spaP Gene Research Articles

Detection of SpaP and vicR Genes in Non-biofilm Producing Streptococcus mutans Isolated from Dental Caries and Plaque

Evidence accumulated over many decades has clearly shown that Streptococcus mutans is a major agent in dental caries. S. mutans SpaP gene (Cell Surface Antigen) mediates the binding of these cariogenic bacteria to tooth surfaces. The aim of this study was to detect the presence of SpaP and vicR genes in non-biofilm forming Streptococcus mutant isolated from dental plaques. In this study, 120 clinical samples were obtained from different dental clinics and hospitals in Baghdad. 26 Bacterial isolates identified as Streptococcus mutans by using selective media (Mitis-salivarius agar) and microscopic examination after gram staining then confirmed S. mutans by molecular detection using SpaP genes. Interestingly all 26 isolates were non-biofilm forming after microtiter plate assay based on the crystal violet staining method, and negatively resulted for the detection of vicR gene which responsible for biofilm formation. 10 antibiotics (Doxycycline, Ofloxacin, Tetracycline, Erythromycin, Vancomycin, Clindamycin, Rifampicin, Imipenem, Amikacin and Cefepime) were used to examine sensitivity of S. mutans isolates, all isolates were multi-drugs resist (MDR) for all 10 antibiotics used. We can conclude that absence of biofilm forming ability does not affecting antibiotic resistance and virulence of Streptococcus mutance.

Assessment of the Antivirulence Potential of Tamarix aphylla Ethanolic Extract against Multidrug-Resistant Streptococcus mutans Isolated from Iraqi Patients.

Halophytes have long been used for medicinal purposes. However, their use was entirely empirical, with no knowledge of the bioactive compounds. The plant Tamarix aphylla L. has not drawn the deserving attention for its phytochemical and bioactive explorations, but available data expressed its needs to be attended for its potential. The Streptococcus mutans SpaP gene (cell-surface antigen) mediates the binding of these bacteria to tooth surfaces. The growing problem of antibiotic resistance triggered the research on alternative antimicrobial approaches. Our study aims to explore the activity of T. aphylla ethanolic extract against the virulence gene found in Streptococcus mutans pathogenic bacteria. Samples that were previously collected and identified in our previous work (in press) were obtained from different dental clinics and hospitals in Baghdad. Three nonbiofilm-forming bacterial isolates having multidrug resistance (MDR) for 10 antibiotics (doxycycline, ofloxacin, tetracycline, erythromycin, vancomycin, clindamycin, rifampicin, imipenem, amikacin, and cefepime) were selected to examine the potential of the T. aphylla ethanolic extract. The ethanolic extract showed high antimicrobial activity against MDR. Minimum inhibition concentration (MIC) for the extract was 17.5 mg/ml, while minimum bactericidal concentration (MBC) was 35 mg/ml. The phytochemical compounds present in the ethanolic extract were determined by using high-performance liquid chromatography (HPLC) which revealed that the leaves contain thirteen different alkaloids, twelve flavonoids, and four vitamins. The extract strongly inhibited a virulence property, the adherence of S. mutans which reduced during critical growth phases. The one-step RT-PCR method was used to study the SpaP gene expression of bacterial isolates which significantly reduced. In conclusion, extraction of T. aphylla leaves showed an antimicrobial effect against MDR S. mutans. The identified phytochemicals in the T. aphylla extract are reported to be biologically important and need further investigation to develop safe and cheap drugs.

The relationship of Streptococcus mutans spaP gene presence to dental caries severity on children in Denpasar City

Oral health problems, especially dental caries, are the most common health problems experienced by children in Indonesia. Streptococcus mutans as the main causative agent of dental caries has the spaP gene which plays a role in the initial process of bacterial invasion. Performing PCR on children's dental plaque samples can determine the presence of the genes spaP S.mutans. Then, the existing data were statistically analyzed using the logistic regression method to assess the relationship of the genes spaP S.mutans to the severity of dental caries. From the research results, 54 samples contained the spaP gene of S.mutans. The prevalence of gene S.mutans spaP in dental plaque in children aged 3-5 years in Denpasar City was 69.2%. The statistical ana-lysis results revealed that the spaP gene of S.mutans (p=0.016) had a p<0.05. It is concluded that the variable presence of the S.mutans spaP had a significant relationship with the severity of dental caries on children aged 3-5 years in Denpasar City.

