Binding properties of Streptococcus gordonii SspA and SspB (antigen I/II family) polypeptides expressed on the cell surface of Lactococcus lactis MG1363

The oral bacterium Streptococcus gordonii expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as Actinomyces naeslundii. To determine if SspA and SspB have differential binding properties, the coding sequences of the sspA and sspB genes were cloned into expression plasmid vector pTREX1-usp45LS to generate pTREX1-sspA and pTREX1-sspB, respectively, and the Ssp polypeptides were displayed on the cell surface of Lactococcus lactis MG1363. Lactococcal cells expressing similar levels of surface SspA or SspB polypeptide were then compared for their abilities to adhere to a range of antigen I/II polypeptide substrates. More than twice as many L. lactis cells expressing SspA bound to immobilized salivary agglutinin glycoprotein (SAG) as did L. lactis cells expressing SspB. In contrast, lactococci expressing SspB adhered twice as well as lactococci producing SspA to collagen type I and to Candida albicans. The binding of A. naeslundii to lactococci was only weakly enhanced by surface expression of Ssp polypeptides. L. lactis(pTREX1-sspB) cells bound in greater numbers to SAG than did Enterococcus faecalis JH2-2 cells expressing SspB from pAM401EB-5. The results suggest that SspA and SspB have markedly different binding affinities for their oral substrates and thus may function to promote site diversity in colonization by S. gordonii.     

Identification of a 100-kilodalton putative coaggregation-mediating adhesin of Streptococcus gordonii DL1 (Challis)

Streptococcus gordonii DL1 (Challis) bears coaggregation-relevant surface proteins which mediate lactose-inhibitable coaggregations with other streptococci. Six spontaneously occurring coaggregation-defective (Cog-) mutants of wild-type strain S. gordonii DL1 unable to coaggregate with wild-type streptococcal partners were characterized. Antiserum raised against wild-type cells and absorbed with Cog- cells specifically blocked lactose-inhibitable coaggregations between S. gordonii DL1 and its streptococcal partner strains; it did not block lactose-noninhibitable coaggregations with actinomyces partners. Surface proteins were released from the cells by mild sonication treatment and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 100-kDa surface protein from S. gordonii DL1 was identified by immunoblot analysis with the mutant-absorbed antiserum. Each of the six Cog- mutants lacked the 100-kDa protein. Several other oral viridans streptococci that exhibit intrageneric lactose-inhibitable coaggregations expressed an immunoreactive protein with about the same size as the 100-kDa putative adhesin. It is proposed that the 100-kDa protein is the adhesin which mediates coaggregation between S. gordonii DL1 and its streptococcal partners. The role of this putative adhesin in accretion of streptococci in early colonization of the tooth surface is discussed.     

Coaggregation of oral lactobacilli with streptococci from the oral cavity

The ability of oral lactobacilli to coaggregate with streptococci and actinomycetes was investigated. Of the 7 species of lactobacilli studied, only two were capable of coaggregation and the coaggregation was restricted to streptococci. Lactobacillus salivarius strains (2/4) coaggregated with Streptococcus salivarius, Streptococcus gordonii, Streptococcus crista and tufted Streptococcus sanguis II strains. Lactobacillus fermentum (2/3) coaggregated with S. gordonii and S. sanguis. The coaggregation between L. salivarius and S. salivarius, S. gordonii or tufted S. sanguis II strains was mediated by a protein on the surface of the lactobacilli and was not inhibited by lactose. The coaggregation between L. fermentum and the streptococci was mediated by protein on the surface of the streptococci and was inhibited by lactose.     

Expression of functional Porphyromonas gingivalis fimbrillin polypeptide domains on the surface of Streptococcus gordonii

Genetically engineering bacteria to express surface proteins which can antagonize the colonization of other microorganisms is a promising strategy for altering bacterial environments. The fimbriae of Porphyromonas gingivalis play an important role in the pathogenesis of periodontal diseases. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to be an important virulence factor, which likely promotes adherence of the bacterium to saliva-coated oral surfaces and induces host responses. Immunization of gnotobiotic rats with synthetic peptides based on the predicted amino acid sequence of fimbrillin has also been shown to elicit a specific immune response and protection against P. gingivalis-associated periodontal destruction. In this study we engineered the human oral commensal organism Streptococcus gordonii to surface express subdomains of the fimbrillin polypeptide fused to the anchor region of streptococcal M6 protein. The resulting recombinant S. gordonii strains expressing P. gingivalis fimbrillin bound saliva-coated hydroxyapatite in a concentration-dependent manner and inhibited binding of P. gingivalis to saliva-coated hydroxyapatite. Moreover, the recombinant S. gordonii strains were capable of eliciting a P. gingivalis fimbrillin-specific immune response in rabbits. These results show that functional and immunologically reactive P. gingivalis fimbrillin polypeptides can be expressed on the surface of S. gordonii. The recombinant fimbrillin-expressing S. gordonii strains may provide an effective vaccine or a vehicle for replacement therapy against P. gingivalis. These experiments demonstrated the feasibility of expressing biologically active agents (antigens or adhesin molecules) by genetically engineered streptococci. Such genetically engineered organisms can be utilized to modulate the microenvironment of the oral cavity.     

