Requirements for surface expression and function of adhesin P1 from Streptococcus mutans

In this report, we define requirements for the successful translocation and functional maturation of the adhesin P1 of Streptococcus mutans. Conformational epitopes recognized by anti-P1 monoclonal antibodies (MAbs) were further characterized, thus facilitating the use of particular MAbs as tools to monitor the locations of various forms of the protein. We show that correct localization of P1 is dependent on structural features of the molecule itself, including a requisite A region-P region intramolecular interaction that occurs within the cell prior to secretion. P1 also was shown to be affected by several members of the protein-folding-secretion-turnover apparatus. It does not achieve a fully functional form in the absence of the trigger factor PPIase homolog RopA, and its translocation is delayed when DnaK levels are limited. In addition, dnaK message levels are differentially altered in the presence of P1 lacking the alanine-rich compared to the proline-rich repeat domains. Lastly, nonsecreted P1 lacking the P region accumulates within the cell in the absence of htrA, implying an intracellular HtrA protease function in the degradation and turnover of this particular internal-deletion polypeptide. However, the opposite effect is seen for full-length P1, suggesting a sensing mechanism and substrate-dependent alteration in HtrA's function and effect that is consistent with its known ability to switch between chaperone and protease, depending on environmental perturbations.     

Differentiation of salivary agglutinin-mediated adherence and aggregation of mutans streptococci by use of monoclonal antibodies against the major surface adhesin P1.

The ability to adhere to salivary agglutinin-coated hydroxyapatite beads and to aggregate in the presence of fluid-phase salivary agglutinin was tested by using 25 isolates of mutants streptococci representing eight serotypes. Both adherence and aggregation activity correlated with expression of the Mr-185,000 cell surface antigen P1 on Streptococcus mutans serotype c, e, and f strains. In addition, it was shown that the P1 molecule itself served as the adhesin of S. mutans serotype c, since adherence was significantly inhibited by the presence of recombinant-specified Mr-150,000 P1. The ability of S. sobrinus strains to adhere or aggregate did not correlate with expression of the P1 cross-reactive antigen SpaA. There was also evidence for interaction with salivary agglutinin, as manifested by aggregation but not adherence of S. rattus serotype b, which does not express a P1 cross-reactive antigen. To understand the interaction of P1 with salivary agglutinin at the molecular level, a panel of 11 anti-P1 monoclonal antibodies was tested for inhibitory activity in adherence and aggregation inhibition assays. Overlapping, but not identical, subsets of monoclonal antibodies were found to inhibit adherence and aggregation, indicating that the interactions of P1 with salivary agglutinin which mediate these two phenomena are different. The localization of functional domains of P1 which may mediate the aggregation and adherence reactions is discussed.     

Surface hydrophobicity, adherence, and aggregation of cell surface protein antigen mutants of Streptococcus mutans serotype c.

The pac gene of the serotype c strain Streptococcus mutans MT8148 encodes a cell surface protein antigen (PAc) of approximate 190 kilodaltons. The serotype c strain S. mutans GS-5 does not produce the 190-kilodalton PAc but produces a lower-molecular-weight protein that reacts with anti-PAc serum. The SphI-BamHI fragment of the pac gene was ligated with the S. mutans-Escherichia coli shuttle vector pSA3. The chimeric shuttle vector was transformed into strain GS-5, and two transformants (TK15 and TK18) were isolated. These transformants produced a large amount of cell-free and cell-bound PAc of 190 kilodaltons. No plasmid was isolated from these transformants, and the EcoRI fragments of their chromosomal DNA hybridized with the erythromycin resistance gene in the shuttle vector DNA, indicating insertion of the chimeric shuttle vector DNA into the chromosomal DNA. The cell hydrophobicity of strains TK15 and TK18 as well as PAc-defective mutants constructed by inserting an erythromycin resistance gene into the pac gene of strain MT8148 was analyzed. Strains MT8148, TK15, and TK18 were hydrophobic. On the other hand, strain GS-5 and PAc-defective MT8148 transformants were hydrophilic. Resting cells of the hydrophobic strains attached in larger numbers to saliva-coated hydroxyapatite than did the hydrophilic strains. Human whole saliva induced the aggregation of cells of the hydrophobic strains but not that of cells of the hydrophilic strains. These results suggest that cell surface PAc of S. mutans serotype c participates in attachment of the streptococcal cell to experimental pellicles.     

