Evidence of Early Emergence of the Primary Dentition in a Northern Plains American Indian Population.

The purposes of this study were to describe primary tooth emergence in an American Indian (AI) population during the first 36 mo of life to compare 1) patterns of emergence between male and female children and 2) tooth emergence between these AI children and other U.S. ethnic groups. Data were derived from a birth cohort of 239 AI children from a Northern Plains tribe participating in a longitudinal study of early childhood caries, with examination data at target ages of 8, 12, 16, 22, 28, and 36 mo of age (±1 mo). Patterns of emergence in AI children were characterized and sex comparisons accomplished with interval-censored survival methodology. Numbers of erupted teeth in AI children at each age were compared via Kruskal-Wallis tests against those in children of the same age, as drawn from a cross-sectional study of dental caries patterns in Arizona; these comparisons were based on the dental examinations of 547 White non-Hispanic and 677 Hispanic children. Characterization of time to achievement of various milestones-including emergence of the anterior teeth, the first molars, and the complete primary dentition-provided no evidence of sex differences among AI children. AI children had significantly more teeth present at 8 mo (median, 3) than either White non-Hispanic (P < 0.0063) or Hispanic (P < 0.0001) children (median, 2 each). This was also true at 12 mo (P < 0.001; medians, 8 vs. 6 and 7, respectively) and 16 mo (P < 0.001; medians, 12 vs. 11 each). Less pronounced differences were seen at 22 mo (P < 0.0001). White non-Hispanic and Hispanic children did not differ at any time considered (P > 0.05). These results provide evidence of earlier tooth emergence in AI children than in the other 2 ethnicities. Although the underlying etiology of the severity of early childhood caries in AI children is likely to be multifactorial, earlier tooth emergence may be a contributing factor. Knowledge Transfer Statement: The findings of this study have practical implications for practitioners providing childhood oral health care to ethnic groups with early tooth emergence. It may be important to provide parents with information on toothbrushing, dentist visits, and other practices supportive of good oral health as early as possible to protect their children's primary dentition.

Streptococcus mutans strains recovered from caries-active or caries-free individuals differ in sensitivity to host antimicrobial peptides.

Antimicrobial peptides (AMPs) are among the repertoire of host innate immune defenses. In the oral cavity, several AMPs are present in saliva and have antimicrobial activities against oral bacteria, including Streptococcus mutans, a primary etiological agent of dental caries. In this study, we hypothesized that unique S. mutans strains, as determined by DNA fingerprinting from sixty 13-year-old subjects with or without experience of caries, would have different susceptibilities to α-defensins-1-3 (HNP-1-3), ß-defensins-2-3 (HBD-2-3) and LL-37. The salivary levels of these peptides in subjects were also measured by enzyme-linked immunosorbent assays. We found that S. mutans strains from children with active caries showed greater resistance to salivary HNP-1-2, HBD-2-3 and LL-37 at varying concentrations than those from caries-free subjects. In addition, combinations of these peptides increased their antimicrobial activity against S. mutans either additively or synergistically. The salivary levels of these peptides were highly variable among subjects with no correlation to host caries experience. However, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. Our findings suggest that the relative ability of S. mutans to resist host salivary AMPs may be considered a potential virulence factor for this species such that S. mutans strains that are more resistant to these peptides may have an ecological advantage to preferentially colonize within dental plaque and increase the risk of dental caries.

Effects of manganese on Streptococcus mutans planktonic and biofilm growth.

Streptococcus mutans, an agent of dental caries, was tested for growth in the presence or absence of manganese (Mn), since studies have linked Mn levels with cariogenic potential. Seven S. mutans serotype c strains were grown in chemically defined medium under different atmospheric conditions: 5% CO2, O2-enriched 5% CO2 (shaking) and anaerobic. There was significant strain variability with respect to Mn requirements under the various conditions tested. Both sucrose-dependent and sucrose-independent biofilm growth by strain UA159 were affected by the absence of Mn. S. mutans strains show highly variable responses to both high and low Mn concentrations.

Manganese affects Streptococcus mutans virulence gene expression.