Pathway for uptake and degradation of X-prolyl tripeptides in Streptococcus mutans VA-29R and Streptococcus sanguis ATCC 10556

The growth of Streptococcus mutans and Streptococcus sanguis in the oral environment requires that these micro-organisms be able to degrade salivary proteins and to assimilate the resulting peptides as an amino nitrogen source. Our research is aimed at the definition of the proteolytic enzyme systems in these oral streptococci which allow them to utilize such substrates. In the present work, the nature of the hydrolytic activity expressed by S. mutans VA-29R and S. sanguis ATCC 10556 against X-Pro4-nitroanilide and X-Pro-Y tripeptide substrates was investigated. This activity was predominantly associated with a cytoplasmic dipeptidyl peptidase which preferentially catalyzes the release of an N-terminal dipeptide from substrates in which proline is the penultimate residue. These streptococci also possess a second cytoplasmic peptidase, pepD, which catalyzes the hydrolysis of X-Pro dipeptides. We found that Gly-Pro-Ala or Ala-Pro-Gly were transported into the bacterial cells only when an energy source such as glucose was present. Peptide uptake was time-dependent, and selective exodus of peptide-derived amino acids from the bacterial cells occurred during peptide uptake. Results from these studies provide evidence that S. mutans VA-29R and S. sanguis ATCC 10556 possess a pathway for the complete degradation of X-Pro tripeptides. Transport of the peptides into cells prior to hydrolysis provides an efficient way by which all amino acids of a peptide may be obtained at an energy expense equivalent to that associated with the transport of just one amino acid. In light of the abundance of proline in salivary polypeptides, this degradative pathway could be an important component in the proteolytic pathway for salivary polypeptide utilization in these oral streptococci.     

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction

Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.     

Tonsillar application of killed Streptococcus mutans induces specific antibodies in rabbit saliva and blood plasma without inducing a cross-reacting antibody to human cardiac muscle

When Streptococcus mutans cells are injected into the skeletal muscle of rabbits, an antibody against human cardiac muscle, as well as an anti-S. mutans antibody, is induced in blood plasma. Our previous study showed that when sheep erythrocytes are applied to palatine tonsils, an antibody against the applied cells is induced both in blood plasma and saliva. This antibody has no activity against cardiac muscle. It is not clear, however, if S. mutans application to the tonsils evokes an antibody response against cardiac muscle. In this study, we immunized rabbits against S. mutans or Streptococcus sobrinus by tonsillar application or by intramuscular injection every 3 days for 6 weeks. Tonsillar applications of formalin-killed cells of S. mutans induced saliva immunoglobulin A (IgA) and blood plasma IgG to the applied cells. In contrast, intramuscular injection of such cells induced only blood plasma IgG. When the route of immunization was intramuscular injection, antibodies in blood plasma cross-reacted with cardiac muscle. By enzyme-immunohistochemistry and Ouchterlony immunodiffusion tests, no cross-reaction to cardiac muscle was observed with the antibody in saliva or in blood plasma after the tonsillar applications. Western blotting of the S. mutans antigen showed that blood plasma from rabbits injected with S. mutans reacted with antigens of 46, 52, 62, and 85 kDa, while that from rabbits subjected to tonsillar application of S. mutans did not react with these bands. Similar results were obtained for S. sobrinus applications. Thus, tonsillar applications of mutants group streptococci induce antibodies differing in antigen specificity and do not induce any cross-reacting antibody to cardiac muscle.     

Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity

The in vitro effect of citric acid application on periodontally involved root surfaces was investigated using scanning electron microscopy. Teeth were selected following specific criteria. The specimens were prepared for observation, and the features of the root surface were studied. The results demonstrated that a 3-minute application of citric acid (pH = 1.0) to the root surface after scaling and root planing demineralized the outer root surface, opened the dentinal tubules, and exposed collagen fibers. Scaling and root planing alone left the surface with a smear layer, scattered islands of cementum, and no collagen fibers or exposed dentinal tubules.     