Identification of a salivary agglutinin-binding domain within cell surface adhesin P1 of Streptococcus mutans.

DNA encoding the alanine-rich region (A-region) of the cell surface adhesin, P1, from Streptococcus mutans was subcloned and expressed as a fusion protein with the maltose-binding protein (MBP) of Escherichia coli. The A-region fusion protein was shown to competitively inhibit both adherence of S. mutans to salivary agglutinin-coated hydroxyapatite and fluid-phase agglutinin-mediated aggregation of this organism. MBP alone or an MBP-paramyosin fusion protein was not inhibitory. Proteolytic cleavage of the fusion protein into its component moieties, MBP and A-region, resulted in breakdown of the A-region into three main fragments. Western immunoblot analysis of calcium-dependent agglutinin binding to this preparation revealed binding specificity for a 28-kDa fragment. Thus, the A-region of P1 is an important domain which interacts directly with salivary agglutinin, and this interaction interferes with both the aggregation and the adherence mechanisms in vitro.     

Molecular cloning and expression of a Streptococcus mutans major surface protein antigen, P1 (I/II), in Escherichia coli.

Antigen P1, also called I/II, is one of the most abundant cell wall proteins of the mutans streptococci. It has been suggested that P1 may be involved in cell adherence to tooth surfaces and in sucrose-induced cell aggregation. As a first step toward fully understanding its biological functions, the P1 gene, which has been designated spaP1, from Streptococcus mutans NG5 (serotype c) has been cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. The recombinant strain expressing P1 carries a 5.2-kilobase DNA insert whose restriction map has been determined. This map is completely different from that of spaA of Streptococcus sobrinus (serotype g), even though P1 and SpaA are antigenically related. Southern hybridization revealed that DNA sequences closely homologous to spaP1 were present in serotypes c, e, and f, and similar sequences also existed in strains of serotypes a and d. The expression of the cloned spaP1 was found to be independent of the lac inducer and the orientation of the DNA insert, suggesting that it carries its own promoter. Western blotting (immunoblotting) revealed at least 20 bands reacting with a mixture of three anti-P1 monoclonal antibodies. The highest-molecular-weight reactive band was comparable in size to the parent P1 (185 kilodaltons [kDa]); however, the major reactive bands were smaller (approximately 160 kDa). Expression of cloned P1 in E. coli LC137 (htpR lonR9) resulted in the increased prominence of the 185-kDa protein reactive band. Ouchterlony immunodiffusion showed partial identity between the parent and cloned P1. In E. coli, P1 was detected primarily in the periplasm and extracellular fluid.     

Isolation and characterization of Streptococcus mutans mutants defective in adherence and aggregation.

A method was developed which enriched for mutants of Streptococcus mutans that exhibit defects in adherence to glass, aggregation, or both. Mutants were isolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) after mutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survival after mutagenesis was kept above 1 to 2% to enhance the probability that mutants resulted from single mutational events. A total of 117 mutants were isolated; they also displayed non-wild-type colony morphology on mitis salivarius agar. These mutants were examined for (i) adherence and aggregation after overnight growth in sucrose-containing medium, (ii) aggregation of nongrowing cells in the presence of 200 microgram of sucrose per ml or 20 microgram of dextran per ml, and (iii) dextranase production on blue dextran agar plates. Although we isolated mutants which exhibited a variation from the parent strain in only one of the traits tested, the majority of mutants exhibited defects in two or more characteristics. Thirty-eight stable mutants of independent origin were categorized into 13 separate phenotypic groups.     

Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein antigen P1 (I/II).

The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or I/II) has been cloned into Escherichia coli (S. F. Lee, A. Progulske-Fox, and A. S. Bleiweis, Infect. Immun. 56:2114-2119, 1988). In the present study, this gene has been disrupted in vitro by insertional inactivation with pVA981, which carries a Tcr marker, and transformed into S. mutans NG8 (serotype c) by electroporation. Upon homologous recombination, the defective spaP was integrated into the genome as demonstrated by Southern hybridization analysis. One Tcr mutant, designated 834, selected by its nonreactivity with anti-P1 monoclonal antibodies, was found to lack the cell surface fuzzy layer which was clearly present on the parent cells. Analysis of extracellular fluids, sodium dodecyl sulfate-solubilized membranes, and cytoplasmic fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 834 had protein profiles identical to the parent. However, a 185-kilodalton protein which reacts with anti-P1 antibodies was missing from the wall of 834, suggesting that spaP has been specifically inactivated. This mutant displayed levels of glucosyltransferase and fructosyltransferase activities similar to those of the parent. It was much less hydrophobic than the parent. S. mutans NG8 aggregated readily in the presence of clarified whole saliva or a high-molecular-weight salivary agglutinin. This strain also adhered to agglutinin-coated hydroxyapatite. The P1-negative mutants, however, did not display these two properties, suggesting that P1 may play a role in saliva-mediated aggregation and adherence.     

