Collagen‐binding proteins of Streptococcus mutans and related streptococci

The ability of Streptococcus mutans to interact with collagen through the expression of collagen‐binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose‐dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra‐oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host.     

PCR detection of Scardovia wiggsiae in combination with Streptococcus mutans for early childhood caries‐risk prediction

The microbial factor is an important determinant in caries risk assessment. This study aimed to use detection, by PCR, of Scardovia wiggsiae, in combination with Streptococcus mutans, for the accurate prediction of caries risk in children. Detection of Lactobacillus, as a caries‐specific species, was also performed. Dental plaque, as well as infected dentine when available, was collected from children who were caries‐free (n = 30) or diagnosed with early childhood caries (n = 30), and the prevalence and abundance of S. wiggsiae and S. mutans were estimated using quantitative PCR. Lactobacillus was amplified by Lactobacillus genus‐specific primers and then sequenced. Both S. wiggsiae and S. mutans were concurrently detected in 19 children diagnosed with early childhood caries, but in none of the caries‐free children. The positive predictive value was 1 in children with S. wiggsiae‐ and S. mutans‐positive test results, compared with 0.58 when only S. mutans was detected and 0.9 when only S. wiggsiae was detected. The abundance of S. wiggsiae and S. mutans in infected dentine was higher than that in dental plaque from children. Diverse Lactobacillus species were observed in dental plaque but none appeared to be caries‐specific. In conclusion, the detection of S. wiggsiae in combination with S. mutans improves the positive predictive value and the specificity of the test.     

Assessment of clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African‐American children was examined using MLST. Serotype and the presence of collagen‐binding proteins (CBPs) encoded by cnm/cbm were also assessed. One‐hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using start 2 and mega . Thirty‐four sequence types were identified, of which 27 were unique to this population. Seventy‐five per cent of the isolates clustered into 16 clonal groups. The serotypes observed were c (n = 84), e (n = 3), and k (n = 11). The prevalence of S. mutans isolates of serotype k was notably high, at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized population studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study, is higher than reported in most populations and is the first report of S. mutans serotype k in a United States population.     

Comparison of immunological and microbiological characteristics in children and the elderly with or without dental caries

The purpose of this study was to determine the relationships among early childhood caries (ECC), root caries (RC), the quantity of Streptococcus mutans in saliva, and the concentrations of total and specific secretory IgA (sIgA). Saliva samples were collected from 70 children, 3–4 yr of age, with and without ECC, and from 43 adults, ≥60 yr of age, with and without RC. The decayed, missing, and filled teeth (dmft) and decayed, missing, and filled surfaces (dmfs) scores of each child, and the root decayed and filled teeth (RDFT) and root decayed and filled surfaces (RDFS) scores of each elderly subject, were determined. The S. mutans levels, total sIgA, and specific sIgA against two virulence antigens of S. mutans in saliva were analysed using quantitative real‐time PCR (qPCR) and ELISAs. The quantity of S. mutans was significantly higher in caries‐positive subjects within the two populations than in the caries‐free subjects; and a positive correlation was found between the quantity of S. mutans and the dmft, dmfs, RDFT, and RDFS scores. In addition, the salivary total sIgA was significantly higher in children with severe early childhood caries (SECC) and in the elderly subjects with RC. Moreover, although the S. mutans level was significantly higher, the concentrations of specific sIgA against S. mutans antigens were significantly lower in samples from elderly subjects than in samples from children. These results support the concept that S. mutans is positively associated with ECC and RC. Furthermore, the levels of S. mutans‐specific antibodies in saliva are too low to prevent infection with cariogenic bacteria and to inhibit development of ECC and RC.     

Identification and characterization of a collagen-binding protein, Cbm, in Streptococcus mutans