Influence of salivary components and extracellular polysaccharide synthesis from sucrose on the attachment of Streptococcus mutans 6715 to hydroxyapatite surfaces

The adsorption of (3)H-labeled Streptococcus mutans 6715 cells to disks of hydroxyapatite (HA) was studied. The number of streptococci that adsorbed was logarithmically related to the concentration of cells available up to at least 2 x 10(8) per ml; equilibrium occurred within 45 min. Assay reliability was verified by direct scanning electron microscopic counts. Untreated HA disks exposed to buffered saline (PBS)-suspended streptococci at a concentration of 1.1 x 10(8) per ml absorbed 3.2 x 10(6) cells per cm(2); approximately 3% of the surface area was, therefore, occupied by adsorbed organisms. The presence of adsorbed salivary components on HA reduced the number of attaching S. mutans cells by half. When S. mutans cells were suspended in saliva to mimic conditions existing in the mouth, the number of streptococci adsorbing to saliva-treated HA was reduced more than 30-fold compared to untreated HA. Approximately one-half of the streptococci adsorbed to untreated or to saliva-treated HA disks could be desorbed over a 4-h period with 0.067 M phosphate buffer. S. mutans cells exposed to sucrose to permit extracellular polysaccharide synthesis before or during adsorption attached in fewer numbers to both saliva-treated and untreated HA than PBS-treated organisms. When S. mutans cells adsorbed on untreated HA were exposed to sucrose, fewer organisms could be desorbed; thus, in situ polysaccharide synthesis promoted their more firm attachment to untreated HA. However, when saliva-suspended streptococci were adsorbed to saliva-treated HA surfaces, exposure to sucrose before or subsequent to adsorption did not promote more firm attachment. Evidently, the powerful adherence-inhibiting and desorptive effects of salivary components overshadowed any promoting effects attributable to glucan synthesis from sucrose. Similarly, no differences were noted in the desorption of S. mutans cells from human teeth after exposure to sucrose, glucose, or PBS relative to a strain of Streptococcus mitis (S. mitior). Thus, no evidence was obtained to support the hypothesis that glucan synthesis from sucrose was essential for, or promoted, the attachment of S. mutans cells to HA surfaces exposed to saliva or to the smooth surfaces of human teeth.     

Cross-reactions of Streptococcus mutans due to cell wall teichoic acid

Antisera to the whole cells of Streptococcus mutans cross-reacted with antigen extracts from four other gram-positive species, as well as with those of three other oral streptococci. Similarly, antisera to these bacteria cross-reacted with extracts from S. mutans and with those from each other. Using a purified phenol extract of the walls of S. mutans, which was identified by chemical, immunochemical, and enzymatic analyses as glycerol teichoic acid, the cross-reactions were shown to be specific for a determinant of the teichoic acid backbone. Results were confirmed in immunodiffusion tests where clear bands of identify were shown. These observations point out the need for caution in sereological research empolying extracts of gram-positive bacteria and may be of interest in investigations of periodontal disease.     

Generation of bovine immune colostrum against Streptococcus mutans and Streptococcus sobrinus and its effect on glucose uptake and extracellular polysaccharide formation by mutans streptococci

Due to potential side-effects of active immunization by cariogenic mutans streptococci, oral administration of passively-derived antibodies could be a more acceptable way to reduce colonization and virulence of these microorganisms in human dentition. The aim of this study was to produce antistreptococcal immunoglobulins into bovine colostrum and explore the possible antibacterial mechanisms of these immunoglobulins against mutans streptococci. Specific serum IgG antibodies to whole cell antigens of both Streptococcus mutans and Streptococcus sobrinus increased rapidly in cows during immunization and were high also in the final whey-product. Low concentration (0.5% w/v) of bovine immune preparation inhibited significantly the incorporation of [14C]glucose by both S. mutans and S. sobrinus. Higher concentration (> 1%) was needed to inhibit the glucosyltransferase or fructosyltransferase activities of these bacteria. No such inhibitory effects were observed with the control preparation from the non-immunized cows. Our results indicate that bovine immune colostrum has a significant inhibitory potential against mutans streptococci, apparently dependent on the presence of specific IgG antibodies against S. mutans and S. sobrinus.     