Different Patterns of Virulence Genes in Streptococcus mutans and Streptococcus sobrinus Originating from Estonian Toddlers—Mothers Cohort

Aims: Mutans streptococci include Streptococcus mutans and Streptococcus sobrinus, which can cause tooth decay. The current study aimed to compare their virulence genes with each other and to correlate them with the clinical data of patients. Materials and methods: Altogether 21 S. mutans and 19 S. sobrinus strains were investigated, originating from 24 children (age 2.7 ± 0.4 years) and 13 mothers (27.3 ± 3.7). The PCR method was applied to detect 11 virulence genes. Caries indices (dmf, decayed/missing/filled; DMFT, decayed/missing/filled teeth) and SM score (Mutans streptococci amount in saliva) were recorded. Results: Most of the S. mutans strains harbored all the virulence genes studied, while S. sobrinus had significantly fewer genes. The genes gbpA, gbpB, wapA and ftf were present in all isolates of S. sobrinus, the spaP, gtfB, vicR, SMU.1037c and SMU.105 genes were present in 41–88% of the isolates, while gtfD and SMU.104 genes were absent in S. sobrinus strains studied. A positive correlation appeared between the biofilm-related vicR and polysaccharide-production-related gtfD genes. In contrast, another polysaccharide-production-related gtfB gene was present in some cases in strains lacking the vicR or gtfD gene. Positive association was found between the presence of adhesion-related spaP gene in pediatric-derived S. sobrinus strains and an increase in SM score. Conclusions: Differences exist between the two common species of mutans streptococci: strains of S. mutans have more virulence genes than that of S. sobrinus, both crucial and virulence enhancing. Deeper research is needed to clarify the mechanisms behind the increased cariogenicity in cohabitation.

Enhancement of cariogenic virulence properties of dental plaque in asthmatics

Objective The aim of this study was to investigate the caries risk of asthmatics in relation to acidogenicity and the expression of caries-related genes in dental plaque. Methods A case-control study composed of 38 asthmatics (cases) and 22 controls with an age range from 6 to 60 years. Characteristics of asthma, use of medications, oral hygiene practices and dietary habits assessed by questionnaires and interviews. The dental plaque maturity evaluated using GC Tri Plaque ID Gel TM. The expression of brpA, gtfB, gbpB, ldh, luxS and spaP genes analyzed using real-time PCR. Results Asthmatics had a higher percentage of mature and acidogenic plaque than immature plaque. In contrast, immature plaque was more evident in controls. Acidogenic plaque commonly occurred in patients using 1 or a combination of two medications. High frequency in meals and sweets were found in asthmatics. Real-time PCR revealed that the expression of spaP, gtfB, gbpB, ldh, brpA and luxS were enhanced in asthmatics compared with the control group. Conclusion An increase in acidogenic and mature plaque is found in asthmatics. The expression of spaP, gtfB, gbpB, ldh, brpA and luxS in dental plaque are upregulated in asthmatics.

Anticariogenic Potential of Korean Native Plant Extracts against Streptococcus mutans.

Numerous chemically synthesized compounds are widely used in oral hygiene products. However, due to their potential risk, there is a need to improve the safety and quality of dental care by seeking alternative control agents such as those naturally found in plant materials. Here we assessed antibacterial potentials of extracts from 100 species of Korean native plants against Streptococcus mutans on cariogenesis. Among those, extracts from five plants (Arctii Fructus, Caryopteris incana, Aralia continentalis, Symplocarpus renifolius, and Lamium amplexicaule) showed a growth inhibition of S. mutans. The five extracts were further individually evaluated for their minimal inhibitory concentration and minimal bactericidal concentration. Interestingly, a synergistic antibacterial activity was observed with the combination of sodium fluoride and the plant extracts. To determine the anti-biofilm activity of plant extracts, S. mutans was treated with increasing concentrations of the extracts in the range from 1250 to 3750 µg/mL. When S. mutans was grown in the defined biofilm medium containing the individual extracts of 47 species, the biofilm amount markedly decreased compared to that of a negative control. Notably, the extract of S. renifolius significantly downregulated the gtf and spaP genes for synthesis of glucan and adhesive proteins in S. mutans, and L.amplexicaule decreased the expression of gtfD gene. Therefore, these results demonstrate that the five plant extracts modulate survival and pathogenesis of S. mutans by growth inhibition and downregulation of the gene(s) implicated in biofilm formation.

