Sequence analysis of the cloned streptococcal cell surface antigen I/II.

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen (I/II) of Streptococcus mutans, serotype c (NG5), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues 1-38) and transmembrane (residues 1537-1556) sequences. The N-terminal region (60-550) has alanine-rich repeats and is predicted to be alpha-helical. However, the C-terminal region (800-1540) is proline-rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutans serotype c. N-Terminal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N-terminal and the latter the C-terminal portions of the intact antigen.

Tn917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes.

Previously, we demonstrated successful Tn917 mutagenesis of the oral pathogen Streptococcus mutans using pTV1-OK (Km(r), repATs), a temperature conditional replicative delivery vector carrying a lactococcal pWVO1Ts backbone. In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn917-lac (em(r), beta-gal(+)) that was employed to isolate transcriptional fusions of the Escherichia coli lacZ reporter gene with streptococcal promoters in S. mutans strain NG8. Tn917-lac transposition occurred at a frequency of ca. 10(-6) with 20% of the resultant em(r) clones displaying varying levels of lacZ expression. Tn917-lac mutants that expressed beta-galactosidase activity under growth conditions of glucose limitation, acidic pH, 35 mM NaCl, and elevated (42 degrees C) temperature were isolated. Further characterization of one of the mutants with increased beta-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion. The beta-gal activity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same medium containing no glucose. Further phenotypic analysis revealed that AS42 had a 30% lower growth yield than the parent strain NG8 when grown in pH 5 medium. Sequence analysis of the region harboring the transposon revealed that the lacZ fusion occurred near the 3'-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters. These findings demonstrate that Tn917-lac mutagenesis can be used to identify environmentally regulated genes in S. mutans and possibly in other medically relevant streptococcal species.

Chemical analyses of membranes isolated from Streptococcus mutans BHT.

Cell membranes of Streptococcus mutans BHT (serotype b) were prepared following glass-bead disruption or mutanolysin digestion of whole cells. Major constituents of purified BHT membranes included: protein (60-65%), fatty acids (10%), glucose (3%), and phosphorus (0.5%). The principal amino acids measured were glutamate, aspartate, lysine, alanine, and leucine. The principal fatty acids measured were octadecenoic, palmitoleic, palmitic, and eicosenoic acids; smaller amounts of eicosanoic acid were also detected. Chemical analyses of membranes from cells grown to four different growth phases revealed no major shifts in composition during batch growth under our experimental conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these four membrane preparations confirmed the apparent compositional stability of cell membranes during growth.

Antigenic variation of indigenous streptococci.

Isolates of Group D streptococci indigenous to the murine oral cavity were studied to detect the occurrence of antigenic variation. Group D streptococci cultured from molar homogenates of Balb/c mice were randomly selected for study on the basis of distinctive colony morphology. Isolates obtained over a 12-week period were biotyped using the API 20S system, and subjected to Lancefield extraction and rocket immunoelectrophoresis for serotyping. All isolates were compared with an arbitrarily selected standard test strain (W1S-1) isolated the first week of the first experimental series. Four biotypes were encountered during the first week of two experimental series. Two very unusual biotypes detected during the first experimental series persisted throughout that series, as did two more common biotypes throughout the second experimental series. Anti-W1S-1 serum produced three precipitin bands (antigens O, D, and K) against W1S-1 Lancefield extract and against the respective biotypes detected during the first week of the two series. Of the three antigens detected, only the group antigen (D) did not vary during either experimental series. Antigenic variants lacking the O or K antigen and bearing these distinctive phenotypes were repeatedly isolated in subsequent weeks. Ultimately, 16% of 190 strains isolated during the first series and 26% of 167 strains isolated during the second series proved to be antigenic variants of the predominant biotypes detected in both series.

Antigens in Streptococcus mutans cross reactive with human heart muscle.

An antigen that cross reacts with mammalian heart tissue has been shown to be present in several strains of S mutans. Inoculation of S mutans into rabbits elicited heart-reactive antibody as part of the immunologic response. This heart-reactive antibody was demonstrated to be tissue-specific in that it only bound heart and smooth muscle tissue. Similar results have been previously reported using group A streptococci. Adsorption studies using sarcolemmal sheaths, group A streptoccal membranes, and fractions of cariogenic streptococci demonstrated the presence of a similar antigen. The aforementioned fractions could remove heart-reactive antibody from both immune rabbit serums and serums from patients with acute rheumatic fever. These findings dictate the necessity for testing any future caries vaccine containing fractions of S mutans for the presence of this cross-reactive antigen to avoid a possible autoimmunization.