Streptococcus mutans, a major pathogen of dental caries, is occasionally isolated from the blood of patients with infective endocarditis. Bacterial attachment of exposed collagen tissue in the impaired endothelium is an important step in the onset of infective endocarditis. In our previous studies, some S. mutans strains were shown to possess collagen-binding activities and most of them had an approximately 120-kDa cell-surface collagen-binding protein called Cnm. However, several strains without Cnm proteins show collagen-binding properties. In the present study, another collagen-binding protein, Cbm, was characterized and its coding gene cbm was sequenced in these strains. The amino acid alignment in the putative collagen-binding domain of Cbm was shown to have approximately 80% identity and 90% similarity to the comparable region of Cnm. Cbm-deficient isogenic mutant strains constructed by insertional inactivation of the cbm gene, lacked collagen-binding properties, which were recovered in the complemented mutant. Analyses of a large number of clinical isolates from Japan, Thailand and Finland revealed that cbm-positive strains were present in all of these countries and that cnm-positive and cbm-positive strains were detected in the oral cavity of approximately 10 and 2% of systemically healthy subjects, respectively. In addition, cnm-positive strains were predominantly identified in the serotype f group, whereas cbm-positive strains were frequently detected in serotype k. These results suggest that Cbm as well as Cnm are major cell surface proteins of S. mutans associated with binding to type I collagen and predominantly identified in serotype k strains.     

PCR detection of Streptococcus mutans and Streptococcus sobrinus in plaque samples from Mongolian mother-child pairs

Early childhood caries results in a considerable burden of pain and suffering as well as poorer general health. Streptococcus mutans (serotypes c, e and f ) and Streptococcus sobrinus (serotype d and g) are the species closely associated with dental caries. The exact age at which their colonization occurs in children is controversial. The objective of this study was to detect S. mutans and S. sobrinus in plaque samples of Mongolian mother-child pairs by PCR and to compare their presence with the caries status. Dental examination and caries risk assessment using the Cariostat^® carried out on 320 children aged 6–30 months and their mothers. The presence of S. mutans and S. sobrinus was checked by PCR. The caries prevalence and DEFT scores of mothers enrolled in the present study were 98% and 11.5±0.7, respectively. In children, the prevalence and deft scores of the 6–18-month-olds were 29% and 1.3±0.2 while those of the 19–30-month-olds were 59% and 3.4±0.4 correspondingly. Twenty nine percent of the 6–18-months old children of high-risk mothers and 53.1% of the 19–30-months old children of high-risk mothers had high caries risk (P<0.001). There was a statistically significant correlation between caries risk of 19–30 month-old children and their mothers (P<0.001). In mothers, the prevalence of S. mutans and S. sobrinus was 79% and 33%, respectively; 54% harbored S. mutans alone, 8% harbored S. sobrinus alone, 25% harbored both strains. In children, 45% were positive for S. mutans alone, 9% were positive for S. sobrinus alone, 18% were positive for both strains. Either or both strains were detected in 67.3% of 6–18-months old children and 76.5% of 19–30-months old children. In conclusion, our results showed that S. mutans and/or S. sobrinus first colonized infants’ teeth from 6–18 months, and the colonization increased with increasing age, so that by 30 months of age, 76.5% of children harbored the bacteria.     

Detection of mutans streptococci in plaque samples from Mongolian preschool and school children

Mutans streptococci, in particular Streptococcus mutans and Streptococcus sobrinus, are generally considered to be the principal microbial pathogen of dental caries. The objective of the study was to isolate S. mutans and S. sobrinus, identify them by PCR, and to compare their presence with the caries status and caries risk in Mongolian preschool and school children. Forty one preschool children aged 3–5 years and 40 school children aged 12–15 years were enrolled in this study. As assessed using Cariostat test, 75.6% of preschool children had high caries risk and 37.5% of school children had high caries risk. In preschool children, the prevalence of S. mutans and S. sobrinus were 100% and 36.6%, respectively; 63.4% were positive for S. mutans alone and 36.6% were positive for both S. mutans and S. sobrinus. In school children, the prevalence of S. mutans and S. sobrinus were 100% and 25.0%, respectively; 75.0% carried S. mutans alone and 25.0% had both S. mutans and S. sobrinus. The percentage of children positive for both S. mutans and S. sobrinus in the high caries risk group were significantly higher than those in the low risk group of either preschool (42.0% vs. 10.0%, P< 0.001) or school children (46.6% vs. 12.0%, P<0.001). Moreover, the caries status of children positive for both S. mutans and S. sobrinus were significantly higher than those positive for S. mutans alone (P< 0.01 for preschool children, and P< 0.05 for school children).     

