Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides

Cell surface proteins SspA and SspB in Streptococcus gordonii and SpaP in Streptococcus mutans are members of the antigen I/II family of polypeptides produced by oral streptococci. These proteins are adhesins and mediate species-specific binding of cells to a variety of host and bacterial receptors. Here we show that antigen I/II polypeptides are involved in the attachment of oral streptococci to collagen and that they also determine the ability of these bacteria to invade human root dentinal tubules. Wild-type S. gordonii DL1 (Challis) cells showed heavy invasion of tubules to a depth of approximately 200 microm, whereas the abilities of cells of isogenic mutant strains OB220 (sspA) and OB219 (sspA sspB) to invade were 50 and >90% reduced, respectively. Likewise, wild-type S. mutans NG8 cells invaded dentinal tubules, whereas cells of isogenic mutant strain 834 (spaP) did not. The invasive abilities of strains OB220 and OB219 were restored by heterologous expression of S. mutans SpaP polypeptide in these strains. The extents of tubule invasion by various wild-type and mutant strains correlated with their levels of adhesion to type I collagen, a major component of dentin. Furthermore, S. gordonii DL1 cells exhibited a growth response to collagen by forming long chains. This was not shown by ssp mutants but was restored by the expression of SpaP in these cells. The production of SspA polypeptide by S. gordonii DL1, but not production of SspB polypeptide by strain OB220 (sspA), was enhanced in the presence of collagen. These results are the first to demonstrate that antigen I/II family polypeptides bind collagen and mediate a morphological growth response of streptococci to collagen. These antigen I/II polypeptide activities are critical for intratubular growth of streptococci and thus for establishment of endodontic infections.     

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.     

Effective immunity to dental caries: passive transfer to rats to antibodies to Streptococcus mutans elicits protection

Rat dams, given intravenous injections of heat-killed Streptococcus mutans 6715, mutant C211 demonstrated significant agglutinin activity to the homologous S. mutans in colostrum, milk, and serum. This antibody activity was associated with the immunoglobulin G (IgG) class. High titers of anti-S. mutans antibody associated with the IgG class were also exhibited in the sera and saliva of the offspring that suckled these dams. After challenge with the homologous, live S. mutans, these offspring developed significantly fewer caries on all molar surfaces than did nonimmunized infected controls. A secretory immune response (manifested by the presence of specific IgA antibody to S. mutans in colostrum and milk) was elicited (i) in rat dams locally injected, in the region of the mammary gland, with heat-killed S. mutans antigen, and (ii) in other rat dams that were provided formalin-killed S. mutans in their drinking water. Offspring suckling these dams were challenged with virulent S. mutans before weaning and developed significantly fewer caries than did their infected controls. These findings clearly suggest that passively derived IgG or IgA antibodies to S. mutans are protective against dental caries.     

The active state of the thin filament is destabilized by an internal deletion in tropomyosin

The function of three of tropomyosin's sequential quasiequivalent regions was studied by deletion from skeletal muscle alpha-tropomyosin of internal residues 49-167. This deletion mutant tropomyosin spans four instead of the normal seven actins, and most of the tropomyosin region believed to interact with troponin is retained and uninterrupted in the mutant. The mutant tropomyosin was compared with a full-length control molecule that was modified to functionally resemble muscle tropomyosin (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466). The tropomyosin deletion suppressed the actin-myosin subfragment 1 MgATPase rate and the in vitro sliding of thin filaments over a heavy meromyosin-coated surface. This inhibition was not reversed by troponin plus Ca2+. Comparable tropomyosin affinities for actin, regardless of the deletion, suggest that the deleted region has little interaction with actin in the absence of other proteins. Similarly, the deletion did not weaken binding of the troponin-tropomyosin complex to actin. Furthermore, Ca2+ had a 2-fold effect on troponin-tropomyosin's affinity for actin, regardless of the deletion. Notably, the deletion greatly weakened tropomyosin binding to myosin subfragment 1-decorated actin, with the full-length tropomyosin having a 100-fold greater affinity. The inhibitory properties resulting from the deletion are attributed to defective stabilization of the myosin-induced active state of the thin filament.     