Role of HtrA in surface protein expression and biofilm formation by Streptococcus mutans

The HtrA surface protease in gram-positive bacteria is involved in the processing and maturation of extracellular proteins and degradation of abnormal or misfolded proteins. Inactivation of htrA has been shown to affect the tolerance to thermal and environmental stress and to reduce virulence. We found that inactivation of Streptococcus mutans htrA by gene-replacement also resulted in a reduced ability to withstand exposure to low and high temperatures, low pH, and oxidative and DNA damaging agents. The htrA mutation affected surface expression of several extracellular proteins including glucan-binding protein B (GbpB), glucosyltransferases, and fructosyltransferase. In addition, htrA mutation also altered the surface expression of enolase and glyceraldehyde-3-phosphate dehydrogenease, two glycolytic enzymes that are known to be present on the streptococcal cell surface. As expected, microscopic analysis of in vitro grown biofilm structure revealed that the htrA deficient biofilms adopted a much more granular patchy appearance, rather than the relatively smooth confluent layer normally seen in the wild type. These results suggest that HtrA plays an important role in the biogenesis of extracellular proteins including surface associated glycolytic enzymes and in biofilm formation of S. mutans.     

Contribution of the alanine-rich region of Streptococcus mutans P1 to antigenicity, surface expression, and interaction with the proline-rich repeat domain

Streptococcus mutans is considered to be the major etiologic agent of human dental caries. Attachment of S. mutans to the tooth surface is required for the development of caries and is mediated, in part, by the 185-kDa surface protein variously known as antigen I/II, PAc, and P1. Such proteins are expressed by nearly all species of oral streptococci. Characteristics of P1 include an alanine-rich repeat region and a centrally located proline-rich repeat region. The proline-rich region of P1 has been shown to be important for the translational stability and translocation of P1 through the bacterial membrane. We show here that (i) several anti-P1 monoclonal antibodies require the simultaneous presence of the alanine-rich and proline-rich regions for binding, (ii) the proline-rich region of P1 interacts with the alanine-rich region, (iii) like the proline-rich region, the alanine-rich region is required for the stability and translocation of P1, (iv) both the proline-rich and alanine-rich regions are required for secretion of P1 in Escherichia coli, and (v) in E. coli, P1 is secreted in the absence of SecB.     

Inactivation of the Streptococcus mutans wall-associated protein A gene (wapA) results in a decrease in sucrose-dependent adherence and aggregation.

A 0.8-kb HindIII-BamHI internal fragment of the Streptococcus mutans wall-associated protein A gene (wapA) was ligated to the 5.1-kb HindIII-BamHI fragment of the chimeric Streptococcus-Escherichia coli plasmid pVA891 (Emr Cmr). The resulting construct was used to transform S. mutans GS-5, and erythromycin-resistant mutants were isolated and analyzed. Directed mutagenesis of the wapA gene by plasmid insertion through homologous recombination was demonstrated by Southern blot hybridization with the wapA and pVA891 probes. Stable mutants were obtained, and the alteration of the wapA gene by insertional inactivation was associated with a significant decrease in S. mutans sucrose-dependent aggregation and binding to smooth surfaces. Thus, WapA may play an important role in the colonization of the tooth surface by S. mutans and in the buildup of dental plaque. These findings provided an explanation for previous studies which indicated that WapA was effective in the prevention of dental caries in animal models. Thus, the use of recombinant WapA in the preparation of a safe and effective human dental vaccine should be investigated further.     

Isolation and characterization of monoclonal antibodies specific for antigen P1, a major surface protein of mutans streptococci.

