Wall-associated protein antigens of Streptococcus mutans.

When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29 000, while B had a molecular weight of 190 000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.     

Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans

Immunization of rabbits with Streptococcus mutans antigens results in the production of serum antibodies that bind in vitro to human, rabbit, and monkey cardiac muscle. Antibodies to heart, however, have also been reported to occur at lower titers in the sera of unimmunized rabbits. In this study, the specificities of heart-reactive antibodies (HRA) in sera of unimmunized and S. mutans-immunized rabbits were compared using indirect immunofluorescence, Western blot, and Bio-Dot immunoassays. Both groups of sera gave striational indirect immunofluorescence-staining patterns on thin sections of native human and monkey cardiac muscle. Western blot analyses revealed that antibodies in normal sera bound 9 to 20 components of human, rabbit, and monkey heart. The major bands had Mr of 205,000, 160,000, 135,000, and 70,000. Several of the normal sera did not have antibody activity to S. mutans antigens, indicating that these HRA do not cross-react with these bacteria. Although immunization of rabbits with S. mutans caused increased titers of HRA (two to three doubling dilutions), Western blot assays using anti-S. mutans sera showed banding patterns qualitatively similar to those of normal sera on heart extracts. Antibodies to skeletal muscle myosin were detected in both serum groups. Of eighteen normal rabbit sera sixteen had antimyosin titers of 10 to 40, whereas all eighteen anti-S. mutans sera had titers of 10 to 160. Affinity-purified antimyosin antibodies isolated from anti-S. mutans serum did not bind to S. mutans components. Conversely, affinity-purified antibodies to S. mutans antigens did not bind to myosin or to other cardiac muscle components. Among these were antibodies to the 185-kDa cell wall protein (also known as B, I/II, IF, Spa A, and P1) previously believed to possess antigenic mimicry. HRA were removed from anti-S. mutans sera by absorption with S. mutans but this effect was not specific, because a non-cross-reactive internal standard antibody was also absorbed to the same extent. Because previous evidence for antigenic mimicry between S. mutans and cardiac muscle was based on serum cross-absorption experiments, this immunologic relationship is not substantiated. These results indicated that naturally occurring antibodies to cardiac muscle components are present in the sera of unimmunized rabbits and that immunization with S. mutans does not stimulate production of new heart-reactive antibody, but rather serves to boost antibody production by preexisting clones of self-reactive B-lymphocytes.     

Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin

Monoclonal antibodies (mAb) generated by immunization of mice with membranes of Streptococcus pyogenes Manfredo (M type 5) were tested for their reactivity with sodium dodecyl sulfate extracts of Streptococcus mutans GS5, Streptococcus rattus BHT (formerly S. mutans, serotype b), and S. mutans Ingbritt 175 in the Western blot. The reaction of the sodium dodecyl sulfate-extracted proteins of whole S. mutans, serotype b), and S. rattus with the mAb revealed that the shared cross-reactive antigens were near m.w. 62,000 to 67,000. Several higher m.w. proteins in S. mutans Ingbritt 175 reacted with the mAb but were not observed in the other two strains of streptococci. Cytoplasmic membranes of S. rattus BHT reacted with these mAb in the enzyme-linked immunosorbent assay and Western blot. The most reactive BHT membrane component detected by these mAb was a 62-kDa polypeptide that was similar in m.w. to the membrane component recognized by these antibodies in purified S. pyogenes membranes. The reactivity of the mAb with BHT membranes in the enzyme-linked immunosorbent assay was absorbed by rabbit skeletal muscle myosin. The patterns of reactivity observed in the BHT membrane immunoblots in this study closely resemble those previously obtained using polyclonal rabbit anti-human heart sera.     

Regulation of ATP-dependent P-(Ser)-HPr formation in Streptococcus mutans and Streptococcus salivarius.

