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Because of the tradition of using honey as an antimicrobial medicament, we investigated the effect of natural honey (NH) on Streptococcus mutans growth, viability, and biofilm formation compared to that of an artificial honey (AH). AH contained the sugars at the concentrations reported for NH. NH and AH concentrations were obtained by serial dilution with tryptic soy broth (TSB). Several concentrations of NH and AH were tested for inhibition of bacterial growth, viability, and biofilm formation after inoculation with S. mutans UA159 in 96-well microtiter plates to obtain absorbance and CFU values. Overall, NH supported significantly less ( P < 0.05) bacterial growth than AH at 25 and 12.5% concentrations. At 50 and 25% concentrations, both honey groups provided significantly less bacterial growth and biofilm formation than the TSB control. For bacterial viability, the results for all honey concentrations except 50% NH were not significantly different from those for the TSB control. NH was able to decrease the maximum velocity of S. mutans growth compared to AH. In summary, NH demonstrated more inhibition of bacterial growth, viability, and biofilm formation than AH. This study highlights the potential antibacterial properties of NH and could suggest that the antimicrobial mechanism of NH is not solely due to its high sugar content.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis identified enolase as a cell surface component of Streptococcus mutans , which was confirmed by enzyme-linked immunosorbent assay, Western blotting, and transmission electron microscopy. Surface enolase was demonstrated to bind to human plasminogen and salivary mucin MG2. The results suggested a role for enolase in S. mutans attachment, clearance, or breach of the bloodstream barrier.

Streptococcus mutansis present in the saliva of most individuals and is modified by salivary components bound to the cells. These saliva-boundS. mutansare swallowed, exposed to high levels of acidity in the stomach, and presented to the common mucosal immune system. Much effort has been directed to identifying the specificS. mutansantigens that the mucosal immune responses are directed against. However, little is known about the host-altered antigenic determinants that the mucosal immune system recognizes. The immunogenicity of gastrically intubated untreatedS. mutanscells, cells coated with whole human saliva, cells treated with HCl (pH 2.0), and saliva-coated and acid-treated cells in mice was investigated. Saliva and serum samples were assayed by enzyme linked immunosorbent assay for immunoglobulin A (IgA) and IgG antibodies, respectively, against the untreated or treatedS. mutanscells. In general, the levels of salivary IgA and serum IgG antibodies to the antigen against which the mice were immunized were significantly higher (P≤ 0.05). In addition, human saliva and serum samples from 12 subjects were assayed for naturally occurring antibody against the untreated or treatedS. mutanscells. In every case, significantly higher reactivity was directed against the saliva-coated and acid-treated cells followed by the saliva-coatedS. mutans. These results provide evidence for the altered immunogenicity of swallowedS. mutansin humans by coating nativeS. mutansantigens with salivary components and/or denaturing surfaceS. mutansantigens in the acidic environment of the stomach, which would lead to an immune response to modifiedS. mutansdeterminants and not to nativeS. mutansantigens.

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.

Identification of inexpensive and technically simple immunological tests useful in predicting the progression to AIDS in human immunodeficiency virus (HIV)-infected patients would be especially welcome in developing countries, in which 80% of HIV-infected patients reside and health budgets are low. In the current study, we evaluated CD4+ and total lymphocyte counts and the concentrations in serum of beta 2-microglobulin, p24 antigen, and immunoglobulin A (IgA) as predictors of disease progression in 74 Panamanian HIV-positive patients and 50 HIV-negative healthy individuals. Total lymphocyte and CD4(+)-cell counts for AIDS patients (1,451 +/- 811 cells/microliters, P < 0.001, and 238 +/- 392 cells/microliters, P < 0.0001, respectively and asymptomatic patients (2,393 +/- 664 cells/microliters, P > 0.05, and 784 +/- 475 cells/microliters, P < 0.001, respectively) were lower than those observed for healthy subjects (2,596 +/- 631 cells/microliters and 1,120 +/- 296 cells/microliters, respectively). The levels of beta 2-microglobulin and IgA in serum were significantly elevated in patients with AIDS (5.7 +/- 3.6mg/liter, P < 0.001, and 541 +/- 265 mg/dl, P < 0.0002, respectively) and asymptomatic infected subjects (3.4 +/- 2.1 mg/liter, P = 0.001, and 436 +/- 216 mg/dl, P < 0.0001, respectively) compared with the levels in healthy subjects (2.2 +/- 0.7 mg/liter and 204 +/- 113 mg/dl, respectively). Nonstatistically significant differences (P > 0.05) for concentrations of p24 antigen between asymptomatic infected patients (29 +/- 13 pg/ml) and AIDS patients (40 +/- 23 pg/ml) were observed. Total lymphocyte counts of 1,750 cells/microliters or less, CD4 counts of 200 cells/microliters or less, beta 2-microglobulin concentrations in serum of 4 mg/liter or higher, concentrations of IgA in serum of 450 mg/dl or higher, and the presence in serum of p24 antigen were correlated with elevated risks for developing AIDS. Monitoring both total lymphocytes and beta 2-microglobulin identified 91% of the AIDS patients; these assays may allow reductions in the annual number of CD4(+)-cell evaluations and the costs associated with monitoring both total lymphocytes and beta 2-microglobulin identified 91% of the AIDS patients; these assays may allow reductions in the annual number of CD4(+)-cell evaluations and the costs associated with monitoring the immune status of HIV-positive patients.