Antibacterial activity of Azadirachta indica, Pongamia pinnata, Psidium guajava and Mangifera indica and their mechanism of action against Streptococcus mutans

Background: Curative plants have reportedly been used to make chewing sticks/toothbrushes intended for the treatment of oral diseases. Objective: The in vitro antibacterial activities of Azadirachta indica, Pongamia pinnata, Psidium guajava, and Mangifera indica were evaluated against Streptococcus mutans, along with the cytotoxicity and antioxidant and synergistic potentials. The effect of M. indica on the expression of crucial virulence genes spaP and gtfB of S. mutans was determined. Materials and Methods: The antibacterial activity was determined using a modified microdilution method. The antioxidant potential was evaluated using diphenyl picrylhydrazyl (DPPH), Griess reagent, and nitroblue tetrazolium calorimetric assays. The synergistic activity was investigated using a modified checkerboard method, while the cytotoxicity was determined according to a cell proliferation 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt assay. Reverse transcription was the chosen method for determining the difference in expression of the spaP and gtfB genes after treatment with the plant sample. Results: M. indica and A. indica had the highest antibacterial activity at concentrations of 0.3 mg/ml and 6.25 mg/ml, respectively. A. indica had the best free radical scavenging of DPPH, exhibiting 50% inhibition at 28.72 μg/ml; while M. indica showed better superoxide scavenging potential than the positive control quercetin. Both M. indica and A. indica had adequate activity against the nitric oxide-free radical (12.87 and 18.89 μg/ml, respectively). M. indica selectively reduced the expression of the gtfB gene, indicating a mechanism involving Glucotranferases, specifically targeting bacterial attachment. Conclusion: The overall good antibacterial activity of M. indica correlated with the general good antioxidant capacity, while showing a potentially unique mechanism of bacterial inhibition, targeting virulence gene expression. Abbreviations used: AA: Ascorbic acid; BHI: Brain–heart infusion; CHX: Chlorhexidine; DPPH: Diphenyl picrylhydrazyl; DMSO: Dimethlysulfoxide; NBT: Nitroblue tetrazolium; NO: Nitric oxide; XTT: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5 -carboxanilide salt.

Effects of sub-minimum inhibitory concentrations of lemon essential oil on the acid tolerance and biofilm formation of Streptococcus mutans

ObjectivesLemon essential oil (LEO) is a kind of secondary metabolite from lemon peels and has been found to inhibit cariogenic bacteria for decades. However, its effects on main cariogenic virulence factors are rarely reported. The present study aimed to investigate the effects of sub-minimum inhibitory concentrations (sub-MICs) of LEO on the acid tolerance and biofilm formation of Streptococcus mutans (S. mutans) and preliminarily reveal the possible underlying mechanisms. DesignsEffects of LEO on the acid tolerance and biofilm formation of S. mutans were investigated by the broth dilution method and crystal violet staining method respectively. The expression of luxS, srtA and spaP gene was also determined to explore the underlying mechanism. In addition, Tea polyphenols (TP), a major natural inhibitor of cariogenic virulence factors, and limonene (LIM), the major component of LEO, were selected as comparisons to evaluate the effects of LEO. ResultsSub-MICs of LEO, LIM and TP exhibited a dose-dependent inhibition of growth of S. mutans at pH ranging from 4.0 to 7.0. The formation of S. mutans biofilm was remarkably inhibited and the inhibitory rates of LEO, LIM and TP were 97.87%, 94.88% and 96.01% respectively at 1/2 MIC. Similarly, a down-regulation was observed in the expression of luxS, srtA and spaP gene at sub-MIC levels. ConclusionsEffects of LEO were similar or slightly stronger than LIM and TP, suggesting that LEO might represent a novel, natural anticarious agent that inhibited the specific genes associated with bacterial acid tolerance and biofilm formation without necessarily affecting the growth of oral bacteria.