A panel of 15 murine monoclonal antibodies (MAbs; 14 immunoglobulin G1, 1 immunoglobulin G2a) directed against antigen P1, a major surface protein of mutans streptococci, was prepared. All of these MAbs reacted by the enzyme-linked immunosorbent assay with solubilized wall material from Streptococcus mutans Ingbritt 175 (a serotype c strain which retains significant amounts of P1 in its cell wall), culture supernatant fluid from Ingbritt 162 (a strain which excretes large amounts of P1 into the culture medium), and purified P1. By Western immunoblotting, these MAbs were observed to react with a high-molecular-weight polypeptide which comigrated with antigen P1. None of these MAbs cross-reacted with human heart tissue or with various eucaryotic proteins. When whole cells of various strains of mutans streptococci were screened against the panel of MAbs, the strongest reactivities were noted with strains of serotype c and e S. mutans, while a serotype f strain of S. mutans, along with S. sobrinus and S. cricetus strains, reacted somewhat more weakly. S. rattus strains were completely negative. Results obtained with bacterial culture supernatants were qualitatively similar. The surface localization of antigen P1 was confirmed by electron microscopy with an indirect immunogold technique. In sectioned S. mutans cells, labeling appeared to be associated with a fibrillar "fuzzy coat" layer, which was far more prominent on cells of Ingbritt 175 than on those of Ingbritt 162.     

Effect of adsorbed protein on hydroxyapatite zeta potential and Streptococcus mutans adherence.

The adherence of Streptococcus mutans PK1 to hydroxyapatite disks pretreated with various acidic and basic proteins in imidazole buffer was studied. Adsorption of a basic protein onto an hydroxyapatite disk enhanced or had no effect on bacterial adherence, whereas adsorption of an acidic protein reduced adherence. The effect of adsorbed protein on bacterial adherence was of both short and long range. The long-range effect of the acidic proteins in reducing the number of bacteria adhering to hydroxyapatite was related to protein adsorption causing an increase in surface net negative charge, as shown by zeta potential measurement. Basic protein produced a net positive surface charge which facilitated adherence. Within the acidic protein group, the acidic residue percentage of the adsorbed protein was negatively correlated with the number of bacteria adhering, whereas the nonpolar residue percentage was positively correlated with bacterial adherence. Within the basic protein group, the basic residue percentage was correlated with the number of cells adhering. These results indicate the involvement of short-range hydrophobic and ionic interactions in bacterial adherence to protein-coated hydroxyapatite.     

Characterization of a P1-deficient strain of Streptococcus mutans that expresses the SpaA protein of Streptococcus sobrinus.

The Streptococcus sobrinus SpaA protein and the Streptococcus mutans P1 protein share 66% sequence homology at the amino acid level. To determine if the SpaA protein can be expressed in S. mutans and functionally replace the P1 protein, the spaA gene of S. sobrinus 6715 was isolated from plasmid pX1303 and inserted into the Escherichia coli-Streptococcus shuttle vector pVA838. The resulting plasmid pX1600 was transformed into the P1-deficient strain S. mutans 834 that has defects in saliva-mediated aggregation and in the ability to adhere to saliva-coated hydroxyapatite surfaces. Western blot (immunoblot) analysis of cellular protein fractions of S. mutans 834 (pX1600) detected in mutanolysin-solubilized cell walls a major protein of 210 kDa with an electrophoretic mobility similar to that of S. sobrinus SpaA protein and a minor 210-kDa protein and a major 64-kDa protein in the extracellular protein fraction. Analysis of virulence traits showed that expression of SpaA protein by S. mutans 834(pX1600) cells had restored the ability of the S. mutans 834 cells to aggregate in the presence of saliva or salivary agglutinin but not to adhere to saliva-coated hydroxyapatite. This cell aggregation was inhibited specifically by antisera to S. sobrinus SpaA protein. These results indicate that SpaA plays a role in the virulence of S. sobrinus by specifically interacting with fluid-phase salivary agglutinin to mediate cell aggregation.     