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.

Streptococcus mutans is the major pathogenic agent causing dental caries (3, 5, 6). The unidentified 65-kDa cell surface protein (SP) of S. mutans is immunodominant (2) and is present in SP preparations from all S. mutans c isolates examined (8). Crude SPs were obtained from 9 liters of S. mutans A32-2 (8) grown in Todd-Hewitt broth (THB) plus 1% dextrose for 18 h at 37°C in 5% CO2 and 95% air. Cells were pelleted at 16,000 × g at 4°C for 10 min, washed once gently in buffer A (10 mM phosphate-buffered saline, 1 mM CaCl2, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2), and stored as a pellet at −20°C overnight. Frozen cells were thawed and then suspended in buffer A, and SP was removed with three 1-min cycles at high speed in a Waring blender. After blending, the sample was centrifuged as above to remove intact cells and cell debris. The proteins in the supernatant were isolated by centrifugation (110,000 × g, 4°C, 2.5 h). The pellet containing the SP was resuspended in buffer A and centrifuged (16,000 × g, 4°C, 10 min) to remove cellular debris. The supernatant was divided into aliquots and stored at −80°C. The protein concentration was determined using the Quantipro bicinchoninic acid protein assay (Sigma Chemical Co., St. Louis, MO). CNBr sequencing (Biochemistry Biotechnology Facility, Indiana University Purdue University at Indianapolis) of the 65-kDa band recovered from separation of SP by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis resulted in two 11-amino-acid (aa) peptides (MSRQAKAVNIP and MQSPTEFNEDK) (Fig. ​(Fig.1).1). The gene encoding this SP (SmaA) was localized to the open reading frame (ORF) at position SMU.609 (1) on the S. mutans UA159 genome. This ORF is designated as a “putative 40K cell wall protein precursor” based on sequence homology to the 40-kDa protein (p40f) from S. mutans OMZ175 (7). Visual comparison of the sequences of these homologous ORFs (Fig. ​(Fig.2)2) revealed that the predicted size difference is actually due to a divergence at a single base, absent in the OMZ175 genome, which produces a reading frameshift and subsequent earlier stop codon in OMZ175. Additional analysis of SMU.609 revealed three copies of a 46-aa repeat region with homology to SH3b, a domain associated with murein hydrolase function (13), and two 88-aa direct repeats of unknown function (Fig. ​(Fig.33). FIG. 1. Protein sequence analysis of the 65-kDa surface protein. A. Coomassie blue-stained 10% SDS-polyacrylamide gel of S. mutans surface proteins. Lane 1, OMZ175; lane 3, A32-2; lanes 2 and 4, molecular mass markers (size in kilodaltons indicated on right). ... FIG. 2. Site of OMZ175 deletion mutation. The sequence surrounding the deletion mutation in the OMZ175 gene encoding p40f is compared to the homologous sequence from UA159 encoding SmaA. This deletion leads to an early stop codon in p40f compared to SmaA. The ... FIG. 3. Comparison of direct repeat regions. A. Short repeats (46 aa), three from S. mutans UA159 SmaA (65-1s, 65-2s, and 65-3s), one from S. mutans OMZ175 p40f (40-1s), and four from homologous Streptococcus agalactiae 60-kDa murein hydrolase protein Bsp (9 ... The OMZ175 deletion mutation was recreated in UA159 by allelic replacement. The Em resistance marker PcEm (860 bp) was kindly provided by D. A. Morrison, University of Illinois at Chicago. Briefly, a 1,920-bp fragment, 40-UP, was amplified from OMZ175 genomic DNA by using primers p65-up-P1 (5′-TTAAAGGGTGAGCTGAG-3′) and p40-up-P2 (5′-GGCGCGCCTGTTTAGTAAAGTGATAGG-3′) (containing an AscI site at the 5′ end). This fragment includes 1,096 bp of sequence immediately upstream from the start site, to the nucleotide which corresponds to the site of the deletion mutation in the homologous gene from OMZ175. Another fragment (65-DN) containing the 960-bp sequence immediately downstream from SMU.609 was amplified from UA159 DNA using primers p65-dn-P3 (5′-GCTGGCCGGCCAGGTTAGGATGACAAAATCCTGAC-3′) (with an FseI site at the 5′ end) and p65-dn-P4 (5′-T GGCGGCTGCATTAGCTCCTGAAC-3′). PcEm (860 bp) was amplified using primers Em-P1 (5′-GGCGCGCCCCGGGCCCAAAATTTGTTTGAT-3′) and