Sugar transport via the phosphoenolpyruvate (PEP) phosphotransferase system involves PEP-dependent phosphorylation of the general phosphotransferase system protein, HPr, at histidine 15. However, gram-positive bacteria can also carry out ATP-dependent phosphorylation of HPr at serine 46 by means of (Ser)HPr kinase. In this study, we demonstrate that (Ser)HPr kinase in crude preparations of Streptococcus mutans Ingbritt and Streptococcus salivarius ATCC 25975 is membrane associated, with pH optima of 7.0 and 7.5, respectively. The latter organism possessed 7- to 27-fold-higher activity than S. mutans NCTC 10449, GS-5, and Ingbritt strains. The enzyme in S. salivarius was activated by fructose-1,6-bisphosphate (FBP) twofold with 0.05 mM ATP, but this intermediate was slightly inhibitory with 1.0 mM ATP at FBP concentrations up to 10 mM. Similar inhibition was observed with the enzyme from S. mutans Ingbritt. A variety of other glycolytic intermediates had no effect on kinase activity under these conditions. The activity and regulation of (Ser)HPr kinase were assessed in vivo by monitoring P-(Ser)-HPr formation in steady-state cells of S. mutans Ingbritt grown in continuous culture with limiting glucose (10 and 50 mM) and with excess glucose (100 and 200 mM). All four forms of HPr [free HPr, P approximately (His)-HPr, P-(Ser)-HPr, and P approximately (His)-P-(Ser)-HPr] could be detected in the cells; however, significant differences in the intracellular levels of the forms were apparent during growth at different glucose concentrations. The total HPr pool increased with increasing concentrations of glucose in the medium, with significant increases in the P-(Ser)-HPr and P approximately HHis)-P-(Ser)-HPr concentrations. For example, while total PEP-dependent phosphorylation [P approximately(His)-HPr plus P approximately (His)-P-(Ser)-HPr] varied only from 21.5 to 52.5 microgram mg of cell protein (-1) in cells grown at the four glucose concentrations, the total ATP-dependent phosphorylation [P-(Ser)-HPr plus P approximately (His)-P-(Ser)-HPr] increased 12-fold from the 10 mM glucose-grown cells (9.1 microgram mg of cell protein (-1) to 106 and 105 microgram mg(-1) in the 100 and 200 mM glucose-grown cultures, respectively. (Ser)HPr kinase activity in membrane preparations of the cells varied little between the 10, 50, and 100 mM glucose-grown cells but increased threefold in the 200 mM glucose-grown cells. The intracellular levels of ATP, glucose-6-phosphate, and FBP increased with external glucose concentration, with the level of FBP being 3.8-fold higher for cells grown with 200 mM glucose than for those grown with 10 mM glucose. However, the variation in the intracellular levels of FBP, particularly between cells grown with 100 and 200 mM glucose, did not correlate with the extent of P-(Ser)-HPr formation, suggesting that the activity of (Ser)HPr kinase is not critically dependent on the availability of intracellular FBP.     

Demonstration of shared antigenic determinants between Streptococcus mutans BHT cell membrane, human heart tissue and myosin using monoclonal antibodies to S. mutans

Monoclonal antibodies (MAb) raised to intact Streptococcus mutans P-4 cells (serotype e) were used to demonstrate the presence of shared antigenic determinant(s) between S. mutans BHT (serotype b) cell membranes and human heart tissue. MAb binding to both BHT membrane and human heart tissue was demonstrated by ELISA. Common antigens were identified by immunoblot analysis following separation of BHT membrane components and human heart antigens by SDS-PAGE. MAb 22C4 recognized three polypeptides from the BHT membrane preparation, having molecular masses of 42, 56 and 85 kDa. MAb 22C4 also recognized an 85 kDa component and a 200 kDa component from human heart tissue. MAb D159 was specific for a single 82 kDa polypeptide in BHT membrane, and also bound to two high molecular mass components in human heart (165 and 200 kDa). When both MAb D159 and 22C4 were first absorbed with S. mutans P-4 cells, subsequent reactivity to the aforementioned BHT membrane components was inhibited, indicating that these cross-reactive components are found in S. mutans P-4 as well as in S. mutans BHT micro-organisms. Competitive binding analysis showed that both MAb D159 and MAb 22C4 bound to myosin, indicating that S. mutans BHT membrane, human heart tissue and myosin share at least one immunodeterminant. This indicates that myosin could be the cross-reactive tissue component in human heart.     