Identification of novel cyanobacterial compounds for oral disease through in vitro and insilico approach

In this research, we have conducted docking study to screen bioactive compounds from Microcystis aeruginosa and Phormidium corium. Among the two blue-green algal species were analyzed for chemical nature of bioactive substances using TLC and GC-MS methods. The antimicrobial compounds identified were phenolics, alkaloids, steroids. In the present study as reported that M. aeruginosa curde extract contained the bioactive compound Microginins, while Cyalobolide B was detected from P. corium. The phytochemical analysis of M. aeruginosa (Microginins), provided ethaneloic acid, Octanal, 3,7,11-Trimethyl-1,6,10-dodecatrien-3-ol (nerolidal), Monomethyl hydrazine and formic acid. The cumulative effect of these phytochemical provided more effective antimicrobial compound in inhibiting microbial growth. The phytochemical analysis of P. corium (Cyalobolide B) exhibited Ethaneloic acid, Dihydrodiplodialide, 3, 7, 11-Trimethyl-1, 6, 10-dodecatrien-3-ol (nerolidal), 1-Dodecanol, 1-Hexadecanol (CAS) and nanonic acid. These compounds together exhibited more effective antimicrobial substance in inhibiting microbial growth. Which members of a community interact with themselves as well as with different host structures and components of Candida albicans and Streptococcus mutans. The pathogenesis of this dental infection is a multi factorial process that results in a serious degenerative disease of the Oral candidiasis. In the present study SAP (secreted aspartyl proteinases in virulence and pathogenesis) is taken as a case study molecule to understand high reactive responses of various drugs administered for the oral candidiasis. The drugs are being compared with the SAP from C. albicans and SpaP (cell surface antigen SpaP gene) from S. mutans. The SAP and SpaP interacted with formic acid, nerolidal, octanol and nonanoic acid using docking methods. The exponentially increasing speed of computational methods makes a more extensive use in the early stages of drug discovery attractive if sufficient accuracy can be achieved.

Molecular characterization of the dextran-binding lectin B gene dblB of Streptococcus criceti in Streptococcus mutans strain GS-5 with mutations in both gbpC and spaP genes.

Streptococcus mutans, a cariogenic agent, has a glucan-binding protein gene, gbpC, and S. criceti possesses four gbpC homologs, including dblA and dblB, as does S. sobrinus. The S. criceti dblB gene encodes a 1,717-amino-acid protein having two repetitive alanine-rich and proline-rich regions and an LPXTG motif, which is recognized by the sortase SrtA, near the C terminus. Reverse transcription-PCR analysis indicated no cotranscription of the dblA and dblB genes of S. criceti. As we could not obtain a dblB mutant of S. criceti, the dblB gene was characterized in S. mutans strain GS-5, which has genetic mutations in both gbpC and spaP genes and shows an inability to agglutinate triggered by dextran. A dextran-induced agglutination assay showed that S. mutans cells carrying dblB agglutinated in the presence of dextran. A hydrophobicity assay showed that the cells containing dblB were hydrophobic. A biofilm formation assay showed that the dblB gene was associated with biofilm formation by cells cultivated in brain heart infusion broth supplemented with glucose and maltose, but not sucrose. Nucleotide sequence analysis of the S. criceti strains studied revealed a frameshift mutation in the srtA gene encoding sortase, but intact dblA and dblB genes were found in dextran-induced agglutination-negative strains, whereas intact dblA, dblB and srtA genes were found in dextran-induced agglutination-positive strains. These results suggest the cell-surface localization of dblA and dblB gene products by SrtA and the responsibility of dblB for dextran-induced agglutination, cell-surface hydrophobicity and biofilm formation in S. criceti.