In silico analysis of the competition between Streptococcus sanguinis and Streptococcus mutans in the dental biofilm

During dental caries, the dental biofilm modifies the composition of the hundreds of involved bacterial species. Changing environmental conditions influence competition. A pertinent model to exemplify the complex interplay of the microorganisms in the human dental biofilm is the competition between Streptococcus sanguinis and Streptococcus mutans. It has been reported that children and adults harbor greater numbers of S. sanguinis in the oral cavity, associated with caries‐free teeth. Conversely, S. mutans is predominant in individuals with a high number of carious lesions. Competition between both microorganisms stems from the production of H₂O₂ by S. sanguinis and mutacins, a type of bacteriocins, by S. mutans. There is limited evidence on how S. sanguinis survives its own H₂O₂ levels, or if it has other mechanisms that might aid in the competition against S. mutans, nonetheless. We performed a genomic and metabolic pathway comparison, coupled with a comprehensive literature review, to better understand the competition between these two species. Results indicated that S. sanguinis can outcompete S. mutans by the production of an enzyme capable of metabolizing H₂O₂. S. mutans, however, lacks the enzyme and is susceptible to the peroxide from S. sanguinis. In addition, S. sanguinis can generate energy through gluconeogenesis and seems to have evolved different communication mechanisms, indicating that novel proteins may be responsible for intra‐species communication.     

Lactobacillus species and genotypes associated with dental caries in Thai preschool children

Lactobacilli have been associated with the presence and progression of dental caries. Nevertheless, the relation between certain species or genotypes of Lactobacillus and caries is unclear and there are no data available for the Thai population. This study aimed to examine the distribution of species and genotypes of oral Lactobacillus among children with rather high caries prevalence, and to investigate whether certain species or genotypes were more related to caries activity than others. One hundred and sixty‐five children were examined for caries status. Saliva samples were collected and the numbers of lactobacilli were counted. A total of 357 Lactobacillus isolates from 59 children were identified to species level by 16S ribosomal RNA genes polymerase chain reaction (PCR) –restriction fragment length polymorphism and 16S ribosomal RNA gene sequencing. Furthermore, 304 isolates from 56 children were genotyped using arbitrarily primed PCR. Significant correlation was found between levels of lactobacilli and dental caries (P < 0.001). Among the 10 identified species of Lactobacillus, L. salivarius was more prevalent in children with moderate to high caries prevalence compared with children with low caries prevalence, while L. fermentum was the most predominant species in all study groups. Moreover, a genetic heterogeneity of Lactobacillus species was found among the children and those with high caries prevalence tended to be colonized with more than one clonal type. In summary, L. salivarius may be a putative caries pathogen among preschool Thai children.     

Antigen I/II mediates interactions between Streptococcus mutans and Candida albicans

Streptococcus mutans and Candida albicans are frequently co‐isolated from dental plaque of children with early childhood caries (ECC) and are only rarely found in children without ECC, suggesting that these species interact in a manner that contributes to the pathogenesis of ECC. Previous studies have demonstrated that glucans produced by S. mutans are crucial for promoting the formation of biofilm and cariogenicity with C. albicans; however, it is unclear how non‐glucan S. mutans biofilm factors contribute to increased biofilm formation in the presence of C. albicans. In this study we examined the role of S. mutans antigen I/II in two‐species biofilms with C. albicans, and determined that antigen I/II is important for the incorporation of C. albicans into the two‐species biofilm and is also required for increased acid production. The interaction is independent of the proteins Als1 and Als3, which are known streptococcal receptors of C. albicans. Moreover, antigen I/II is required for the colonization of both S. mutans and C. albicans during co‐infection of Drosophila melanogaster in vivo. Taken together, these results demonstrate that antigen I/II mediates the increase of C. albicans numbers and acid production in the two‐species biofilm, representing new activities associated with this known S. mutans adhesin.     

Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans

We report the whole genome sequence of the serotype e Cbm⁺ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra‐oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall‐anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram‐positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.     

Comparative genotyping of Streptococcus mutans by repetitive extragenic palindromic polymerase chain reaction and multilocus sequence typing