Sequence analysis of the wall-associated protein precursor of Streptococcus mutans antigen A

The nucleotide sequence has been determined for the Streptococcus mutans wall-associated protein A (wapA) gene from serotype c strains Ingbritt and GS5. The nucleotide sequence for each wapA gene was virtually identical, although the gene from strain GS5 contained a 24 base pair deletion. A 29 amino acid signal peptide was specified by each wapA gene with a mature protein of 424 amino acids (Mr, 45,276) for strain Ingbritt and 416 amino acids (Mr, 44,846) for strain GS5. In the C-terminal region of the wall-associated protein A, considerable sequence similarity was found with the membrane anchor region of proteins from other Gram-positive organisms such as the group A streptococcal M protein and the group G streptococcal IgG binding protein. Adjacent to the proposed membrane anchor is a highly hydrophilic region which may span the cell wall; both sequence data and experimental evidence indicate the existence of a region immediately outside the wall at which proteolytic cleavage occurs to release antigen A of Mr 29,000 into the culture supernatant. Thus, the wall-associated protein A is a precursor of the 29,000 Mr antigen A.     

Human IgG and Streptococcus mutans SR protein contain cross-reactive epitopes

Antigen B, a glycoprotein present on the cell surface of "mutans streptococci," mediates bacterial adherence to teeth surfaces and has been implicated in cross-reactivity with human heart components. Elevated levels of anti-IgG antibodies were generally found in sera of rabbits immunized with protein SR, a B-related protein from Streptococcus mutans serogroup f, or recombinant protein SR (rSR). These anti-IgG antibodies could be involved in the previously mentioned heart cross-reactivities. Results from immunoblots and ELISA analyses demonstrate that these anti-IgG antibodies recognize common epitopes on SR, rSR, and human IgG2 and IgG4 probably located on the Fab region. Furthermore, control experiments with biotinylated human IgG show that the cross-reactions between IgG and SR were not mediated by an FcR mechanism. Direct competition between rSR and human IgG in binding to anti-IgG or anti-SR antibodies confirm that S. mutans SR protein possesses Ag mimicry with human IgG. Our studies provide some evidence that S. mutans SR protein and human IgG H chains share autoimmune epitopes which could play a role in the induction of anti-IgG antibodies and therefore could explain the enhancement of anti-IgG antibody levels observed in rabbits immunized with either S. mutans whole cells or purified B-related Ag.     

Regulation of glucosyl- and fructosyltransferase synthesis by continuous cultures of Streptococcus mutans

Streptococcus mutans strains Ingbritt, and its derivative B7 which had been passaged through monkeys, have been used to investigate how the synthesis of extracellular glucosyl- and fructosyltransferases is regulated. The most active enzyme from carbon-limited continuous cultures was a fructosyltransferase; enzymes catalysing the formation of water-insoluble glucans from sucrose were relatively inactive. Dextransucrase (EC 2.4.1.5), which catalyses soluble glucan synthesis, was most active in the supernatant fluid from cultures grown with excess glucose, fructose or sucrose, but full activity was detected only when the enzyme was incubated with both sucrose and dextran. Little dextransucrase activity was detected in carbon-limited cultures. It is concluded that glucosyl- and fructosyltransferases are constitutive enzymes in that they are synthesized at similar rates during growth with an excess of the substrate or of the products of the reactions which they catalyse. Although the Ingbritt strain was originally isolated from a carious lesion, it is now a poor source of glucosyltransferase activity. Glucosyltransferases were extremely active in cultures of a recent clinical isolate, strain 3209, and were apparently induced during growth with excess glucose.     

