Identification of essential amino acids in the Streptococcus mutans glucosyltransferases

A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp491 or His561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.     

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.     

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.     

Paleomicrobiological study in dental calculus: Streptococcus mutans

Morphological types of bacterial remains preserved in ancient tartar of teeth from extinct human groups, which included some communities of coastal gatherers, fishermen, hunters, and farmers, and those practicing a mixed economy, were analyzed. Previous studies have shown the presence of bacteria in ancient tartar. The aim of this work was to determine whether Streptococcus mutans was present in ancient populations (500-12,000 years old). Teeth samples were from ancient skulls obtained from different anthropological collections: the north and south of Chile (before the Spanish conquest), Palencia, Spain, and an eastern Mediterranean region (Levant). Optical microscopy showed Gram positive and Gram negative bacteria. Scanning electron microscopy identified morphological types of bacteria. Transmission electron microscopy enabled categorization of bacterial structures. Fluorescence microscopy helped label and identify S. mutans, using polyclonal antibodies. Bacterial morphotypes were related to different subsistence patterns. Hunters, fishermen, and gatherers had a less diverse flora with bacillary and coccal morphotypes. Agricultural groups showed greater diversity with additional filamentous and spiral morphotypes. The best preserved ultrastructural feature was the cell wall. The existence and colonization capacity of the mutans-like streptococci preserved in tartar was established for the ancient populations studied, with the exception of Cerro Sotta (south of Chile). Hence, their occurrence could not be related to diet or subsistence pattern.     

Recovery of Streptococcus mutans after amino acid deprivation

The recovery of Streptococcus mutans FA-1 in a complete, chemically defined medium was examined after 1, 3, and 6 h of essential amino acid deprivation. Amino acids could be divided into two groups based on their effect on the relative rates of recovery: those amino acids (leucine and cystine) that are precursors of protein only, and amino acids (glutamate/glutamine or lysine) that are incorporated into both protein and cell wall peptidoglycan. Culture turbidity, deoxyribonucleic acid, ribonucleic acid, protein and cell wall peptidoglycan measurements indicated rapid recovery after leucine/cystine starvation periods. However, a 6-h leucine/cystine deprivation resulted in a slower exponential rate of growth (180-min doubling time compared to the normal doubling time of 85 to 90 min) after recovery. Glutamate/glutamine starvation, on the contrary, resulted in greatly extended recovery periods, especially after 3- and 6-h amino acid deprivations. Macromolecular synthesis was most severely affected by 6-h glutamate/glutamine starvation and required 6 to 10 h for recovery of an exponential rate. A delay in the recovery of deoxyribonucleic acid and cell wall peptidoglycan synthesis beyond that of the other macromolecules was observed after 1 and 3 h of deprivation with either leucine/cystine or glutamate/glutamine. However, after a 6-h amino acid deprivation, deoxyribonucleic acid synthesis recovered more rapidly than that of the other macromolecules studied. The results are discussed in terms of the nutritional environment of the oral cavity and its effect on the growth and survival of S. mutans.     

L-(+)-lactate dehydrogenase deficiency is lethal in Streptococcus mutans

The previously cloned gene for L-(+)-lactate dehydrogenase (LDH) from Streptococcus mutans was mutagenized in vitro. An Escherichia coli transformant which expressed a thermolabile LDH activity was identified. The ldh(Ts) gene was introduced into S. mutans on a suicide vector to create a heterodiploid expressing both wild-type and thermolabile LDH activities. Self-recombinants which had only one ldh gene were isolated. One of these clones expressed only the thermolabile LDH activity. This isolate grew well at 30 degrees C but did not grow at 42 degrees C under a variety of cultivation conditions, thereby proving that LDH deficiency is lethal in S. mutans in the absence of compensatory mutations.     

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.     

Role of the charged tail in localization of a surface protein antigen of Streptococcus mutans

To make clear the role of the C terminus of a surface protein antigen (PAc) of Streptococcus mutans, stepwise truncations beginning at the C terminus of PAc were performed by utilizing site-directed mutagenesis. A remarkable increase in the amount of cell-free PAc was observed upon deletion of four or more amino acid residues at the C terminus. On the other hand, the amount of cell surface PAc gradually decreased when increasing numbers (four or more) of amino acid residues were deleted at the C terminus, and deletion of six amino acids involving both the total charged tail and Leu, an amino acid residue immediately upstream of the charged tail, resulted in a drastic reduction in the amount of cell surface PAc. These results indicate that the cytoplasmic charged tail and Leu residue are required for cell surface localization of PAc in S. mutans.     