Intracellular alpha-amylase of Streptococcus mutans

Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose.     

The effect of antibody to Streptococcus mutans in saliva on caries activity and S. mutans adhering

An indirect enzyme-linked immunosorbent assay (ELISA) was used for measuring IgA antibody to whole cell of Streptococcus mutans (serotype c) in saliva. 36 parotid salivary samples of human were collected from two groups: Caries free (CF) and caries sensitive (CS). The result shows that the IgA antibody to S. mutans in CF group was higher than those in CS group (P < 0.05). The saliva antibody was gained from the rabbits by injection with S. mutans (serotype c), and the adhesion of S. mutans--3H on the surface of hydroxylapatite beads treated by rabbit's saliva was measured. The results show that the saliva with immunity could inhibit the S. mutans to adhere on the HA beads (P < 0.05). It means saliva antibody may prevent caries through inhibition of S. mutans from adhesion.     

Critical closing pressure explains cerebral hemodynamics during the Valsalva maneuver

Immunization with a particulate fraction of blood-stage antigens was shown previously to protect mice against Plasmodium yoelii malaria. To identify antigens inducing the protective response, sera from immunized mice were used to screen a P. yoelii cDNA expression library. Sequence analysis of one 2.6-kb cDNA clone indicated that the identified gene, pypag-1, encoded a novel plasmodial antigen. Two nonoverlapping regions of pypag-1 were expressed in Escherichia coli. The first recombinant antigen, pAg-1N, contained the N-terminal 337 residues, which included a putative transmembrane domain and a region relatively rich in tryptophan residues. The second recombinant antigen, pAg-1C, contained the remaining C-terminal 211 residues, which included 31 copies of a 5-amino-acid degenerative repeat. Immunoblot studies using rabbit antiserum raised against recombinant pAg-1N showed that the native pypAg-1 protein migrated at approximately 98 kDa, considerably slower than its predicted molecular mass of 66 kDa. Immunofluorescence studies localized the expression of the native pypAg-1 protein both to the cytoplasm and at the surface of P. yoelii-infected erythrocytes. Immunization with either pAg-1N or pAg-1C induced a four- to sevenfold reduction in P. yoelii blood-stage parasitemia. As such, pypAg-1 appears to contain at least two distinct protective epitopes. To our knowledge, this is the first characterization of a protective antigen of P. yoelii that is associated with the erythrocyte membrane.     

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.     

Emergence of an S gene mutant during thymosin alpha1 therapy in a patient with chronic hepatitis B

The presence of a hepatitis B virus S gene mutant was investigated in a patient being treated with thymosin alpha1. He was seropositive for hepatitis B e antigen throughout therapy but was intermittently seronegative for hepatitis B surface antigen (HBsAg) by an RIA. Sequence analysis revealed an S gene mutant in HBsAg-seronegative serum with two consecutive amino acid substitutions: threonine115-to-isoleucine and threonine116-to-asparagine, whereas no amino acid substitution or deletion was found in the pre-S region. A site-directed mutagenesis experiment confirmed that these mutations were responsible for the failure to detect HBsAg. In summary, an S gene mutant was identified in an HBsAg-seronegative patient. The mutations were located outside the putative "a" determinant. The emergence of an S gene mutant during thymosin alpha1 treatment suggests that enhanced host immunity against HBsAg may play a role in its antiviral activity.     

