-1 x kg-1). Milk was collected at rest, the breasts were emptied, and samples obtained 10, 30, and 60 min following either exercise or 30-min control rest periods. IgA concentrations were established by enzyme-linked immunosorbent assay. The results indicated that samples taken 10 and 30 min after the exercise period had significantly lower (P < or = 0.05) milk IgA concentrations (21.0 +/- 1.8 and 21.8 +/- 1.4 microg x mL-1, respectively) than the corresponding control resting samples (52.8 +/- 3.5 and 79.3 +/- 7.7 microg x mL-1). The exercise samples were similar to the control samples at 60 min (134.0 +/- 24.6 and 116.0 +/- 15.4 microg x mL-1, respectively), indicating that by 1 h, milk IgA production had recovered. The IgA1 data showed a similar significant decrease (P < or = 0.05) at 10 min postexercise, which also returned to control concentrations by the 30- and 60-min collection intervals. There was no significant change in the milk IgA2 concentrations at any of the time points studied. Milk IgA concentrations increased significantly in both exercise and resting control groups after the breasts were emptied, suggesting that breast emptying stimulated milk IgA synthesis. The results provide evidence that exercise alters milk IgA and IgA1 concentrations for 10-30 min after exhaustive exercise, but recovers by 1 h, and provide additional support for exercise effects on the mucosal immune system.

Effect of nicotine on secretory component synthesis by secretory epithelial cells.

Previously, we reported that secretory component (SC), lactoferrin (LF), and lysozyme (LY) levels were significantly lower in saliva from smokeless tobacco (ST) users than in saliva from control non-tobacco users. However, the levels of salivary immunoglobulin A were significantly higher, albeit with an altered attachment of SC, in ST users than in control subjects. SC, LF, and LY are synthesized by secretory epithelial cells at mucosal sites adjacent to lymphocyte regions. In the present report, HT-29 human epithelial cells, cultured with various concentrations of an ST aqueous extract or pure nicotine (0 to 1 mg/ml) or cotinine (0 to 5 mg/ml), exhibited significantly lower levels of cell-associated cell lysate (CL) and secreted culture supernatant (CS) SC, LF, and LY than cells cultured without ST components. Nicotine significantly decreased (P < or = 0.05) the synthesis of SC by 20 to 100%, LF by 20 to 60%, and LY by 5 to 75% of CL and CS control values. Studies also indicated significant decreases (P < or = 0.05) in SC, LF, and LY levels in both CL and CS of cells cultured with ST aqueous extract or cotinine. Total cell numbers and metabolic activity significantly decreased primarily when cells were incubated with higher concentrations of ST extract, nicotine, or cotinine. The addition of human recombinant interleukin-4 or gamma interferon diminished the effects ST had on HT-29 cell synthesis of SC, LF, and LY. Our data indicate that nicotine, cotinine, and ST have an adverse effect on synthesis and secretion of SC, LF, and LY. These effects were below ST concentrations found to be cytotoxic for secretory epithelial cells. Furthermore, addition of interleukin-4 or gamma interferon reduced the suppressive effect of ST on synthesis or secretion of SC, LF, or LY.

Characterization of preparations enriched for Streptococcus mutans fimbriae: salivary immunoglobulin A antibodies in caries-free and caries-active subjects.

The ability of bacteria to adhere to salivary pellicle-coated enamel tooth surfaces is a critical step in oral bacterial colonization. Oral bacteria adhere to receptors of host origin in salivary pellicle. Streptococcus mutans has been identified as the major etiological agent of human dental caries and composes a significant proportion of the oral streptococci in carious lesions. Bacterial fimbriae are small (100 to 300 nm) hairlike appendages emanating from the cell surface. Preparations enriched for S. mutans fimbriae were isolated by a shearing technique and alternating high- and low-speed centrifugations. A representative fimbrial preparation had two distinct double bands comprising four proteins of approximately 100 to 200 kDa and one faint band at 40 kDa on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblots and had demonstrable glucosyltransferase activity. Rabbit antisera raised against the preparation specifically stained the fuzzy coat of S. mutans, demonstrating short fimbria-like structures protruding 100 to 200 nm from the cell surface. Controls without antifimbria antibody did not exhibit this staining. There were significantly higher (P < or = 0.05) levels of salivary immunoglobulin A, but not serum immunoglobulin G, antibodies to the enriched S. mutans fimbria preparation by enzyme-linked immunosorbent assay from caries-free subjects than from caries-active subjects. The results suggest that S. mutans fimbriae may be an important adherence factor to which caries-free subjects mount a protective salivary immune response.

IgA antibodies to Streptococcus mutans in caries-resistant and -susceptible children.

Previous studies have shown a positive correlation between salivary IgA antibody levels to Streptococcus mutans and caries resistance in adults. In this study, enzyme-linked immunosorbent assay (ELISA) was used to compare IgA antibody levels with S. mutans in whole and parotid saliva from 20 caries-susceptible (CS; DMFS > 5) and 20 caries-resistant (CR; DMFS < or = 1) children (aged 7-11 years). Whole salivary S. mutans numbers were significantly greater (P < or = 0.05) in the CS group (mean of 31.2% of total oral streptococci) than in the CR group (mean of 1.6% of total oral streptococci). Whole saliva, but not parotid saliva, from CR children had significantly higher (P < or = 0.05) levels of IgA antibodies to S. mutans than saliva from CS children. These results suggest that salivary IgA antibodies to S. mutans may play a role in natural protection from dental caries in children and that the source of increased salivary IgA antibody in CR children may be either the minor, submandibular, or sublingual salivary glands.