Cloning and inactivation of the gene responsible for a major surface antigen on Streptococcus mutans.

To understand more fully the biological function(s) and investigate the reported cross-reactivity with heart tissue of antigen P1 (I/II) of Streptococcus mutans (serotype c), this molecular biological study of the responsible gene, spaP, was undertaken. A 5.2 kb Hin dIII fragment of strain NG5 was cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. Recombinant SM2949 expressed a P1 fusion protein under the control of the streptococcal promoter. Southern analysis revealed hybridization of pSM2949 with DNA from Strep. mutans (serotypes c, e, f), Strep. cricetus (a) and Strep. sobrinus (d), but not Strep. sobrinus (g), Strep. rattus (b) or Strep. downei (h). Recombinant (r) antigen was detected in E. coli periplasm, indicating the presence of a signal sequence. This product (of Mr 155K) showed partial identity to the native streptococcal P1 antigen by Ouchterlony double-diffusion analysis. The N-terminal 28 amino acid residues of rP1 were determined by Edman degradation analysis and an end-labelled oligonucleotide probe corresponding to residues 8-13 was used to determine the 5'-3' orientation of spaP by Southern hybridization with restriction enzyme digests of pSM2949. Rabbit antisera made against native and rP1 did not cross-react with human heart tissue. Isogenic mutants of strain NG8 were isolated after transformation with insertionally inactivated spaP. Each mutant was non-reactive with anti-P1 antisera. Selected mutants were shown to have a defective spaP gene incorporated into their chromosomal DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequencing and characterization of the 185 kDa cell surface antigen of Streptococcus mutans.

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen I/II of Streptococcus mutans, serotype c (strain NG5) has been sequenced. The deduced amino acid sequence of antigen I/II (1561 residues) includes a putative signal peptide (residues 1-38), as well as a transmembrane region (residues 1537-1556). The N-terminal part of the protein (residues 39-550) is particularly rich in alanine and includes three tandem repeats of a sequence of 82 residues. This region is predicted to be alpha-helical, adopting a coiled-coil formation, and may account for the cell surface hydrophobicity associated with expression of antigen I/II. In contrast the C-terminal region (residues 800-1549) is proline-rich, favouring an extended conformation. Comparison with the sequence determined from Strep. mutans strain MT8148 showed that antigen I/II is highly conserved with the exception of a short central region (residues 750-805). N-terminal sequencing of purified antigens I and II components indicated that antigen I extends from the amino-terminus of the intact Mr 185,000 surface antigen while antigen II extends from residue 996.

Molecular, immunological and functional characterization of the major surface adhesin of Streptococcus mutans.

In the 15 years since the last major NIH conference that dealt with anti-caries vaccines, we have learned much. Certainly, whole bacteria or bacterial fractions may not be proper immunogens due to the possibility of inducing tissue cross-reactivity. Our own experience (van de Rijn et al., 1976) illustrates that pitfall. But even in the era of genetically engineered vaccines, we first must understand the biological functions of our chosen immunogen before employing that pure protein in a vaccine. Our recent work (Brady et al., 1991c) indicates that antigen P1, a ubiquitous protein found on several oral streptococci, may possess different, but possibly overlapping, functional domains influencing reactions with fluid-phase salivary agglutinin (aggregation) versus fixed agglutinin (adherence). A proper vaccine would induce antibodies against the latter domain(s) thereby retarding colonization. An improper vaccine that induces antibodies against aggregation-related domains on P1 would lessen the host's ability to clear those bacteria from the oral cavity. After carefully identifying appropriate functional domains and obtaining sub-clones of the larger gene that yield truncated polypeptides typical of adherence-specific regions that are also immunogenic, we may be in a position to create the most effective vaccine. In studies employing the polymerase chain reaction (PCR) and standard cloning procedures, we have already begun to produce such polypeptides. Once a library of polypeptides is assembled, they may be tested for functional activity and for lack of induction of cross-reactivity with nonpathogenic streptococci (i.e., S. gordonii). Certain of these recombinant-specified polypeptides could serve as the basis for an anti-caries vaccine. Alternatively, peptides may be synthesized that resemble these sub-molecular regions for use in a vaccine or as competitive inhibitors of adherence but not aggregation. Clearly, a vaccine against dental caries remains a real possibility for the future.