变形链球菌表面蛋白可变区真核载体质粒pEGFP-N1一SrV+的构建及在293T细胞的表达

目的 构建变形链球菌(变链菌)表面蛋白可变区(extended-v)真核表达质粒pEGFP-NI-SrV+,并转染哺乳动物细胞293T,为进一步研究该区功能奠定基础.方法 根据已报道变链菌OMZ175的sry+基因序列,化学合成srV+的编码基因srv+,将真核载体质粒pEGFP-N1和srv+基因片段分别用Kpn Ⅰ/Xho Ⅰ双酶切,连接获取重组质粒pEGFP-NI-SrV+,并对其进行PCR、酶切和测序鉴定.通过脂质体法瞬时转染哺乳动物细胞293T.运用荧光显微镜观察、Western blot及real-time PCR法检目的 蛋白在293T细胞中的表达情况.结果 通过基凶化学合成技术,获得1136 bp的目的 基因srv+,经测序鉴定与模板目的 基因序列一致;成功构建真核表达质粒pEGFP-N1-SrV+;PCR扩增榆测可获得1.33 kb目的 基因片段;Kpn Ⅰ/Xho Ⅰ双酶切可见4.7 kb和1.1 kb两条电泳条带,证实重组质粒pEGFP-N1-SrV+携带目的 基因;通过荧光显微镜观察到重组质粒pEGFP-N1-SrV+融合GFP后的表达,目的 细胞的转染效率达到80%以上;Western blot检测到相对分子质量(M1)为72 × 103处有特征条带,其大小和Srv+融合蛋白(42 × 103+28 × 103=70 × 103)相吻合;real-time PCR 数值分析显示:在293T细胞中,srv+基因的过表达效果显著.结论 成功构建了真核表达载体质粒pEGFP-N1-SrV+,并能够在哺乳动物细胞293T中表达.

Abstract：

Objective To construct the recombinant plasmid pEGFP-N1-SrV+ and evaluate the expression of SrV+in mammalian 293T cells.nethods srv+.a gene encoding the vailable region of the surface protein of the Streptococcus mutans OMZ175.was cloned chemically based on its reported nucleotide sequence.The eukaryotic expression plasmid,pEGFP-N1-SrV+,was constructed by introducing the srv+ gene into the Kpn Ⅰ/Xho Ⅰ site of pEGFP-NI.The recombinant plasmid pEGFP-N1-SrV+was transfected into 293T cells with lipofectamine and the expression level of SrV+was evaluated.Results The eukaryotic expression plasmid pEGFP-N1-SrV+was constructed successfully.GFP was observed by green fluorescent microscope.and a 72 × 1 03 protein was detected bv Westem blot.Real-time RT-PCR analysis revealed that the expression of the pEGFP-N1-SrV+in 293T was excellent and significant compared the control group. Conclusion The recombinant plasmid pEGFP-N1-SrV+was successfully constructed.which could encode the expression of SrV+after transfected into the mammalian 293T Cells.     

Involvement of sortase anchoring of cell wall proteins in biofilm formation by Streptococcus mutans

Streptococcus mutans is one of the best-known biofilm-forming organisms associated with humans. We investigated the role of the sortase gene (srtA) in monospecies biofilm formation and observed that inactivation of srtA caused a decrease in biofilm formation. Genes encoding three putative sortase-dependent proteins were also found to be up-regulated in biofilms versus planktonic cells and mutations in these genes resulted in reduced biofilm biomass.     

Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae.

Pneumococcal surface adhesin A (PsaA), with a molecular mass of approximately 37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotypes of Streptococcus pneumoniae. S. pneumoniae serotype 6B genomic DNA was amplified to generate a DNA fragment carrying the full-length psaA sequence and was cloned into a baculovirus expression system. We expressed either cell-associated or cell-free nonfusion PsaA polypeptides using two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High-Five). Recombinant PsaA (rPsaA) polypeptides were partially purified by partitioning in PBS/Triton X-114 buffers and by weakly basic ion exchange filter chromatography. Membrane-bound 'hydrophobic rPsaA' (hrPsaA) expressed by either Sf9 or High-Five cells had a molecular mass of approximately 38 kD by SDS-PAGE and partitioned in a Triton X-114 phase, it reacted with both rabbit polyclonal and five monoclonal anti-PsaA antibodies by dot blot or Western blot analysis. High-Five-cell-expressed 'soluble rPsaA' (srPsaA) with a molecular mass of approximately 37 kD by SDS-PAGE, was isolated from the serum-free culture medium and did not partition in the Triton X-114 phase; it reacted with anti-PsaA rabbit polyclonal and mouse monoclonal antibodies by ELISA and Western blot analysis. Both rPsaA polypeptide forms were immunogenic in Swiss-Webster adult female mice. In an infant mouse model of bacteremia, survival rates for mice given mouse anti-rPsaA immune serum (from mice immunized with High-Five-expressed srPsaA; 20 microl, 1:50,000 titer) 24 h before bacteremic challenge were greater than for the control group (48 h postchallenge, 20 vs. 90% survival rates) when challenged with S. pneumoniae serotype 6B. These results indicate that rPsaA is immunogenic and elicits protective antibody in mice similar to native protein.     