The genetic diversity of Streptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic palindromic‐polymerase chain reaction (rep‐PCR) is a genotyping approach used for screening large numbers of bacterial isolates. This two‐part study used multilocus sequence typing (MLST) analysis to evaluate genotypes previously identified as unique using rep‐PCR. In part one, an isolate was selected from each of the 22 S. mutans rep‐PCR genotype groups representing 8000 clinical isolates. For part two, four additional isolates were selected from the six most commonly occurring genotype groups (GG) for further analysis. Real‐time PCR was performed using eight housekeeping S. mutans gene loci and the amplicons were sequenced. Sequence data analysis was performed using CLC DNA Workbench and alleles were compared with the PubMLST database for Oral Streptococcus using the Nakano scheme. Concatenated sequences were evaluated with MEGA using a minimum evolution method with bootstrap. All 22 rep‐PCR genotypes were unique by MLST analysis. Within rep‐PCR GGs, MLST matched rep‐PCR in three groups demonstrating clonality; three groups exhibited more diversity with MLST. The discovery of three clonal groups is unique to this study and suggests that S. mutans genotypes are shared between unrelated subjects. Furthermore, MLST defined 19 new alleles and 26 new sequence types that have been confirmed and registered with PubMLST. Methods for processing were streamlined and a process for using MLST with rep‐PCR is suggested. In conclusion, MLST verified that rep‐PCR is a reliable and cost‐effective method for screening large numbers of S. mutans strains for epidemiological study.     

Invasive Streptococcus mutans induces inflammatory cytokine production in human aortic endothelial cells via regulation of intracellular toll‐like receptor 2 and nucleotide‐binding oligomerization domain 2

Streptococcus mutans, the primary etiologic agent of dental caries, can gain access to the bloodstream and has been associated with cardiovascular disease. However, the roles of S. mutans in inflammation in cardiovascular disease remain unclear. The aim of this study was to examine cytokine production induced by S. mutans in human aortic endothelial cells (HAECs) and to evaluate the participation of toll‐like receptors (TLRs) and cytoplasmic nucleotide‐binding oligomerization domain (NOD) ‐like receptors in HAECs. Cytokine production by HAECs was determined using enzyme‐linked immunosorbent assays, and the expression of TLRs and NOD‐like receptors was evaluated by real‐time polymerase chain reaction, flow cytometry and immunocytochemistry. The involvement of TLR2 and NOD2 in cytokine production by invaded HAECs was examined using RNA interference. The invasion efficiencies of S. mutans strains were evaluated by means of antibiotic protection assays. Five of six strains of S. mutans of various serotypes induced interleukin‐6, interleukin‐8 and monocyte chemoattractant protein‐1 production by HAECs. All S. mutans strains upregulated TLR2 and NOD2 mRNA levels in HAECs. Streptococcus mutans Xc upregulated the intracellular TLR2 and NOD2 protein levels in HAECs. Silencing of the TLR2 and NOD2 genes in HAECs invaded by S. mutans Xc led to a reduction in interleukin‐6, interleukin‐8 and monocyte chemoattractant protein‐1 production. Cytokine production induced by invasive S. mutans via intracellular TLR2 and NOD2 in HAECs may be associated with inflammation in cardiovascular disease.     

Salivary antimicrobial proteins associate with age‐related changes in streptococcal composition in dental plaque

Secretion of antimicrobial proteins (AMPs) and salivary antibodies can modify biofilm formation at host body surfaces. In adolescents, associations have been reported between dental caries and salivary AMPs. AMPs demonstrate direct antimicrobial effects at high concentrations, and at lower more physiological concentrations they mediate changes in host cell defenses, which may alter the local environment and indirectly shape local biofilm formation. The expression of salivary AMPs in preschool children, at an age when the oral bacteria are known to change, has not been investigated. We sought to investigate salivary AMP expression in the context of previously well‐documented changes in the oral cavities of this age group including salivary immunoglobulin A (IgA), oral bacteria and dental caries. Dental plaque and saliva were collected from 57 children aged 12–24 months at baseline, of whom 23 children were followed‐up at 3 years of age. At each time, saliva was assessed for LL37, human neutrophil peptides 1–3, calprotectin, lactoferrin, salivary IgA, total plaque bacteria and Streptococcus mutans. Over time, concentrations of AMPs, S. mutans and bacteria‐specific salivary IgA increased. Caries experience was also recorded when children were 3 years old. Concentrations of AMPs were highest in the saliva of 3‐year‐old children with the greatest burden of S. mutans. These data suggest that salivary AMPs are variable over time and between individuals, and are linked with bacterial colonization. At follow up, the majority of children remained caries free. Larger longitudinal studies are required to confirm whether salivary AMP levels are predictive of caries and whether their modulation offers therapeutic benefit.     