BACKGROUND/AIMS: Studies of trace metals in drinking water and tooth enamel have suggested a caries-promoting potential for manganese (Mn). Additionally, Mn has been shown to be essential for the expression of mutans streptococci virulence factors such as the glucan-binding lectin (GBL) of Streptococcus sobrinus. The Streptococcus mutans glucan-binding protein (Gbp) GbpC is the functional analogue of the S. sobrinus GBL. S. mutans Gbps have been shown to contribute to biofilm architecture and virulence. This study was undertaken to examine the effects of Mn on the transcription of genes encoding S. mutans Gbps, including gbpC, along with other critical S. mutans virulence genes. METHODS: Microarray analyses suggested the potential for an Mn effect on Gbp genes. Further investigation of the Mn effects on selected genes was undertaken by performing Northern blots, Western blots, and RT-PCR under conditions of planktonic and biofilm growth in Mn-depleted media or in media containing 50 mircoM Mn. RESULTS: Mn resulted in increased expression of gbpC and gtfB, and decreased expression of wapA, in both planktonic and biofilm cultures. The expression levels of gbpA and gbpD were also decreased in the presence of Mn, but only in biofilms. The expression of gtfC was increased in the presence of Mn only in planktonic cultures. The spaP gene was expressed more highly in Mn-supplemented planktonic cultures but less in Mn-supplemented biofilms. CONCLUSION: Mn availability affects the expression of multiple S. mutans genes involved in adhesion and biofilm formation. Furthermore, these effects depend on the growth state of the organism.

Glucan-binding proteins of the oral streptococci.

The synthesis of extracellular glucan is an integral component of the sucrose-dependent colonization of tooth surfaces by species of the mutans streptococci. In investigators' attempts to understand the mechanisms of plaque biofilm development, several glucan-binding proteins (GBPs) have been discovered. Some of these, the glucosyltransferases, catalyze the synthesis of glucan, whereas others, designated only as glucan-binding proteins, have affinities for different forms of glucan and contribute to aspects of the biology of their host organisms. The functions of these latter glucan-binding proteins include dextran-dependent aggregation, dextranase inhibition, plaque cohesion, and perhaps cell wall synthesis. In some instances, their glucan-binding domains share common features, whereas in others the mechanism for glucan binding remains unknown. Recent studies indicate that at least some of the glucan-binding proteins modulate virulence and some can act as protective immunogens within animal models. Overall, the multiplicity of GBPs and their aforementioned properties are testimonies to their importance. Future studies will greatly advance the understanding of the distribution, function, and regulation of the GBPs and place into perspective the facets of their contributions to the biology of the oral streptococci.

Lipoteichoic acid inhibits interleukin-2 (IL-2) function by direct binding to IL-2.

Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved.

An in vitro model for studying the contributions of the Streptococcus mutans glucan-binding protein A to biofilm structure.

The method described here for analyzing biofilms was sensitive enough to allow the detection of differences formed by pure cultures of S. mutans or a GbpA knockout strain. Other strains have also been tested, and the differences in biofilm structure were sometimes even more extensive (data not shown). The advantages of this method are that it is quick, inexpensive, and adaptable to almost any laboratory setting. The constant rotation of the cultures, which was employed to simulate salivary flow, appears to be a critical element for establishing biofilm differences. An analysis of protein profiles confirmed that the biofilm bacteria were metabolically distinct from the planktonic phase bacteria. For the strains tested, the variations in biofilm architecture could be visualized with or without magnification. Staining of the bacteria was not required, though we typically stained the biofilms with either crystal violet or Schiff's reagent. Altogether, this in vitro method for generating biofilms allowed the evaluation of visual, quantitative (confocal microscopy), and functional (antimicrobial susceptibility) differences. We have employed these methods in a reductionist approach to understanding the contribution of individual proteins to dental plaque development. These methods may also be useful in the screening of mutants that would be of greatest for testing in multispecies biofilms, animal models, or more complex biofilm models.

Modulation of glucan-binding protein activity in streptococci by fluoride.

Glucan-binding lectin (GBL) activity of Streptococcus sobrinus was significantly reduced by fluoride in the growth medium. Approximately 1.5 mM fluoride was required for a 50% reduction in GBL activity. In addition to the GBL, several other glucan-binding proteins were reduced when the bacteria were grown in subinhibitory fluoride. Fluoride had no effect on glucosyltransferases (GTFs), enzymes capable of converting sucrose into alpha-1,6-glucans. All the proteins were detected by use of enhanced chemiluminescence (ECL of fluorescein-labeled dextran) and Western blotting of renatured SDS-PAGE gels. The effects of fluoride on the bacteria were abrogated when the manganous ion was included in the growth medium. It thus appears that one mechanism of action of fluoridated water is its effects on glucan-binding proteins. The fluoride may be reducing metabolism of the mangano aquo ion, essential for expression of the glucan-binding proteins.