Concentrated bovine colostral whey proteins from Streptococcus mutans/Strep. sobrinus immunized cows inhibit the adherence of Strep. mutans and promote the aggregation of mutans streptococci

The aim of this study was to examine the effect of bovine colostral whey proteins from cows immunized with Streptococcus mutans/Strep. sobrinus on the adherence and aggregation of caries-inducing bacteria, i.e., mutants streptococci. Both adherence and aggregation are important phenomena in the bacterial colonization of the human oral cavity. In all adherence experiments there was a significant difference between treatments by immune product (IP; from immunized cows) and a control product (CP; a similar product from non-immunized cows). The adherence of 35S-labelled Strep. mutans cells (serotype c) to parotid saliva-coated hydroxyapatite (SHA) was dose-dependently inhibited by both IP and CP if SHA was coated with either product before exposure to bacteria, but markedly lower concentrations of IP than CP were effective. When instead of SHA the bacterial cells were pretreated with IP or CP, only IP strongly and dose-dependently inhibited streptococcal adherence. When bacteria, IP or CP, and SHA were incubated simultaneously, a significant difference between IP and CP treatments was again found. Further, IP effectively aggregated both Strep. mutans and Strep. sobrinus cells, whereas hardly any effect was seen with CP. Both IP and CP aggregated the control bacterium Strep. sanguis, which affected the adherence of the pretreated bacteria.     

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol. 1991, 65, 3106-3113). Pretreatment of MA-104 cells with the VP4-peptide before infection with rotavirus prevented both the attachment of 35S-labelled virus and plaque formation in vitro. In vivo studies using a murine rotavirus model demonstrated that intragastric administration of VP4-peptide protected subjects from challenge with virulent rotavirus. These results clearly indicate the importance of this epitope in virus-cell interactions and their potential as a rotavirus vaccine candidate.     

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.     

Decreased oral colonization of Streptococcus mutans during aging of Sprague-Dawley rats

The colonization by streptomycin-resistant Streptococcus mutans strains of the teeth of conventional and ex-germfree Sprague-Dawley rats of various ages fed either a high-sucrose or a high-glucose diet was studied. Bacterial colonization occurred with increasingly greater difficulty as the rats became older. This was observed in studies of the implantation of the test organism after oral inoculation with different cell numbers as well as its transmission between infected and uninfected rats. With rat fed sucrose diet, the effect of age could not be demonstrated until they were age 3 months or older; the results from rats fed a glucose diet suggest that changes may already have occurred early after weaning. Changes in susceptibility to colonization during aging manifested themselves as a decrease in the proportions of rats which became infected as well as lower population levels in infected rats. The possible mechanism(s) involved as well as the possible significance of the findings was discussed.     

Antigenicity of janin, a new protein from smooth muscle

The exact nature of smooth muscle autoantibodies is still unclear. The antigen(s) which they are directed against remain unknown. Janin, a so far unknown smooth muscle protein, was obtained from smooth muscle tissue extracts and purified by means of three different procedures. Its mol. w. was estimated at 60,000 daltons. It stimulated a specific anti-janin antibody in rabbits as shown by tube test, double diffusion in agar and indirect immunofluorescence. The new protein absorbed eighteen out of 104 human smooth muscle positive sera that were not absorbed with smooth muscle actin, myosin, heavy meromyosin, light meromyosin, tropomyosin and brain tubulin. This would vindicate a view that smooth muscle autoantibodies differ from patient to patient with regard to the autoantigen(s) involved.     

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.     

Serologic reactivity of a synthetic peptide from human immunodeficiency virus type 1 gp41 with sera from a Mexican population

The reactivities of 1,172 serum samples obtained from asymptomatic human immunodeficiency virus type 1 (HIV-1)-positive and HIV-1-negative individuals residing in Mexico to a synthetic disulfide-looped peptide from the HIV-1 gp41 (amino acids 602 to 616 [IWGCSGKLICTTAVP] were examined by an enzyme-linked immunoadsorbent assay (ELISA) procedure. Antibodies to the synthetic peptide were detected in 261 of 268 serum samples from HIV-positive individuals (sensitivity, 97.4%). The peptide also reacted with 12 of 904 serum samples from control HIV-negative individuals (specificity, 98.7%). Western blots (immunoblots) of four of the seven serum samples that produced false-negative results in the ELISA showed that three of them reacted weakly with gp41 and strongly with gp120, p55, and/or p24. Potential diagnostic difficulties raised by the reported C1q binding capacity of this peptide were also evaluated: few and weak false-positive results were found among sera from patients with rheumatoid arthritis (1 of 31) and neurocysticercosis (2 of 111). In fact, strong reactivity with the peptide spotted an undetected HIV infection underlying clinical neurocysticercosis.     

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.     

Cloning and sequence analysis of the gbpC gene encoding a novel glucan-binding protein of Streptococcus mutans

We have isolated dextran-aggregation-negative mutants of Streptococcus mutans following random mutagenesis with plasmid pVA891 clone banks. A chromosomal region responsible for this phenotype was characterized in one of the mutants. A 2.2-kb fragment from the region was cloned in Escherichia coli and sequenced. A gene specifying a putative protein of 583 amino acid residues with a calculated molecular weight of 63,478 was identified. The amino acid sequence deduced from the gene exhibited no similarity to the previously identified S. mutans 74-kDa glucan-binding protein or to glucan-binding domains of glucosyltransferases but exhibited similarity to surface protein antigen (Spa)-family proteins from streptococci. Extract from an E. coli clone of the gene exhibited glucan-binding activity. Therefore, the gene encoded a novel glucan-binding protein.