Complexes of the alpha1C and beta subunits generate the necessary signal for membrane targeting of class C L-type calcium channels

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha1C subunit with different accessory beta subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha1C subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta subunits also exhibited intracellular staining, co-expression of the alpha1C subunit with either the wild-type beta2a subunit, a palmitoylation-deficient beta2a(C3S/C4S) mutant or three other nonpalmitoylated beta isoforms (beta1b, beta3, and beta4 subunits) resulted in the redistribution of both the alpha1C and beta subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha1C was co-expressed with an N- and C-terminal truncated mutant beta2a containing only the central conserved regions. However, when the alpha1C subunit was co-expressed with an alpha1 beta interaction-deficient mutant, beta2aBID-, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta2a that was unable to interact with the alpha1C subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha1C subunit with the largely peripheral accessory alpha2 delta subunit was ineffective in recruiting alpha1C to the plasma membrane, while co-distribution of all three subunits was observed when beta2a was co-expressed with the alpha1C and alpha2 delta subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha1 and beta subunits.     

The structural basis for pseudoreversion of the H95N lesion by the secondary S96P mutation in triosephosphate isomerase

The structural basis for the 3000-fold decrease in catalytic efficiency of the H95N mutant chicken triosephosphate isomerase and the 60-fold regain of catalytic efficiency in the double mutant, H95N.S96P, have been analyzed. The results from a combination of X-ray crystallography and Fourier transform infrared spectroscopy experiments indicate that the predominant defect in the H95N mutant isomerase appears to be its inability to bind the substrate in a coplanar, cis conformation. The structures of each mutant isomerase were determined from X-ray crystallography of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog with the isomerase, and each was solved to a resolution of 1.9 A. The PGH appeared to be in two different conformations in which the enediol-mimicking atoms, O2-N2-C1-O1, of the PGH were not coplanar. No density was observed that would correspond to the coplanar conformation. Two bands are observed for the dihydroxyacetone phosphate carbonyl in the H95N mutant FTIR spectrum, and these can be explained if the O1 of DHAP, like the O1 of PGH in the crystal structure, is in two different positions. Two ordered water molecules are located between O1 of PGH and N delta of N95. Comparison of the structure of the pseudorevertant, H95N.S96P with that for the H95N single mutant, shows that S96P mutation causes the double mutant to regain the ability to bind PGH predominantly in the coplanar, cis conformation. Electron density for a single ordered water molecule bridging the N95 amide side chain and the O2 of PGH is observed, but the density was weak, perhaps indicating that the water molecule is somewhat disordered. Whether or not a water molecule is hydrogen bonded to O2 of PGH may explain the two carbonyl stretching frequencies observed for the GAP carbonyl. Together, the crystal structures and the FTIR data allow a complete explanation of the catalytic properties of these two mutant isomerases.     

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.     

Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants

In the present study, we compared the ability of the soluble adjuvants concanavalin A (ConA), muramyl dipeptide (MDP), and peptidoglycan (PG) to enhance immune responses to orally administered particulate antigens of Streptococcus mutans 6715 in gnotobiotic rats. The isotype and levels of antibody in saliva and in serum from experimental rats were determined by an enzyme-linked immunosorbent assay using S. mutans whole cells (WC) as the coating antigen. The specificities of salivary and serum immunoglobulin A (IgA) antibodies to particulate S. mutans antigens, lipoteichoic acid, S. mutans serotype g carbohydrate, and dextran were also determined. When 50 micrograms of ConA was used as the oral adjuvant with S. mutans 6715 WC immunogen, a slight enhancement of immune responses was obtained. A higher dose of ConA suppressed humoral responses to the immunogen. Enhanced immune responses, especially of the IgA isotype, in both serum and saliva were induced in gnotobiotic rats given MDP and either S. mutans 6715 WC or purified cell walls (CW) by gastric intubation. Elevated IgA antibody levels to CW, lipoteichoic acid, and carbohydrate were observed in rats given S. mutans WC and MDP by gastric intubation, whereas oral immunization with S. mutans CW and MDP resulted in higher antibody levels to CW and carbohydrate and lower levels to lipoteichoic acid when compared with the antibody levels in rats given antigen alone. Rats orally immunized with either S. mutans WC or CW and MDP and challenged with virulent S. mutans 6715 exhibited significantly (P less than or equal to 0.05) lower plaque scores, numbers of viable S. mutans in plaque, and caries scores than did rats immunized with antigen alone or in infected-only controls. In another series of experiments, a PG fraction derived from S. mutans 6715 CW was assessed for adjuvant properties. The oral administration of PG and either S. mutans WC or CW induced good salivary and serum IgA antibody responses. The specificity of the antibodies was similar to that obtained in rats given antigen and MDP. Rats receiving either S. mutans WC or CW and PG and challenged with virulent S. mutans 6715 had lower plaque scores, fewer numbers of viable S. mutans in plaque, and lower caries activity than did infected rats receiving S. mutans WC or CW immunogen alone. These results provide evidence that soluble adjuvants derived from the gram-positive bacterial CW, e.g., MDP and PG, are effective oral adjuvants and augment IgA immune responses to particulate S. mutans antigens which are protective against the mucosally associated disease, dental caries.     