Acid tolerance response and survival by oral bacteria

Using 21 species of oral bacteria, representing six acidogenic genera, we undertook to determine whether the pH-limiting exponential growth is related to the ability of the organisms to generate an acid-tolerance response that results in enhanced survival at low pH. The lower pH limit of exponential growth varied by more than two units with that of Neisseria A182 at pH 6.34; growth of Lactobacillus casei RB1014 stopped at pH 3.81, with species of Actinomyces, Enterococcus, Prevotella and Streptococcus falling between these limits. The working hypothesis was that the organisms with the higher pH limits for growth are unable to respond to acidic environments in order to survive, whereas the more aciduric organisms would possess or acquire acid tolerance. Adaptation to acid tolerance was tested by determining whether the prior exposure of exponential-phase cells to a low, sub-lethal pH would trigger the induction of a mechanism that would enhance survival at a pH killing pH 7.5 control cells. The killing pH varied from pH 4.5 for Prevotella intermedia ATCC 25611 to pH 2.3 for the three Lactobacillus casei strains in the study, with the three Streptococcus mutans strains killed at pH 3.0 for 3 h. The adaptation experiments revealed three groups of organisms: non-acid-responders, generally representing strains with the highest terminal pH values; weak acid-responders in the middle of the pH list, generating low numbers of survivors at one or two pH values, and the aciduric, strong responders generating a high number of survivors at pH values in the range 6.0 to 3.5, but not at pH 7.5. Predominant among the latter group were the S. mutans and Lactobacilli casei strains, with the most significant adaptive response exhibited by S. mutans LT11 and S. mutans Ingbritt, involving a process that required protein synthesis. Time course experiments with the latter organisms indicated that 90-120 min was required after exposure to the triggering pH before the acid response was fully functional. These results indicate that the sudden exposure of strains of oral streptococci and lactobacilli, as well as Enterococcus faecalis, to pH values between 6.0 and 3.5 results in the induction of an acid tolerance response that enhances the survival of these strains at or below pH 3.5.     

Efficacy of ondansetron in prophylaxis of postoperative nausea and vomiting in patients following infratentorial surgery: a placebo-controlled prospective double-blind study

Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent Streptococcus gordonii DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-microg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to S. gordonii DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.     

A giant protease with potential to substitute for some functions of the proteasome

An alanyl-alanyl-phenylalanyl-7-amino-4-methylcoumarin-hydrolyzing protease particle copurifying with 26S proteasomes was isolated and identified as tripeptidyl peptidase II (TPPII), a cytosolic subtilisin-like peptidase of unknown function. The particle is larger than the 26S proteasome and has a rod-shaped, dynamic supramolecular structure. TPPII exhibits enhanced activity in proteasome inhibitor-adapted cells and degrades polypeptides by exo- as well as predominantly trypsin-like endoproteolytic cleavage. TPPII may thus participate in extralysosomal polypeptide degradation and may in part account for nonproteasomal epitope generation as postulated for certain major histocompatibility complex class I alleles. In addition, TPPII may be able to substitute for some metabolic functions of the proteasome.     

Signals that regulate astroglial gene expression: induction of GFAP mRNA following seizures or injury is blocked by protein synthesis inhibitors

The purpose of this study was to develop methods for the consistent production of biofilms of S. mutans containing reporter gene fusions, and to examine the expression of genes involved in sucrose metabolism in adherent populations of this organism. Three strains of S. mutans harboring reporter gene fusions to the gene promoter regions of the gtfBC genes, ftf, and scrA were grown in a Rototorque biofilm fermenter in a tryptone-yeast extract-sucrose medium. Quasi-steady-state levels of reporter gene activity were measured after the biofilms were grown for either 48 hrs of 7 days. Also, induction of gene expression by the addition of sucrose to biofilm cells was monitored. Reporter gene activity was measurable from all gene fusion strains. This study (i) establishes the feasibility of doing detailed molecular and physiologic studies on immobilized populations of S. mutans, (ii) demonstrates that the polysaccharide synthesis machinery of S. mutans is differentially expressed in biofilms, and (iii) opens the way for a more detailed analysis of the environmental signals and signal transduction pathways governing the regulation of gene expression by S. mutans cells that are immobilized on a solid surface.     