Ligand-binding properties of the carboxyl-terminal repeat domain of Streptococcus mutans glucan-binding protein A.

Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypothesis and to quantitate the ligand-binding specificities of the GbpA GBD, several fusion proteins were generated and tested by affinity electrophoresis or by precipitation of protein-ligand complexes, allowing the determination of binding constants. It was determined that the 16 YG repeats in GbpA comprise its GBD and that GbpA has a greater affinity for dextran (a water-soluble form of glucan) than for mutan (a water-insoluble form of glucan). Placement of the GBD at the carboxyl terminus was necessary for maximum glucan binding, and deletion of as few as two YG repeats from either end of the GBD reduced the affinity for dextran by over 10-fold. Interestingly, the binding constant of GbpA for dextran was 34-fold higher than that calculated for the GBDs of two S. mutans GTFs, one of which catalyzes the synthesis of water-soluble glucan and the other of which catalyzes the synthesis of water-insoluble glucan.

Inactivation of the gbpA gene of Streptococcus mutans alters structural and functional aspects of plaque biofilm which are compensated by recombination of the gtfB and gtfC genes.

Inactivation of the gbpA gene of Streptococcus mutans increases virulence in a gnotobiotic rat model and also promotes in vivo accumulation of organisms in which gtfB and gtfC have recombined to reduce virulence (K. R. O. Hazlett, S. M. Michalek, and J. A. Banas, Infect. Immun. 66:2180-2185, 1998). These changes in virulence were hypothesized to result from changes in plaque structure. We have utilized an in vitro plaque model to test the hypothesis that the absence of GbpA alters S. mutans plaque structure and that the presence of gtfBC recombinant organisms within a gbpA background restores a wild-type (wt)-like plaque structure. When grown in the presence of sucrose within hydroxyapatite-coated wells, the wt S. mutans plaque consisted primarily of large aggregates which did not completely coat the hydroxyapatite surface, whereas the gbpA mutant plaque consisted of a uniform layer of smaller aggregates which almost entirely coated the hydroxyapatite. If 25% of the gbpA mutants used as inoculum were also gtfBC recombinants (gbpA/25%gtfBC), a wt-like plaque was formed. These changes in plaque structure correlated with differences in susceptibility to ampicillin; gbpA plaque organisms were more susceptible than organisms in either the wt or gbpA/25%gtfBC plaques. These data allow the conclusion that GbpA contributes to S. mutans plaque biofilm development. Since the changes in plaque structure detailed in this report correlate well with previously observed changes in virulence, it seems likely that S. mutans biofilm structure influences virulence. A potential model for this influence, which can account for the gtfBC recombination compensating gbpA inactivation, is that the ratio of glucan to glucan-binding protein is a critical factor in plaque development.

Circular dichroism analysis of the glucan binding domain of Streptococcus mutans glucan binding protein-A.

The glucan binding domain (GBD) of the glucan binding protein-A (GBP-A) from the cariogenic bacterium Streptococcus mutans was studied using circular dichroism (CD) analysis, Chou-Fasman-Rose secondary structure prediction, and absorption and fluorescence spectroscopy. Our data show that the binding domain undergoes a conformational shift upon binding to the ligand dextran. The CD spectrum shows two positive bands at 280 nm and 230 nm which were assigned to aromatic residues. The 230-nm band was seen at 20 degrees C and 30 degrees C, lost intensity at 40 degrees C, and was eliminated at 45 degrees C coinciding with complete denaturation. The protein was stable at physiological pH, but precipitated at pH 5. A pH of 10 changed the secondary structure but had no effect on the 230-nm band. Analysis of the CD data in the far UV using the SELCON computer program revealed a high content of beta-sheets and a lack of alpha-helical structures. Secondary structure prediction based on the amino acid sequence of GBD agreed with the CD analysis. The fluorescence emission maximum at 339 nm suggested that the majority of the tryptophans were located in the interior of the protein. This maximum shifted to higher energy upon binding to the ligand dextran.

Inactivation of the gbpA gene of Streptococcus mutans increases virulence and promotes in vivo accumulation of recombinations between the glucosyltransferase B and C genes.

Glucan-binding protein A (GbpA) of Streptococcus mutans has been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenic gbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutans was grown in the presence of [3H] thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. The gbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB and gtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.