Em-P2 (5′-GCTGGCCGGCCAGTCGGCAGCGA-3′) with AscI and FseI sites engineered into their 5′ ends, respectively. The amplicons were subjected to restriction enzyme digestion and subsequent ligation to produce a 40-UP::PcEm::65-DN fragment. The ligated product was directly transformed into S. mutans wild-type UA159 using the biofilm transformation technique (4). Following double-crossover homologous recombination, the region of SMU.609 from nucleotide 825 to the 3′ end was replaced by the Em cassette (PcEm). The transformant UA159D65::fPcEm was selected on THB agar plus Em (10 μg/ml). Confirmation of the Emr marker at the desired locus was determined by PCR. Murein hydrolase activity of the SP of S. mutans was examined using the peptidoglycan zymogram assay (Fig. ​(Fig.4).4). Crude SP-enriched fractions of S. mutans A32-2, UA159, UA159D65::fPcEm, NG8, SrtA− (a sortase-knockout mutant of NG8 obtained from Song Lee, Dalhousie University, Halifax, Nova Scotia, Canada), PC3370 (an antigen I/II-knockout mutant of NG8 obtained from L. J. Brady, University of Florida), and OMZ175 (obtained from Suzanne Michalek, University of Alabama at Birmingham) were used as the enzyme sources. The peptidoglycan was obtained from a 3-liter (THB plus 1% dextrose) overnight culture of S. mutans washed in 10 mM HEPES, pH 6.8, and boiled in 4% SDS and 10 mM HEPES for 45 min (12). The mixture was cooled to room temperature and centrifuged at 70,000 × g for 40 min at 20°C, and the pellet was washed with 2 M NaCl. The pellet was weighed and resuspended in 2 M NaCl to a concentration of 185 mg/ml and sonicated for 5 min, on ice, at the maximum setting. The peptidoglycan was treated with 48% hydrofluoric acid (HF) at 4°C overnight and then washed 3× with distilled H2O and resuspended in the original volume of 2 M NaCl. The HF-treated peptidoglycan (0.5 ml) was added to a 7.5% SDS-polyacrylamide gel prior to solidification. The nondenatured SPs were separated on the gel at 150 V for 1 h. FIG. 4. Zymogram activity gel. HF-treated peptidoglycan from S. mutans UA159 was incorporated into a 7.5% SDS-polyacrylamide gel. Nondenatured surface proteins were separated on the gel. Lane 1, UA159; lane 2, UA159D65::fPcEm; lane 3, NG8; lane 4, SrtA− ... Two bands of activity, corresponding to 125 and 90 kDa, respectively, were present in SPs from A32-2, NG8, UA159, and PC3370. UA159D65::fPcEm, OMZ175, and SrtA− SP preparations contain the upper (125-kDa equivalent band) but not the lower (90-kDa) band of autolytic activity. The autolytic activity associated with the lower band but not the upper band is present in the supernatant of SrtA− cultures (data not shown). The loss of the 90-kDa autolytic band in the sortase mutant SrtA− suggests that the enzyme or enzyme complex uses this transpeptidase cell wall anchoring system (10). Sequence analysis suggests a 5-aa sequence, LPAQG, at nucleotide 296 as a possible candidate for the LPXTG canonical recognition sequence for sortase. Two bacteriolytic bands of similar sizes (107 and 79 kDa) were recently reported using whole-cell S. mutans extracts (14) separated on HF-treated peptidoglycan. Subsequently, the upper band was reported to be associated with AltA (homologous to SMU.689), while the lower band was predicted to be a proteolytic product of AltA (11). Our data suggest that there are two separate autolytic activities present in the SP fractions of S. mutans, one of which is inactivated by a mutation in SMU.609 and is linked to the cell surface via the sortase system.

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.

Streptococcus mutans has been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination of S. mutans surface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected with S. mutans at 18 to 20 days of age, were intranasally immunized with a mixture of S. mutans surface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly ( P < 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recovered S. mutans cells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture of S. mutans surface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries.