Deletion of the central proline-rich repeat domain results in altered antigenicity and lack of surface expression of the Streptococcus mutans P1 adhesin molecule

Members of the family of surface adhesins of oral streptococci, including P1 of Streptococcus mutans, contain two highly conserved repeat domains, one rich in alanine (A region) and the other rich in proline (P region). To assess the contribution of the P region to the biological properties of P1, an internal deletion in spaP was engineered. In addition, the P region was subcloned and expressed as a fusion partner with the maltose binding protein of Escherichia coli and liberated by digestion with factor Xa. Results of Western blot experiments in which recombinant polypeptides were probed with a panel of 11 monoclonal antibodies indicated that the P region is a necessary component of conformational epitopes within the central portion of P1. Antibodies reactive with the P region were detected in a polyclonal rabbit antiserum generated against whole S. mutans cells but not in two rabbit antisera generated against purified P1 (Mr approximately 185,000), suggesting that this domain is immunogenic on the surface of intact bacteria but not as part of a soluble full-length molecule. Finally, transformation of a spaP-negative mutant with a shuttle vector containing an internally deleted spaP lacking P-region DNA resulted in a complete absence of surface-localized P1 and substantially less P1 in sonicated cells compared to the case for the mutant complemented with the full-length gene. These results suggest that the P region is an integral component contributing to the conformation of the central region of P1 and indicate that its presence is necessary for surface expression of the molecule on S. mutans.     

Detection of Streptococcus mutans by PCR amplification of the spaP gene in teeth rendered caries free

OBJECTIVES: To investigate the degree of association between tactile and optical criteria as used to assess the carious status of the enamel-dentine junction (EDJ) during cavity preparation, assessment with a caries detector dye and detection of Streptococcus mutans using culture and polymerase chain reaction (PCR) techniques. METHODS: Twenty-nine teeth, extracted within the previous 30 min, and 15 teeth prepared under rubber dam in vivo, were clinically assessed at the EDJ after the removal of evident carious tissue. Demineralisation was then assessed using a caries detector dye (1% acid red in propylene glycol; Cavex). A rosehead bur was used to remove tissue at the EDJ for culture and PCR analysis. Culture was carried out on a tryptone yeast cystine sucrose bacitracin selective medium, and PCR used to amplify a sequence (192 bp) of the spaP gene, which encodes the surface protein antigen I/II of S. mutans. RESULTS: Demineralised tissue at the EDJ, as shown using the dye, was found in 52% of teeth. Removed tissue was culture and PCR positive for S. mutans in 2 and 47% of teeth, respectively. A highly significant association (77% of cases; P < 0.001) was shown between dye and PCR assessment methods. No association was found between any other combination of assessment methods. CONCLUSIONS: Culture methods may underestimate the presence of S. mutans. Removal of sufficient dye-stained tissue is therefore recommended to prevent further carious assault from residual S. mutans.     

Expression of jumper ant (Myrmecia pilosula) venom allergens: post-translational processing of allergen gene products

N-terminal analyses of electrophoretically-separated allergenic polypeptides of the venom of the jumper ant M. pilosula showed that five out of the six allergenic polypeptides identified are homologous with the cloned major allergen Myr p I and may be derived from a single precursor polypeptide. The sixth polypeptide is homologous with a second cloned major allergen, Myr p II which is expressed as a single precursor polypeptide but exists in its native form as a disulphide bond-linked complex.     

Transforamtion of twitching strains of Streptococcus sanguis

Ninety-five strains of S. sanguis, 90 of which were twitching, were screened for competence in transformation with DNA from the "Challis" strain. Seventy-two strains, 68 of sero-group H and 4 of the provisional group 10043, were competent. Fourteen of the competent strains and all strains which appeared to be incompetent were tested with DNA from 3 other strains. The 14 competent strains were transformed by all the 3 DNAs. One of the apparently incompetent strains was transformed by autologous DNA only. Among 8 reference strains (including ATCC 10577, Type II of Washburn et al.) 5 were competent. Three of these did not show spreading or twitching. Among 16 non-spreading strains of alphahaemolytic streptococci which did not possess either the H or the 10043 group antigen, only one showed competence. The results indicate that twitching mobility is not a prerequisite for competence.     

Secretion of Porphyromonas gingivalis fimbrillin polypeptides by recombinant Streptococcus gordonii

The fimbriae of Porphyromonas gingivalis plays an important role in the pathogenesis of periodontal disease. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to promote adherence of the bacteria to host surfaces and also induce an immune response. Biologically active domains of fimbrillin responsible for adherence or eliciting immune responses have been determined. In a previous study, we engineered the human oral commensal organism Streptococcus gordonii to express such biologically active domains on the surface of the bacteria as a vaccine delivery system. In this study we report an alternative approach of secreting fimbrillin polypeptide domains into the medium by modification of the surface-expression system described earlier. Such recombinant S. gordonii, in addition to being a source for antigen presentation to trigger a protective immune response, may have the added advantage of directly blocking the fimbriae-mediated adherence of P. gingivalis to the oral cavity following implantation. This approach can also be utilized for secreting other biologically important therapeutic molecules on mucosal surfaces for modulating local microenvironments.