Characterization of transmembrane movement of glucose and glucose analogs in Streptococcus mutants Ingbritt.

The transmembrane movement of radiolabeled, nonmetabolizable glucose analogs in Streptococcus mutants Ingbritt was studied under conditions of differing transmembrane electrochemical potentials (delta psi) and pH gradients (delta pH). The delta pH and delta psi were determined from the transmembrane equilibration of radiolabeled benzoate and tetraphenylphosphonium ions, respectively. Growth conditions of S. mutants Ingbritt were chosen so that the cells had a low apparent phosphoenolpyruvate (PEP)-dependent glucose:phosphotransferase activity. Cells energized under different conditions produced transmembrane proton potentials ranging from -49 to -103 mV but did not accumulate 6-deoxyglucose intracellularly. An artificial transmembrane proton potential was generated in deenergized cells by creating a delta psi with a valinomycin-induced K+ diffusion potential and a delta pH by rapid acidification of the medium. Artificial transmembrane proton potentials up to -83 mV, although producing proton influx, could not accumulate 6-deoxyglucose in deenergized cells or 2-deoxyglucose or thiomethylgalactoside in deenergized, PEP-depleted cells. The transmembrane diffusion of glucose in PEP-depleted, KF-treated cells did not exhibit saturation kinetics or competitive inhibition by 6-deoxyglucose or 2-deoxyglucose, indicating that diffusion was not facilitated by a membrane carrier. As proton-linked membrane carriers have been shown to facilitate diffusion in the absence of a transmembrane proton potential, the results therefore are not consistent with a proton-linked glucose carrier in S. mutans Ingbritt. This together with the lack of proton-linked transport of the glucose analogs suggests that glucose transmembrane movement in S. mutans Ingbritt is not linked to the transmembrane proton potential.     

Inducible and constitutive formation of fructanase in batch and continuous cultures of Streptococcus mutans

The production of extracellular beta-D-fructanase by several strains of Streptococcus mutans was studied in continuous culture. When glucose was the limiting nutrient, S. mutans K1-R and OMZ176 accumulated fructanase to maximum levels at low growth rates (dilution rate 0.05-0.10 h-1), due to the longer residence times of the bacteria in the culture vessel under these conditions. Extracellular fructanase activity was greater than has been previously reported for batch cultures. The rate of fructanase production for both S. mutans strains K1-R and OMZ176 increased with increasing growth rate when glucose was limiting. Under conditions of glucose sufficiency, the rate of fructanase production was always lower than in cultures where glucose was limiting, irrespective of the growth rate. Cultures of S. mutans Ingbritt (serotype c) grown with sorbitol- or glucose-limitation synthesized fructanase at a very low basal rate. When fructose was the limiting carbohydrate the enzyme was induced with a maximum rate of production occurring at a dilution rate of 0.40 h-1. Strains of S. mutans from other serotypes (a, d, d/g) were either not affected by changing the limiting sugar from glucose to fructose or else fructanase activity was slightly decreased in the fructose-limited medium. Fructanases from various strains of S. mutans readily hydrolysed (2----6)-beta-D-fructans, but all possessed the ability to hydrolyse (2----1)-beta-D-fructans to varying degrees.     