A mechanism for extremely weak SpaP-expression in Streptococcus mutans strain Z1

Background: Streptococcus mutans surface-protein antigen (SpaP, PAc, or antigen I/II) has been well known to play an important role in initial attachment to tooth surfaces. However, strains with weak SpaP-expression were recently reported to be found in natural populations of S. mutans. The S. mutans gbpC-negative strain Z1, which we previously isolated from saliva and plaque samples, apparently expresses relatively low levels of SpaP protein compared to S. mutans strains MT8148 or UA159.Objective: To elucidate the mechanism for weak SpaP-expression in this strain, the spaP gene region in strain Z1 was amplified by polymerase chain reaction (PCR) and analyzed.Methods: Allelic exchange mutants between strains Z1 and UA159 involving the spaP gene region were constructed. The SpaP protein expressed in the mutants was detected with Coomasie Brilliant Blue (CBB)-staining and Western blot analysis following SDS-PAGE.Results: The 4689 bp spaP gene coding sequence for Z1 appeared to be intact. In contrast, a 20 bp nucleotide sequence appeared to be deleted from the region immediately upstream from the Z1 spaP gene when compared to the same region in UA159. The 216 bp and 237 bp intergenic fragments upstream from the spaP gene, respectively, from Z1 and UA159 were isolated, modified, and transformed into the other strain by allelic replacement. The resultant UA159-promoter region-mutant exhibited extremely weak SpaP-expression similar to that of strain Z1 and the Z1 complemented mutant expressed Spa protein levels like that of strain UA159.Conclusion: These results suggest that weak SpaP-expression in strain Z1 resulted from a 20 bp-deletion in the spaP gene promoter region.

Manganese Affects Streptococcus mutans Virulence Gene Expression

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

Peptide Mapping of a Novel Discontinuous Epitope of the Major Surface Adhesin from Streptococcus mutans

Guy's 13 is a mouse monoclonal antibody that specifically recognizes the major cell-surface adhesion protein SA I/II of Streptococcus mutans, one of the major causative agents of dental caries. Passive immunization with Guy's 13 prevents bacterial colonization in humans. To help elucidate the mechanism of prevention of colonization conferred by this antibody, the SA I/II epitope recognized by Guy's 13 was investigated. It was previously established that the epitope is conformational, being assembled from two non-contiguous regions of SA I/II. In the current study, using recombinant fragments of SA I/II and, ultimately, synthetic peptides, the discontinuous epitope was localized to residues 170-218 and 956-969. This work describes the mapping of a novel discontinuous epitope that requires an interaction between each determinant in order for epitope assembly and recognition by antibody to take place. Guy's 13 binds to the assembled epitope but not to these individual epitope fragments. The assembled epitope results from the interaction between the individual antigenic determinants and can be formed by mixing together determinants present on separate polypeptide chains. The data are consistent with one of the epitope fragments adopting a polyproline II-like helical conformation.

Detection of potentially cariogenic strains of Streptococcus mutans using the polymerase chain reaction

Streptococcus mutans is a pathogen related to the occurrence of human dental caries. The determination of total amounts of mutans streptococci, as well as the proportion related to other oral bacteria, is of interest when assessing the risk of developing caries. In this context, it is better to use a sensitive, specific and non-time consuming method such as the polymerase chain reaction (PCR), than to use culture and biochemical identification methods. In this work we identified potentially cariogenic strains of S. mutans and assessed the relationship with the dmft, DMFT or dmft/DMFT index. Using DNA isolated from dental plaque, a 192 bp sequence was identified and amplified from the spaP gene and a 722 bp sequence from the dexA gene. The results suggest that it is important to evaluate the presence of cariogenic S. mutans strains in plaque content rather than the accumulation of plaque itself However, other factors like diet, hygiene, genetic background, the flow rate of saliva and the presence of specific antibodies, also play a key role in the development of caries.

Detection of Streptococcus mutans by PCR amplification of the spaP gene in teeth rendered caries free

Objectives: To investigate the degree of association between tactile and optical criteria as used to assess the carious status of the enameldentine junction (EDJ) during cavity preparation, assessment with a caries detector dye and detection of Streptococcus mutans using culture and polymerase chain reaction (PCR) techniques. Methods: Twenty-nine teeth, extracted within the previous 30 min, and 15 teeth prepared under rubber dam in vivo, were clinically assessed at the EDJ after the removal of evident carious tissue. Demineralisation was then assessed using a caries detector dye (1% acid red in propylene glycol; Cavex ™). A rosehead bur was used to remove tissue at the EDJ for culture and PCR analysis. Culture was carried out on a tryptone yeast cystine sucrose bacitracin selective medium, and PCR used to amplify a sequence (192 bp) of the spaP gene, which encodes the surface protein antigen I/II of S. mutans. Results: Demineralised tissue at the EDJ, as shown using the dye, was found in 52% of teeth. Removed tissue was culture and PCR positive for S. mutans in 2 and 47% of teeth, respectively. A highly significant association (77% of cases; P < 0.001) was shown between dye and PCR assessment methods. No association was found between any other combination of assessment methods. Conclusions: Culture methods may underestimate the presence of S. mutans. Removal of sufficient dye-stained tissue is therefore recommended to prevent further carious assault from residual S. mutans.