Interaction of second and third domains of Japanese quail ovomucoid with ten mammalian trypsins

Second and third domains were prepared from Japanese quail ovomucoid and association equilibrium constants, Kas, were measured at 25 degreesC and pH 8 for these domains with trypsins from ten mammalian species: cat, cow, dog, guinea pig, hog, horse, man, rabbit, rat, and sheep. The values ranged from 108 M-1 to 1010 M-1 for the second domain-trypsin associations and from 106 M-1 to 108 M-1 for the third domain-trypsin associations. Changes in Ka values for the interactions between the trypsins and each domain are attributed to slight changes in surface conformation caused by the residue changes in the inhibitor-binding region other than the S1 pocket of the trypsin species. The representative of such residue changes is assumed to be the one observed at residue 217 of trypsin molecule. Concerning each trypsin, the Ka value with the second domain was always higher than that with the third domain. However, the ratios between the two equilibrium constants varied from 3 to 60 depending upon trypsin species. This means that amino acid changes in enzyme-contact residues other than the P1 site of the Kazal-type inhibitor can make it possible to recognize even a slight difference in inhibitor-binding surface among the enzymes with the same S1 pocket and highly similar overall three-dimensional structure.     

Embryonic development of the Drosophila brain. I. Pattern of pioneer tracts

The neuropile of the late embryonic Drosophila brain can be subdivided into a vertical component (cervical connective), a transverse component (supraesophageal commissure), and a horizontal component for which we propose the term protocerebral connective. The core of each neuropile component is formed by numerous axon fascicles, the trajectory of which follows an invariant pattern. In the present study we have used an antibody against the adhesion molecule Fasciclin II (FasII) that is expressed in a large number of early differentiating neurons of the Drosophila embryo to follow the development of the axon tracts of the brain. The FasII antigen appears on the surface of clusters of neuronal somata prior to axon outgrowth. These clusters, for which we propose the term fibre tract founder clusters, are laid out in a linear pattern that forms an almost uninterrupted longitudinal track reaching from the ventral nerve cord to the "tip" of the brain. After expressing FasII on their soma, neurons of the fibre tract founder clusters extend axons that grow along the surface of the founder clusters and form a simple system of pioneer tracts for each of the components of the brain neuropile. We have reconstructed the FasII-positive fibre tract founder clusters and their axons from optical sections and generated digital 3-D models that illustrate the spatial relationships of the pioneer tracts. Three fibre tract founder clusters, D/T, P1, and P3m, pioneer the cervical connective. P21 and P2m form a transverse track that pioneers the supraesophageal commissure. P4m and P41/P51/VP5m form two tracts that pioneer a medial and a lateral component of the protocerebral connective, respectively. Because FasII expression continues uninterruptedly into the larval period when the "rudiments" of many parts of the adult neuropile are readily identifiable, it was possible to assign several of the embryonic pioneer tracts to definitive neuropile components, including the median bundle, antennocerebral tract, mushroom body, and posterior optic tract.     