To evaluate the feasibility of mucosal immunization against Pneumocystis carinii (Pc) experimental infection, female BALB/c mice were intranasally immunized three times with soluble Pc antigens plus cholera toxin fraction B (Pc-CTB); control groups received either Pc antigen, CTB, or phosphate-buffered saline (PBS) alone. Two weeks after the last immunization, five animals from each group were sacrificed, and cellular and humoral immune responses were evaluated. The remaining five mice were CD4 depleted using a monoclonal antibody against mouse CD4 and inoculated with viable Pc. Significantly higher specific lymphoproliferative responses from tracheobronchial lymph node cells, immunoglobulin M (IgM) and IgG antibody levels in serum, and bronchoalveolar lavage (BAL)-derived IgA antibody concentrations were observed in the Pc-CTB group of mice relative to control groups ( P < 0.01). Five weeks after challenge, no Pc organisms were observed in the lung smears of the Pc-CTB group, while the animals receiving antigen, adjuvant, or PBS had progressively higher numbers of Pc microorganisms. By Western blot analysis, a strongly reactive 55- to 60-kDa antigen was recognized by BAL IgA and by serum IgG. In summary, mucosal immunization elicited specific cellular and humoral immune responses and protected against Pc lung infection after immunosuppression.

Ingestion of a vaccine containing killed Streptococcus mutans, originally isolated from each volunteer, daily for 10 consecutive days induced increased levels of specific secretory immunoglobulin A (sIgA) antibodies to S. mutans cells and two cell surface proteins, glucosyltransferase and surface antigen I/II, in parotid saliva and tears of four healthy males and in parotid saliva, tears, colostrum, and milk of a pregnant woman. In addition, these antibodies inhibited glucosyltransferase activity. Both IgA1 and IgA2 antibodies were induced. The levels of IgA antibodies in all secretions remained significantly above preimmunization levels for more than 50 days after oral administration of antigen. A second series of immunizations for 7 consecutive days resulted in even higher levels of sIgA antibodies, which peaked earlier and persisted longer than those observed after the primary immunizations. No increase in levels of antibodies in serum were detected in any subject. Antibodies reactive with human heart and kidney antigens could not be detected in saliva, tears, colostrum, milk, or serum samples collected at any time during the immunization regimen. The numbers of viable S. mutans organisms in dental plaque and whole saliva decreased after each series of immunizations, which correlated with increased levels of IgA antibodies in saliva, suggesting that IgA antibodies in saliva were responsible for the reduced adherence of this bacterium. These results indicate that ingested S. mutans antigen induces secretion of specific IgA1 and IgA2 antibodies in saliva, tears, colostrum, and milk, providing further evidence for the existence of a common mucosal immune system.

Streptococcus mutans 6715 ribosomes disrupted in a Braun homogenizer were isolated in sodium dodecyl sulfate by differential centrifugation. This preparation contained 80% RNA and 20% protein, and carbohydrate was not detected by phenol-sulfuric acid and methyl pentose assays. The sedimentation coefficient of the ribosomes was 70S. After dialysis in 0.01 M phosphate buffer containing 10(-4) M MgCl2, the ribosomes dissociated into 54S and 32S particles. Leukocytes from rabbits immunized intramuscularly with the ribosomal preparation showed transformation and migration indices of 13.0 and 0.71, which were significantly different (P less than 0.05) from the respective indices of 0.9 and 0.98 in nonimmunized animals. Hyperimmune serum from these rabbits agglutinated representative Formalin-killed strains of all seven serotypes of S. mutans, inhibited adherence of live S. mutans 6715 to glass, and agglutinated S. mutans 6715 ribosomes adsorbed upon erythrocytes. These findings suggested that animals immunized with S. mutans ribosomes may be protected from caries caused by any of the seven serotypes of this organism.

Antibodies induced in sera and saliva of rats and rabbits immunized with ribosomal preparations from Streptococcus mutans 6715 inhibited transport of glucose by the phosphotransferase system by greater than 60%, acid production from sucrose by greater than 95%, and growth of the homologous S. mutans by greater than 59%. Inhibition of growth and acid production by immune sera and saliva were abrogated by prior adsorption with S. mutans 6715 whole cells, glucosyltransferase, lipoteichoic acid, or alpha 1-6 or alpha 1-3 dextran. These results indicate that antibodies induced to an S. mutans ribosomal preparation react with cell surface determinants and suggest that the antibodies inhibit sucrose-induced acid formation and growth of virulent S. mutans by neutralizing the glucose-phosphotransferase system.