Genotypic diversity of Streptococcus mutans and Streptococcus sobrinus in 3-4-year-old children with severe caries or without caries

International Journal of Paediatric Dentistry 2011; 21: 422–431 Background. The genotypic diversity of both Streptococcus mutans and Streptococcus sobrinus in children with different caries experience remains unclear. Aim. To investigate the genotypic diversity of S. mutans and S. sobrinus in children with severe early childhood caries (SECC) and in caries‐free (CF) children. Methods. Stimulated saliva of 87 SECC and 91 CF children aged 3–4 years was collected and submitted to cultivation, and MS colonies were enumerated. The genomic fingerprint analysis of S. mutans and S. sobrinus was carried out using AP‐PCR. Results. One to five genotypes of S. mutans were colonized in an oral cavity of SECC and CF children; 85.5% SECC children and 57.9% CF children harboured more than one genotype of S. mutans. One to three genotypes of S. sobrinus were detected from each SECC child; 31.25% SECC children harboured more than one genotype of S. sobrinus. And one genotype was colonized in each CF child. S. mutans isolates from different individuals displayed distinctive DNA fingerprints. Conclusions. DNA fingerprints of S. mutans and S. sobrinus isolates from 3‐ to 4‐year‐old children displayed genetic polymorphism, and S. mutans has greater genetic diversity than S. sobrinus. SECC children harboured more genotypes of S. mutans and S. sobrinus than CF children.     

Presence of Streptococcus mutans or Streptococcus sobrinus in Cariostat®-inoculated plaque samples from Japanese mother-child pairs

The aim of this study was to determine the presence of Streptococcus mutans or Streptococcus sobrinus in Cariostat-inoculated plaque samples obtained from Japanese mother-child pairs through a conventional PCR technique and to establish the presence of these bacteria and caries risk. Oral examination and caries risk assessment using the Cariostat^® were carried out on 168 children, aged 6–31 months, and their mothers. The presence of S. mutans and S. sobrinus in Cariostat-inoculated plaque samples was checked through PCR and tested for relevance with caries risk. A significant correlation (P < 0.001) was found between caries risk of mothers and presence of S. mutans or S. sobrinus in plaque samples from their children in the 19–31-month-old age range. However, no significant relationship found between the presence of either strain in the plaque of younger children (6–18 months) and caries risk of mothers. Likewise, high caries risk was seen in 49.1% of the 19–31-month-old children of highrisk mothers (P < 0.001) and 27% of the 6–18-month-old children of high-risk mothers (P < 0.05). The effectiveness of the Cariostat method for prediction of caries risk can be improved by detecting the presence of S. mutans and S. sobrinus in plaque samples obtained from mothers and their children through conventional PCR techniques.     

Genotypic and phenotypic analysis of Streptococcus mutans from different oral cavity sites of caries‐free and caries‐active children

Introduction: Streptococcus mutans exhibits extensive genotypic diversity, but the role of this variation is poorly understood. This study aimed to determine the number and distribution of genotypes of S. mutans isolated from caries‐active and caries‐free children and to evaluate some of their phenotypic traits. Methods: Stimulated saliva, tongue surface and biofilms over sound and carious teeth surfaces were sampled from 10 caries‐free and 11 caries‐active children aged 5–8 years. A total of 339 isolates of S. mutans were genotyped by arbitrarily primed polymerase chain reaction using OPA2 primer. One isolate from each genotype was tested for its acid susceptibility and its ability to form a biofilm. Results: Fifty‐one distinct genotypes were determined, one to three genotypes in each oral sample. A single genotype was detected in seven children, whereas the remaining 14 children exhibited two to seven genotypes. There were no significant differences in the number of genotypes detected in caries‐free and caries‐active children. No correlation was observed between the number of genotypes and the mutans streptococci salivary levels. Five of the six high biofilm‐forming genotypes were obtained from caries‐active children, although the differences in biofilm formation between isolates from caries‐free and caries‐active children were not statistically significant. Genotypes with low susceptibility to acid challenge were statistically more frequent among isolates from caries‐active children than among those from caries‐free children. Conclusion: The present data suggested that there were differences in the distribution of genotypes of S. mutans according to the oral site and that S. mutans populations differ in their acid susceptibility and ability to form biofilms, factors allowing their colonization of sucrose‐rich environments.     

