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.     

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.     

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.     

Lysis of grouped and ungrouped streptococci by lysozyme

Thirty strains of streptococci were tested for lysis with lysozyme, and 29 of these could be lysed by the following method: (i) suspension of the cells to a Klett reading of 200 units (no. 42 filter) in 0.01 m tris(hydroxymethyl)aminomethane buffer, pH 8.2, after washing twice with the buffer; (ii) addition of lysozyme to a final concentration of 250 mug/ml with incubation for 60 min at 37 C; (iii) addition of sodium lauryl sulfate (SLS) to a final concentration of 0.2% and incubation up to an additional 15 min at 37 C. Significant lysis was obtained only after the addition of SLS. (Strains of groups A, E, and G were treated with trypsin at a concentration of 200 mug/ml for 2 hr at 37 C before exposure to lysozyme.) These parameters for optimal lysis of streptococci by lysozyme were established by testing the group D Streptococcus faecalis strain 31 which lyses readily with lysozyme and the group H strain Challis which is less susceptible to the action of the enzyme. Viability of S. faecalis decreased 96% after 3 min of exposure to 250 mug of lysozyme per ml, whereas the more resistant strain Challis retained 27% of the initial viability after the same period. After 60 min, there was almost total loss of viability in each case. Variations of three methods of lysing streptococci with lysozyme were compared with respect to the decrease in turbidity and the release of protein and deoxyribonucleic acid (DNA) effected by each variation. The method presented in this paper allowed the greatest release of these cytoplasmic constituents from S. faecalis and strain Challis. Transformation experiments using DNA obtained from strain Challis (streptomycinresistant) by this method showed that the DNA released is biologically active.     

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.     

Phenotypic stability of the cell wall of Streptococcus mutans Ingbritt grown under various conditions.

Quantitative analyses of cell walls from Streptococcus mutans Ingbritt grown under carbohydrate limitation in the chemostat showed that growth conditions had no statistically significant effect on the composition of polysaccharide, peptidoglycan, or the proportion of polysaccharide in the cell wall. Lysis of cell wall preparations with a muramidase supported this conclusion and further indicated that there was little difference in their overall structure. In contrast, there was a consistent difference between the rates of lysis by this enzyme of organisms grown in 0.2% glucose and 0.5% glucose. Extremes of pH or dilution rate essentially did not influence the immunogenicity of type c antigen in whole organisms irrespective of whether the carbohydrate source was glucose or sucrose. However, differences were found in the immunogenicity of lipoteichoic acid under similar circumstances. The results indicated there was an inherent phenotypic stability in the cell walls of S. mutans Ingbritt despite changes in pH, generation time, and carbohydrate source, and that any changes that did occur were probably due to associated cell-surface components.     

The diagnostic process and perioperative and anesthetic management of an undiagnosed congenital cyanotic cardiac defect in an adult for trauma surgery

A 39-year-old patient awaiting emergency surgery due to a crush foot injury, with an undiagnosed cyanotic cardiac lesion that was diagnosed later as a complete atrioventricular canal defect, is presented. Complete atrioventricular canal defects usually present in the first few months of life and can be fatal if not treated in the first few years. Adult patients with congenital cardiac malformations seem to be at increased risk for noncardiac surgery. The diagnostic process, perioperative management, and anesthetic implications are discussed.     

Restriction fragment length polymorphisms and sequence variation within the spaP gene of Streptococcus mutans serotype c isolates.

A restriction fragment length polymorphism study was undertaken to determine the extent and location of heterogeneity within spaP encoding the Mr 185,000 cell surface protein P1 (antigen I/II) of Streptococcus mutans serotype c isolates. The gene was found to be highly conserved except for a central variable (V) region predicted to encode less than 150 amino acids. Sequence analysis identified two V-region variants. These differences were independent of the geographic source of the isolates. Southern analysis using synthetic oligonucleotide probes indicated that nonretention of P1 (I/II) by some isolates is not due to a deletion of the 3'-terminal DNA necessary to encode an intact carboxy terminus.     

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.