Inhibition of interleukin-2 by a Gram-positive bacterium, Streptococcus mutans.

Generation of an effective cellular immune response is key to the successful development of both humoral and cellular immune defences against most pathogens. However, while the type of cellular immune response elicited by any given pathogen is dictated by the entire array of antigens and molecules which comprise that pathogen, most studies of human immune responses to bacterial pathogens tend to focus on selected antigens. This is a result, in part, of a desire to find those antigens that will generate a desired immune response, as well as limited technology for monitoring the complex array of responses generated by an intact organism. Utilizing Streptococcus mutans as a model Gram-positive organism, a novel flow cytometric assay that permits the identification of individual cells within a responding population, and highly sensitive cytokine assays, we show for the first time that CD8 T cells and natural killer (NK) cells comprise a significant component of the response to this organism in humans. This is despite the fact that CD8 T cells are traditionally thought to respond to endogenously derived antigens only. In addition, we provide the first evidence that a Gram-positive organism can actively inhibit interleukin-2 (IL-2), an important autocrine growth factor for T cells. The latter observation could represent an additional mechanism by which Gram-positive organisms evade host defences.

The regulation of Streptococcus mutans glucan-binding protein A expression.

The S. mutans GBP-A is hypothesized to be constitutively expressed and to contribute to the sucrose-dependent colonization of S. mutans. To investigate GBP-A expression, a reporter gene encoding chloramphenicol acetyltransferase (CAT) was placed downstream of the gbpA promoter and CAT activity was measured under conditions that would be associated with the sucrose-dependent colonization of S. mutans. Expression of GBP-A was optimal under anaerobiosis and neutral pH conditions, and correlated with optimal growth. The addition of sucrose to the growth medium did not elevate the expression of GBP-A.

Multiple glucan-binding proteins of Streptococcus sobrinus.

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.

The cloning, expression and sequence analysis of a second Porphyromonas gingivalis gene that codes for a protein involved in hemagglutination.

It has been suggested that Porphyromonas gingivalis may possess more than one hemagglutinin. We have previously reported the cloning of a gene (hagA) that encodes a hemagglutinin. In this study we report the cloning, characterization, and sequencing of a second gene (hagB) that encodes a protein that also appears to be involved in hemagglutination. Antiserum to the clone (ST 7) was found to inhibit hemagglutination by P. gingivalis 381, and hemagglutinating inhibition activity of anti-P. gingivalis antiserum was reduced by adsorption of the antiserum with cells of clone ST 7. Restriction mapping and Southern analysis indicates there is little or no DNA homology between this cloned 4.8-kb HindIII DNA fragment and a cloned hemagglutinin gene we have previously described. Minicell analysis of the cloned P. gingivalis chromosomal DNA fragment revealed that the major gene product is a 49-kDa protein. Immunoaffinity chromatography using purified rabbit immunoglobulin G against the cloned protein resulted in the purification of a major reactive 49- to 50-kDa protein from a P. gingivalis cell lysate. Nucleotide sequence analysis revealed the hagB open reading frame to be 1053 nucleotides in length with a mol% G+C of 59.9% coding for a protein of 350 residues with a calculated molecular weight of 39.375 kDa. This protein was also determined to be basic and hydrophilic and to contain a potential signal peptide. Comparison of both the nucleotide and derived amino acid sequences with computer-based databases did not reveal any significant homologies between habB and any other previously sequenced genes.

Analysis of a primer-independent GTF-I from Streptococcus salivarius.

A glucosyltransferase (GTF) gene, designated gtfL, from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.

Identification and sequence analysis of a methylase gene in Porphyromonas gingivalis.

A gene from the periodontal organism Porphyromonas gingivalis has been identified as encoding a DNA methylase. The gene, referred to as pgiIM, has been sequenced and found to contain a reading frame of 864 basepairs. The putative amino acid sequence of the encoded methylase was 288 amino acids, and shared 47% and 31% homology with the Streptococcus pneumoniae DpnII and E. coli Dam methylases, respectively. The activity and specificity of the pgi methylase (M.PgiI) was confirmed by cloning the gene into a dam- strain of E. coli (JM110) and performing a restriction analysis on the isolated DNA with enzymes whose activities depended upon the methylation state of the DNA. The data indicated that M.PgiI, like DpnII and Dam, methylated the adenine residue within the sequence 5'-GATC-3'.