Shared epitopes between Mycoplasma pneumoniae major adhesin protein P1 and a 140-kilodalton protein of Mycoplasma genitalium

Previous serological data have demonstrated cross-reactive antigens between two pathogenic species of mycoplasmas, M. pneumoniae and M. genitalium. Preliminary analysis of sera and monoclonal antibodies (MAbs) to protein antigens of these species showed an immunodominance of adhesin P1 (165 kilodaltons [kDa]) of M. pneumoniae in mice and hamsters and a 140-kDa protein of M. genitalium in mice and experimentally infected chimpanzees. To further characterize these two proteins, we assayed multiple anti-P1 and anti-140-kDa protein MAbs by enzyme-linked immunosorbent assay, immunoblot, and radioimmunoprecipitation techniques. The 140-kDa M. genitalium protein was shown to be surface accessible and insensitive to levels of trypsin which readily degrade protein P1. Peptide mapping was used to identify a unique class of MAbs which bound a cross-reactive molecule common to both the major adhesin protein P1 of M. pneumoniae and the 140-kDa protein of M. genitalium. MAbs generated against both M. pneumoniae and M. genitalium which were reactive with this determinant blocked M. pneumoniae attachment to chicken erythrocytes.     

Identity of Streptococcus mutans surface protein antigen III and wall-associated protein antigen A.

Preparations of Streptococcus mutans surface proteins AgIII and antigen A from different laboratories were compared with regard to amino acid composition, N-terminal amino acid sequence, electrophoretic mobility, and antigenic similarity. Despite previous observations of differences in physical properties, data indicate that these two preparations represent the same protein.     

Characterization of recombinant, ureolytic Streptococcus mutans demonstrates an inverse relationship between dental plaque ureolytic capacity and cariogenicity

Dental caries results from prolonged plaque acidification that leads to the establishment of a cariogenic microflora and demineralization of the tooth. Urease enzymes of oral bacteria hydrolyze urea to ammonia, which can neutralize plaque acids. To begin to examine the relationship between plaque ureolytic activity and the incidence of dental caries, recombinant, ureolytic strains of Streptococcus mutans were constructed. Specifically, the ureABCEFGD operon from Streptococcus salivarius 57.I was integrated into the S. mutans chromosome in such a way that the operon was transcribed from a weak, cognate promoter in S. mutans ACUS4 or a stronger promoter in S. mutans ACUS6. Both strains expressed NiCl(2)-dependent urease activity, but the maximal urease levels in ACUS6 were threefold higher than those in ACUS4. In vitro pH drop experiments demonstrated that the ability of the recombinant S. mutans strains to moderate a decrease in pH during the simultaneous metabolism of glucose and urea increased proportionately with the level of urease activity expressed. Specific-pathogen-free rats that were infected with ACUS6 and fed a cariogenic diet with drinking water containing 25 mM urea and 50 microM NiCl(2) had relatively high levels of oral urease activity, as well as dramatic decreases in the prevalence of smooth-surface caries and the severity of sulcal caries, relative to controls. Urease activity appears to influence plaque biochemistry and metabolism in a manner that reduces cariogenicity, suggesting that recombinant, ureolytic bacteria may be useful to promote dental health.     

In vitro and in vivo complementation of Streptococcus mutans mutants defective in adherence

Previous studies have shown that adherence-defective mutants of Streptococcus mutans PS14, serotype c, can be grouped into several different phenotypic groups. In this study a method was developed to test for complementation between pairs of nonadhering mutants which possess different genotypic defects. Mutant strains UAB95 and a spectinomycin-resistant derivative of UAB95 (UAB516) were found to exhibit increased levels of adherence when grown together with UAB230 in media containing sucrose as compared to the adherence of each strain grown separately. An increase in caries was also observed in gnotobiotic rats mixedly infected with the two mutants as compared to either strain alone. Tests revealed that UAB95 produced more water-insoluble glucan than its parent strain but had a defect in glucan binding. UAB230 was found to produce levels of a defective glucan that could not be bound by mutant or wild-type cells. Our results suggest that UAB95 produces a water-insoluble glucan which is bound by UAB230, thus allowing complementation for adherence and caries production.