灰色链霉菌RX-17溶菌酶R1的纯化及性质研究

通过硫酸铵分级沉淀,CM-Sephadex C50、CM-Sepharose Fast Flow离子交换层析及Sephadex G-75凝胶过滤层析,从灰色链霉菌（Streptomyces griseus）RX17的发酵上清液中得到了电泳纯的溶菌酶R1,回收率6.89%。测得该酶分子量和等电点分别为16.8kD和9.10,作用于变链球菌（Streptococcus mutans）Ingbritt的最适温度和pH分别为70℃和6.6。R1酶在50℃以下及pH6～9的范围内保持稳定,60℃保温1h,残存酶活20.3％。Mg2+对酶有激活作用,而Zn2+、Cu2+、Fe2+、Cd2+、Pb2+则使酶完全丧失活性,螯合剂、盐酸羟胺、碘乙酸抑制酶活,β-巯基乙醇及表面活性剂则对溶菌有部分促进作用。R1酶溶菌谱广泛,对多种卵清溶菌酶不能作用的G+、G细菌均有溶解能力,对变链球菌、金黄色葡萄球菌（Staphylococcus aureus）、乳杆菌(Lactobacillus)等则呈现高活性。     

Antibody responses to antigens of Streptococcus mutans in monkeys (Macaca fascicularis) immunized against dental caries

Immunization of rabbits or monkeys with walls prepared from Streptococcus mutans by a procedure including extraction with SDS at room-temperature induced antibodies to three antigens (A, B and C) detectable by crossed immunoelectrophoresis. Antigens A and B have previously been characterized as proteins of molecular weight 29 000 and 190 000, respectively. Antigen C was characterized as having a molecular weight of 70 000 and was purified by immunosorbent affinity chromatography and hydrophobic interaction chromatography. Another wall protein, antigen D, of molecular weight 13 000, was extracted from walls with Triton X-100. Immunization of monkeys with walls prepared from cultures of S. mutans grown at a high (D = 0.5 h-1) or low (D = 0.05 h-1) dilution rate in a chemostat showed that only the latter induced protection against dental caries. There was a positive correlation between levels of antibody to antigens A and C and induction of protection and a negative correlation between protection and the level of antibody to antigen B. No antibody to antigen D was detected in protected monkeys and an experiment in which monkeys were immunized with pure antigen D confirmed that it does not induce protection.     

Comparison of extracellular protein profiles of seven serotypes of mutans streptococci grown under controlled conditions

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.     

Effect of growth conditions on levels of components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans and Streptococcus sobrinus grown in continuous culture.

The membrane-bound, sugar-specific enzyme II (EII) component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Streptococcus mutans Ingbritt is repressed by growth on glucose under various conditions in continuous culture. Compared with optimal PTS conditions (i.e., glucose limitation, dilution rate [D] of 0.1 h-1, and pH 7.0), EII activity for glucose (EIIGlc) and mannose (EIIMan) in cells grown at a D of 0.4 h-1 and pH 5.5 with the same glucose concentration was reduced 24- to 27-fold. EII activity with methyl alpha-glucoside and 2-deoxyglucose was reduced 6- and 26-fold, respectively. Growth with excess glucose (i.e., nitrogen limitation) resulted in 26- to 88-fold repression of EII activity with these substrates. The above conditions of low pH, high dilution rate, and excess glucose also repressed EII activity for fructose (EIIFru), but to a lesser extent (two- to fivefold). Conversely, growth of S. mutans DR0001 at a D of 0.2 h-1 and pH 5.5 resulted in increased EIIGlc and EIIMan activity. Unlike the EII component, the HPr concentration in S. mutans Ingbritt varied only twofold (5.5 to 11.4 nmol/mg of protein) despite growth at pH 5.5 with limiting and excess glucose. The HPr concentrations in S. mutans DR0001 and the glucose-PTS-defective mutant DR0001/6 were similar. In a companion study, the soluble components of the PTS (i.e., HPr, EI, and EIIILac) in Streptococcus sobrinus grown on limiting lactose in a chemostat were not influenced significantly by growth at various pHs (7.0 and 5.0) and growth rates (D of 0.1, 0.54, and 0.8 h-1). However, growth on lactose resulted in repression of both EIIGlc and EIIFru, confirming earlier results with batch-grown cells. Thus, the glucose-PTS in some strains of S. mutans is regulated at the level of EII synthesis by certain environmental conditions.     