Role of the phosphoenolpyruvate-dependent glucose phosphotransferase system of Streptococcus mutans GS5 in the regulation of lactose uptake.

When Streptococcus mutans GS5 was grown in equimolar (5 mM) amounts of glucose and lactose, a classical diauxic growth curve was obtained. Glucose was taken up during the first growth phase, followed by a 60-min lag, and then lactose was transported. Synthesis of lactose phosphotransferase system (PTS) enzymes was repressed until the complete exhaustion of glucose, indicative of an inducer exclusion mechanism of repression. The enzyme phospho-beta-galactosidase, however, was found in small amounts even in the presence of glucose. Repression was not observed when GS5 was grown in equimolar amounts of fructose and lactose. Although fructose was taken up preferentially, synthesis of the lactose PTS occurred from the onset of growth in these sugars. It is proposed that a component of the glucose PTS may be a regulatory factor in lactose transport. Glucose PTS- mutants did not display diauxic growth in glucoselactose mixtures and, in fact, transported the disaccharide preferentially.

Transport of glucose and mannose by a common phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus mutans GS5.

Decryptified cells of Streptococcus mutans GS5 transport glucose, mannose, and fructose by constitutive phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). Although the non-metabolizable glucose analog 2-deoxyglucose is transported by a PTS, alpha-methylglucose is not taken up by strain GS5. The transport of [14C]mannose and [14C]glucose was almost totally blocked by the heterologous sugars, indicating that these substrates may share a common PTS permease. [14C]fructose transport, however, was not inhibited by large excesses of glucose, indicating the existence of a separate fructose PTS. All "tight" glucose PTS- mutant clones studied were also unable to transport mannose, whereas some "leaky" glucose PTS- clones also were leaky for mannose phosphorylation. Fructose transport in most of these mutant strains was unimpaired, indicating that genetic lesions did not involve soluble (cytoplasmic) PTS components.

Glucose phosphoenolpyruvate-dependent phosphotransferase system of Streptococcus mutans GS5 studied by using cell-free extracts.

The glucose phosphotransferase system (PTS) of Streptococcus mutans GS5 has been partially characterized, using fractions derived from cells treated with the muramidase mutanolysin. Membranes retained functional PTS enzymes for the phosphoenolpyruvate-dependent phosphorylation of glucose, fructose, and mannose. This was confirmed by assaying membranes directly for enzyme I (EI) and enzyme IIglc (EIIglc) by employing specific phosphoryl-exchange reactions for each factor. Membranes prepared from glucose PTS- mutants, however, were either deficient in glucose phosphorylation or reflected the "leakiness" displayed by whole cells. Mutant membranes were unable to catalyze the glucose:glucose 6-phosphate transphosphorylation reaction, indicating a defective EIIglc in these fractions. Although total cellular EI activities in the mutant clones were about the same as that measured for the wild-type strain by employing the pyruvate:phosphoenolpyruvate phosphoryl-exchange reaction, mutant membranes were found to possess less than 10% of the specific EI activity of wild-type membranes. The cytoplasmic fractions of mutants, however, displayed markedly increased specific activities for this enzyme when compared with wild-type extracts. These results strongly suggest a molecular association of EI with a normal membrane protein, perhaps EIIglc, that is absent in mutants. This would explain the absence of fructose PTS activity in glucose PTS- mutant membranes despite the fact that whole cells of these clones are normal for this transport function.

Chemical, immunochemical, and structural studies of the cross-reactive antigens of Streptococcus mutans AHT and B13.