Differential participation of hippocampal formation in cocaine-induced cortical electroencephalographic desynchronization and penile erection in the rat

Rts1 RepA and P1 RepA are trans-acting proteins essential for initiation of replication of Rts1 and P1 plasmids, respectively. We recently found that P1 RepA bound in vitro to the Rts1 replication origin as strongly as Rts1 RepA and activated the origin in vivo. However, the ori activation was quite inefficient. This study shows that by replacing a small region of P1 RepA with the corresponding region of Rts1 RepA, the efficiency of Rts1 ori activation increased markedly. Interestingly, the same subregion of P1 RepA was found to be important for in vivo activation of the P1 origin. Thus, a region essential for efficient activation of the replication origin was assigned to the P1 RepA molecule as well as to the Rts1 RepA molecule. The region was distinct from a domain necessary for in vitro binding to the origin, although both regions were required for in vivo activation of the respective origin.     

Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli

Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114. A rabbit polyclonal antiserum raised against these proteins was used to screen an S. japonicum expression cDNA library. Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae [Hawn et al., J. Biol. Chem. 268 (1993) 7692-7698]. The S. japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins. The Chinese strain S. japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone. Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing. Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S. japonicum. Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography. Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions. The presence of conserved Ca(2+)-binding motifs predicted from the primary sequence, together with the Ca(2+)-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.     

Magnesium reversal of digoxin-facilitated ventricular rate during atrial fibrillation in the Wolff-Parkinson-White syndrome

The purpose of this investigation was to determine whether pyrophosphate, the anticalculus component of tartar-control dentifrices, exerts antimicrobial activity against oral bacteria commonly found in supragingival plaque. Minimal inhibitory concentrations of pyrophosphate were determined for Streptococcus sanguis, Streptococcus mutans (serotype c), Actinomyces viscosus and Actinomyces naeslundii. All of the bacteria tested were susceptible to pyrophosphate with identical minimal inhibitory concentrations of 0.67% wt/vol (25 mM). Bactericidal kinetics assays revealed that both S. mutans and A. viscosus were killed by pyrophosphate, with the latter being considerably more susceptible. The mechanism of killing was not due to high ionic strength, as comparable controls showed no loss in numbers of viable cells. Brief exposure (two 5-min incubations) of S. mutans to pyrophosphate and sodium dodecyl sulfate caused pronounced inhibition of growth over the 24-h test period. Under the constraints of the conditions used, these studies indicate that pyrophosphate and sodium dodecyl sulfate can substantially inhibit the growth of oral bacteria. These compounds may affect the oral microflora of patients who routinely use tartar-control dentifrices and mouthrinses.     

The role of the Streptococcus mutans glucosyltransferases in the sucrose-dependent attachment to smooth surfaces: essential role of the GtfC enzyme

Previous results have indicated that the glucosyltransferase activities of mutans streptococci are required for sucrose-dependent colonization of tooth surfaces. We have constructed mutants of Streptococcus mutans GS5 that are altered in varying combinations of the three gtf genes present in this organism. A quantitative in vitro sucrose-dependent attachment system was used to demonstrate that the inactivation of the gtfC gene drastically reduced adherence to smooth surfaces. By contrast, inactivation of the gtfB gene resulted in a smaller, but significant, reduction in attachment while the gtfD mutant was only marginally affected. Furthermore, production of only the glucosyltransferase C enzyme allowed for attachment although at reduced levels compared to the wild-type organism. The results from reintroduction of single copies of each of the gtf genes into a mutant of strain GS5 lacking glucosyltransferase activity also demonstrated the crucial role of the glucosyltransferase C enzyme in colonization. These results suggest a unique role for the glucosyltransferase C enzyme in the sucrose-dependent colonization of tooth surfaces by S. mutans strains.