Role of the C terminus in antigen P1 surface localization in Streptococcus mutans and two related cocci.

The C terminus of the major surface protein P1 from Streptococcus mutans is composed of a hydrophilic domain, an LPNTGV motif, a hydrophobic domain, and a charged tail. These features are shared by surface proteins from many gram-positive coccal bacteria. To investigate the role of the C-terminal domains in antigen P1 surface localization, full-length and truncated P1 gene constructs, which were expressed on the shuttle vector pDL276, were transformed into the P1-negative mutant S. mutans SM3352, Streptococcus gordonii DL-1, and Enterococcus faecalis UV202. Transformants were tested for expression of P1 by enzyme-linked immunosorbent assaying and Western blotting. The results showed that full-length P1 was expressed by transformants of all three bacteria and was localized on the cell surface. A fusion protein composed of the Staphylococcus aureus fibronectin binding protein C terminus and the P1 protein N terminus was found to surface localize in S. mutans. Deletion of the entire C-terminal domains resulted in P1 being expressed in the culture supernatant. A P1 truncation, which carried only the hydrophilic domain at its C terminus, was found partially associated with the cell surface. This truncated P1 was readily removed from the isolated cell wall by hot sodium dodecyl sulfate-mercaptoethanol extraction. In contrast, the full-length P1 remained associated with the isolated cell wall after similar treatment, suggesting covalent linkages between the full-length P1 and the cell wall. The results described above showed that antigen P1 was anchored to the cell wall by its C-terminal domains probably via covalent linkages with the cell wall. The results also support a universal mechanism involving the C-terminal domains for protein surface localization among this group of gram-positive bacteria.     

Concentration-dependent repression of the soluble and membrane components of the Streptococcus mutans phosphoenolpyruvate: sugar phosphotransferase system by glucose.

Growth of Streptococcus mutans Ingbritt in continuous culture (pH 7.0, dilution rate of 0.1 h-1) at medium glucose concentrations above 2.6 mM resulted in repression of the sugar-specific membrane components, enzyme IIGlc (EIIGlc) and EIIMan, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In one experiment, significant repression (27-fold) was observed with 73 mM glucose when the glycolytic capacity of the cells was reduced by only 2-fold and when the culture was still glucose limited. In a more comprehensive experiment in which cells were grown in continuous culture at eight glucose concentrations from 2.6 to 304 mM, in addition to repression of specific EII activities for glucose, mannose, 2-deoxyglucose, and fructose, synthesis of the general protein, EI, was repressed at all glucose levels above 2.6 mM to a maximum of 4-fold at 304 mM glucose when the culture was growing with excess glucose (i.e., nitrogen limited). The other PTS general protein, HPr, was less sensitive to the exogenous glucose level but was nevertheless repressed fourfold under glucose-excess conditions. The Km for glucose for EIIGlc increased from 0.22 mM during growth at 3.6 mM glucose (glucose limited) to 0.48 mM at 271 mM glucose (glucose excess). The shift from heterofermentation to homofermentation during growth with increasing glucose levels suggests the involvement of glycolytic intermediates, ATP, or another high-energy phosphate metabolite in regulation of the synthesis of the PTS components in S. mutans.     

Insoluble glucan synthesis by mutansucrase as a determinant of the cariogenicity of Streptococcus mutans

Five strains of Streptococcus mutans were grown in continuous culture with either a limited supply or an excess of glucose. Proteins secreted into the extracellular fluid by strains C67-1, 3209 and K1 rapidly catalysed the synthesis of insoluble glucan from sucrose (mutansucrase activity). The culture fluid from strains Ingbritt or C67-25 catalysed the synthesis of soluble glucan (dextransucrase activity) and fructan, but little or no mutansucrase activity was detected. The strains which secreted active mutansucrase readily colonized a smooth hard surface during growth in batch culture and were more cariogenic in pathogen-free rats than those which secreted little mutansucrase activity. There was no similar correlation between fructosyltransferase, dextransucrase or total glucosyltransferase activity and either adherence or cariogenicity. We conclude that the ability to catalyse insoluble glucan synthesis is a major determinant of the cariogenicity of S. mutans strains.     