Two antigenic polysaccharides were extracted from cell walls of the cross-reactive strains Streptococcus mutans AHT (a) and S. mutans B13 (d). The antigens extracted from walls by the hot formamide method, were purified by affinity chromatography on columns containing the galactose-specific lectin from the castor bean and were found to be diheteroglycans consisting of galactose and glucose. Antigenic specificities of both the serotype-specific and the cross-reactive sites on each polymer were studied: the AHT (a) antigen is determined by D-galactose linked 1 leads to 6 to adjacent sugar, the B13 (d) antigen is determined by D-glucose similarly linked to o its neighbor, and the cross-reactive (a--d) site present on both polymers consists of D-galactose linked 1 leads to 6 to a subterminal sugar moiety. Methylation analysis revealed structural similarities between the purified polysaccharides that may reflect the nature of the cross-reactive sites and differences that may reflect the natures of the specific haptenic regions. Based on these studies, a partial hypothetical structural model is proposed.

Ultrastructural, physiological, and cytochemical characterization of cores in group D streptococci.

Cores are large, rod-shaped structures that have been found almost exclusively in group D streptococci, measure 0.1 to 0.16 mum in diameter, and extend the width or length of cells. This study has shown that cores are produced in the cells at a reproducible point in early stationary growth after extensive mesosomal formation and after the pH has dropped below 6.5. When cells containing cores were introduced into a fresh medium with a pH above 6.5, the structures disappeared within 5 min. The structures were not found in young, logarithmically growing cells but formed in these cells upon autolysis or treatment with penicillin. Cores that were forming or disintegrating appeared to have a lamellar substructure. When chloramphenicol was added to the medium before the culture reached stationary phase, no cores were found in the cells. Cytochemical studies indicated that cores contain protein and are not composed of cell wall material or other polysaccharides that contain 1,2-glycol groups.

Multilevel control of extracellular sucrose metabolism in Streptococcus salivarius by sucrose.

Standardized experimental conditions were established to test the role of sucrose in the regulation of control of its metabolism in Streptococcus salivarius. A fresh isolate of S. salivarius was used. The extracellular dextranase activity of cells grown on sucrose was 10-fold higher than that of cells grown on glucose, fructose or galactose. This activity increased in less than 5 min following the addition of sucrose to galactose-grown cells, a phenomenon which was affected by neither rifampicin nor chloramphenicol which inhibit transcription and translation, respectively. Extracellular fructanase activity was 2-fold higher when cells were grown on sucrose than when they were grown on the other sugars. This increase also occurred within 5 min, but was diminished by transcriptional and translational inhibitors. De novo synthesis was required for the production of extracellular glucosyltransferase (GTF) activity which, upon the addition of sucrose, became associated with the cell surface. Conversely, cell-associated fructosyltransferase (FTF) activity appeared to require genetic induction for its production and cell-surface association, but required sucrose for its release from the surface framework. Versatility in the control mechanisms of this complex set of enzymes allows their expression and function to be regulated at several widely separated stages in the life histories of these proteins.

Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.

We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth.

Antigens of Streptococcus mutans: characterization of a polysaccharide antigen from walls of strain GS-5.

A cell wall-associated polysaccharide antigen was isolated from Streptococcus mutans GS-5 and appeared to determine serotype c specificity. Ouchterlony double-diffusion analysis of crude formamide extracts derived from purified cell walls of two serotype c strains (GS-5 and JC-2) showed complete identify when reacted with anti-GS-5 sera. Immunoelectrophoresis of this extract demonstrated the typical mobility for this serotype as described by others. Column chromatography on BioGel P-100 of the crude formamide extracts derived from GS-5 walls resulted in a single antigenic peak being resolved. This material, when loaded onto a diethylaminoethylcellulose column and eluted with a linear gradient of ammonium carbonate (0.0 to 0.2 M), was resolved further into two serologically reactive peaks (I and II). Only two consituents, rhamnose and glucose, were detected in the purified column fractions. Peak 1 had a rhamnoseto-glucose molar ratio of 0.9:1.0, and peak II, the major resolvable fraction, had a molar ratio of 1.7:1.0, The peak II ratio was very similar to that found in the formamide extract residue pellet (1.6:1.0)9 Ouchterlony analysis of the crude formamide extract and the purified fractions revealed only partial identify between peaks I and II but complete identify between peak II and the crude extract. Likewise, immunoelectrophoresis showed no differences in mobility of peak II and the crude extract, whereas peak I moved towards the cathode. Possible structural relationships between the two antigenic fractions are discussed below. Hapten inhibition studies suggested that an alpha-glucosyl group is at the immunodeterminant site of the antigen.