Detection of Streptococcus mutans by PCR amplification of spaP gene.

Synthetic oligonucleotide primers were used in the polymerase chain reaction (PCR) to amplify a sequence of the spaP gene, which encodes the surface protein antigen I/II of Streptococcus mutans. A DNA fragment of c. 192 bp was amplified from lysed S. mutans cells or isolated DNA. With S. mutans cells, the lower limit of detection was 4-40 cfu. With these primers, 13 reference and 50 clinical strains of S. mutans were identified. Amplification of the 192-bp product was not demonstrated when 41 strains of other streptococcal and non-streptococcal species were tested. The spaP gene PCR has potential for the rapid diagnosis of S. mutans infections.

Role of a cell surface-associated protein in adherence and dental caries

Insertional inactivation of the Streptococcus mutans spaP gene was used to construct an isogenic mutant (834) of strain NG8 (serotype c) which lacked the major cell surface-associated protein referred to as P1 (15). Results of several studies suggest that P1 is involved in the adherence of S. mutans to saliva-coated apatite surfaces. With an in vitro model system of hydroxyapatite (HA) beads coated with parotid saliva (PS) and additional HA surfaces coated with PS and in situ-formed glucan, it was observed that mutant 834 adhered poorly to the PS/HA surfaces. In contrast, both parent and mutant strains bound to the PS-glucan/HA surface. Groups of intact and desalivated rats were infected with each strain to determine relative capacities to induce dental caries. Rats were fed a highly cariogenic diet containing 56% sucrose for 3 to 5 weeks. Each strain colonized the rodent model and caused similar levels of smooth-surface caries under these dietary conditions. It was concluded that P1 influences the ability of organisms to adhere to saliva-coated surfaces and possibly affects primary colonization of the oral cavity in the absence of a glucan surface but has no effect on glucan-mediated adherence in vitro or in vivo.

Conservation of the gene encoding streptococcal antigen I/II in oral streptococci

The spaP gene of Streptococcus mutans serotype c encodes a major cell surface protein, streptococcal antigen (SA) I/II, with an Mr of 185,000, that is thought to be involved in bacterial adhesion to teeth. Proteins with significant amino acid sequence homology to SA I/II have also been found in S. sobrinus and S. sanguis. The objectives of this study were to investigate the conservation of the spaP gene in the mutans groups of streptococci and to determine whether homologous genes were present in other species of alpha-hemolytic streptococci. DNA extracted from representative strains of 19 streptococcal species was examined by Southern hybridization and partial DNA sequence analysis. A series of five overlapping DNA probes from the spaP gene were amplified by the polymerase chain reaction and used in the Southern hybridizations. The entire gene was found to be well conserved in all strains of S. mutans serotypes c, e, and f investigated. A probe from the 3' region of the gene, which encodes residues 857 to 1207 of the SA I/II protein, hybridized with DNA from a number of mutans streptococci, as well as with DNA from nonmutans alpha-hemolytic streptococci. Conservation within this region was further demonstrated by sequencing gene fragments of two strains of S. intermedius and S. oralis. The results show that some regions of the spaP gene are highly conserved not only in the mutans group of streptococci but also in other nonmutans alpha-hemolytic streptococci. This suggests that a family of cell surface proteins which, by analogy with the 185,000-Mr SA I/II of S. mutans, could be involved in bacterial adhesion might exist.

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.

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 HindIII 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 M r 155 K) 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. Rabbit antisera against isogenic mutants (and against Strep. rattus) reacted with human heart tissue by ELISA to the same extent as anti-NG8 antisera, indicating that the heart cross-reactive antigen of mutans streptococci may not reside in the wall. Isogenic P1 − mutants had similar growth rates and surface enzymatic properties to the parent, but lacked P1 in their cell walls as shown by Western immunoblot and electron microscopy. Mutants failed to agglutinate with salivary agglutinin or bind agglutinin-coated hydroxyapatite beads, indicating that the 185 K native surface antigen may play a role in adherence to teeth or colonization of dental plaque.