Comparative study of new alpha-galactosidases in transglycosylation reactions

We have studied the potential of several newly cloned alpha-galactosidases to catalyze the regioselective synthesis of disaccharides using 4-nitrophenylgalactoside as a donor. The kinetics of the reactions were followed by in situ NMR spectroscopy. The following thermophilic enzymes have been tested: Aga A and an isoenzyme Aga B obtained from the strain KVE39 and Aga 285 from the strain IT285 of Bacillus stearothermophilus; Aga T is an alpha-galactosidase from Thermus brockianus (strain IT360). Two other non-thermophilic alpha-galactosidases have also been evaluated: Aga 1 (Streptococcus mutans, strain Ingbritt) and Raf A (Escherichia coli, strain D1021). For all of the enzymes studied, high regioselectivity was observed leading to two (1 --> 6)-disaccharides: 4-nitrophenyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside and methyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside, which were obtained in 54% (Aga B) and 20% (Aga T) yields, respectively.     

Tissue form of type VII collagen from human skin and dermal fibroblasts in culture

The triple-helical domain of type VII collagen was isolated from human placental membranes by mild digestion with pepsin, and polyclonal antibodies were raised in rabbits against this protein. After affinity purification the antibodies specifically recognized type VII collagen in both the triple-helical and the unfolded state. They also reacted with the fragments P1 and P2, derived from the triple-helical domain by further proteolysis with pepsin, but did not crossreact with other biochemical components of the dermal connective tissue. In skin the presence of a fragment of type VII collagen, similar to that isolated from placenta, was demonstrated by SDS-PAGE and immunoblotting. Type VII collagen represented less than 0.001% of the total collagen extracted by pepsin digestion from newborn or adult skin. The tissue form of type VII collagen was obtained from dermis after artificial epidermolysis with strongly denaturing buffers under conditions reducing disulfide bonds. The protein was identified by immunoblotting with the antibodies. The molecule was composed of three polypeptides with an apparent molecular mass of about 250 kDa, each. Similar large-molecular-mass chains could be identified by immunoblotting in extracts of human fibroblasts in culture.     

Streptococcus mutans ribosomal preparations: purification and properties

Ribosomal preparations were obtained from Streptococcus mutans. Sucrose density gradient analyses showed the ribosomes to be 70S and dissociated subunits to be 56S and 34S. The ribosomal preparation contained 57.4% RNA and 42.6% protein and gave an absorption maximum at 260 nm and a minimum at 235 nm and ribosomal particles were approximately 150-180 X 190-220 A as determined by electron microscopy. Immunodiffusion analysis of pooled antiserum raised by injecting the ribosomal preparation into rabbits disclosed precipitin lines with glucosyltransferase and lipoteichoic acid preparations from S. mutans. Gas chromatography showed rhamnose and glucose to be present in the ribosomal preparation indicating the presence of nonribosomal carbohydrate materials. The ribosomes were able to synthesize precipitable polypeptides when exogenous mRNA and tRNA were added and anti-ribosomal antibodies reduced this activity. Protease treatment rendered the ribosomal preparation less immunogenic in rats and less antigenic when the ribosomal preparation was used to coat erythrocytes for passive haemagglutination assays, while RNase treatment of the ribosomal preparation had no effect, suggesting that a protein(s) is the principal immunogenic moiety of the ribosomal antigen. Polyacrylamide gel electrophoresis of the ribosomal preparation revealed 27 protein bands of which five were found to react with hyperimmune rabbit antisera to the S. mutans ribosomal preparation by Western blot analysis. Washing the ribosomal preparation with 1 M NH4Cl did not remove any of the five immunogenic ribosomal protein antigens indicating that these were innate ribosomal proteins.