DMBT1 involvement in the human aortic endothelial cell response to Streptococcus mutans

Streptococcus mutans is a causative organism of dental caries and has been reported to be associated with the development of cardiovascular disease (CVD). Previous studies have demonstrated that S. mutans invades human aortic endothelial cells (HAECs) and HAECs invaded by S. mutans produce higher levels of CVD–related cytokines than non‐invaded HAECs. DMBT1 (deleted in malignant brain tumors 1), also known as salivary agglutinin or gp‐340, belongs to the scavenger receptor cysteine–rich superfamily. DMBT1 is expressed in epithelial and non‐epithelial tissues and has multiple functions. The interaction between S. mutans and DMBT1 has been demonstrated in cariogenesis, but DMBT1 involvement in CVD has not been examined. In this study, we investigated DMBT1 expression in HAECs stimulated with S. mutans and examined the role of DMBT1 in the interaction between S. mutans and HAECs. All of the tested S. mutans strains induced higher production levels of DMBT1 in HAECs than those in unstimulated HAECs. More S. mutans cells adhered to DMBT1 knock down HAECs than to DMBT1–producing HAECs. Invasion of DMBT1 knock down HAECs by S. mutans was stronger than that of DMBT1–producing HAECs, and externally added DMBT1 reduced bacterial invasion. Cytokine production by DMBT1 knock down HAECs by S. mutans stimulation was higher than that by DMBT1–producing HAECs. These phenomena seemed to be due to the effect of released DMBT1, namely, the inhibition of bacterial adherence to HAECs by DMBT1. These results suggest that DMBT1 plays a protective role against the S. mutans–induced CVD process in HAECs.     

Deficiency of BrpA in Streptococcus mutans reduces virulence in rat caries model

Our recent studies have shown that BrpA in Streptococcus mutans plays a critical role in cell envelope biogenesis, stress responses, and biofilm formation. In this study, a 10‐species consortium was used to assess how BrpA deficiency influences the establishment, persistence, and competitiveness of S. mutans during growth in a community under conditions typical of the oral cavity. Results showed that, like the wild‐type, the brpA mutant was able to colonize and establish on the surfaces tested. Relative to the wild‐type, however, the brpA mutant had a reduced ability to persist and grow in the 10‐species consortium (P < .001). A rat caries model was also used to examine the effect of BrpA, as well as Psr, a BrpA paralog, on S. mutans cariogenicity. The results showed no major differences in infectivity between the wild‐type and the brpA and psr mutants. Unlike the wild‐type, however, infection with the brpA mutant, but not the psr mutant, showed no significant differences in both total numbers of carious lesions and caries severity, compared with the control group that received bacterial growth medium (P > .05). Metagenomic and quantitative polymerase chain reaction analysis showed that S. mutans infection caused major alterations in the composition of the rats’ plaque microbiota and that significantly less S. mutans was identified in the rats infected with the brpA mutant compared with those infected with the wild‐type and the psr mutant. These results further suggest that BrpA plays a critical role in S. mutans pathophysiology and that BrpA has potential as a therapeutic target in the modulation of S. mutans virulence.     

Cnm is a major virulence factor of invasive Streptococcus mutans and part of a conserved three‐gene locus

Cnm, a collagen‐ and laminin‐binding protein present in a subset of Streptococcus mutans strains, mediates binding to extracellular matrices (ECM), intracellular invasion and virulence in the Galleria mellonella model. Antibodies raised against Cnm were used to confirm expression and the cell surface localization of Cnm in the highly invasive OMZ175 strain. Sequence analysis identified two additional genes (cnaB and cbpA) encoding putative surface proteins immediately upstream of cnm. Inactivation of cnaB and cbpA in OMZ175, individually or in combination, did not decrease the ability of this highly invasive and virulent strain to bind to different ECM proteins, invade human coronary artery endothelial cells (HCAEC), or kill G. mellonella. Similarly, expression of cnaB and cbpA in the cnm^? strain UA159 revealed that these genes did not enhance Cnm‐related phenotypes. However, integration of cnm in the chromosome of UA159 significantly increased its ability to bind to collagen and laminin, invade HCAEC, and kill G. mellonella. Moreover, the presence of antibodies against Cnm nearly abolished the ability of OMZ175 to bind to collagen and laminin and invade HCAEC, and significantly protected G. mellonella against OMZ175 infection. We concluded that neither CnaB nor CbpA is necessary for the expression of Cnm‐related traits. We also provided definitive evidence that Cnm is an important virulence factor and a suitable target for the development of novel preventive and therapeutic strategies to combat invasive S. mutans strains.