Multiple factors are involved in regulation of extracellular membrane vesicle biogenesis in Streptococcus mutans

Streptococcus mutans, a major etiological agent of human dental caries, produces membrane vesicles (MVs) that contain protein and extracellular DNA. In this study, functional genomics, along with in vitro biofilm models, was used to identify factors that regulate MV biogenesis. Our results showed that when added to growth medium, MVs significantly enhanced biofilm formation by S. mutans, especially during growth in sucrose. This effect occurred in the presence and absence of added human saliva. Functional genomics revealed several genes, including sfp, which have a major effect on S. mutans MVs. In Bacillus sp. sfp encodes a 4′‐phosphopantetheinyl transferase that contributes to surfactin biosynthesis and impacts vesiculogenesis. In S. mutans, sfp resides within the TnSmu2 Genomic Island that supports pigment production associated with oxidative stress tolerance. Compared to the UA159 parent, the Δsfp mutant, TW406, demonstrated a 1.74‐fold (p < .05) higher MV yield as measured by BCA protein assay. This mutant also displayed increased susceptibility to low pH and oxidative stressors, as demonstrated by acid killing and hydrogen peroxide challenge assays. Deficiency of bacA, a putative surfactin synthetase homolog within TnSmu2, and especially dac and pdeA that encode a di‐adenylyl cyclase and a phosphodiesterase, respectively, also significantly increased MV yield (p < .05). However, elimination of bacA2, a bacitracin synthetase homolog, resulted in a >1.5‐fold (p < .05) reduction of MV yield. These results demonstrate that S. mutans MV properties are regulated by genes within and outside of the TnSmu2 island, and that as a major particulate component of the biofilm matrix, MVs significantly influence biofilm formation.     

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.     

Streptococcus oralis maintains homeostasis in oral biofilms by antagonizing the cariogenic pathogen Streptococcus mutans

Bacteria residing in oral biofilms live in a state of dynamic equilibrium with one another. The intricate synergistic or antagonistic interactions between them are crucial for determining this balance. Using the six‐species Zürich “supragingival” biofilm model, this study aimed to investigate interactions regarding growth and localization of the constituent species. As control, an inoculum containing all six strains was used, whereas in each of the further five inocula one of the bacterial species was alternately absent, and in the last, both streptococci were absent. Biofilms were grown anaerobically on hydroxyapatite disks, and after 64 h they were harvested and quantified by culture analyses. For visualization, fluorescence in situ hybridization and confocal laser scanning microscopy were used. Compared with the control, no statistically significant difference of total colony‐forming units was observed in the absence of any of the biofilm species, except for Fusobacterium nucleatum, whose absence caused a significant decrease in total bacterial numbers. Absence of Streptococcus oralis resulted in a significant decrease in Actinomyces oris, and increase in Streptococcus mutans (P < .001). Absence of A. oris, Veillonella dispar or S. mutans did not cause any changes. The structure of the biofilm with regards to the localization of the species did not result in observable changes. In summary, the most striking observation of the present study was that absence of S. oralis resulted in limited growth of commensal A. oris and overgrowth of S. mutans. These data establish highlight S. oralis as commensal keeper of homeostasis in the biofilm by antagonizing S. mutans, so preventing a caries‐favoring dysbiotic state.     

A clinical,randomized study on the influence of dental whitening on Streptococcus mutans population

Background

Dental whitening with peroxides has been popularized through the at‐home technique, which employs low concentrations of peroxide applied in individual trays. However, there are few clinical trials reporting the effects of its continuous use on oral microbiota. Thus, the purpose of the present clinical, randomized study was to evaluate the influence of at‐home whitening treatment on Streptococcus mutans in saliva, buccal mucosa, and subgingival and supragingival plaque.

Methods

Thirty volunteers were randomly divided into two study groups (N = 15) according to the whitening therapy: G CP, whitening using 10% carbamide peroxide 4 h daily for 21 days; and G HP, whitening using 6% hydrogen peroxide 1.5 h daily for 21 days. Samples from the predetermined locations were collected at three evaluation periods: T1, before; T2, immediately after; and T3, 30 days after the beginning of the treatment. The microbiological evaluation was made using conventional and molecular methods.

Results

Student's t‐test demonstrated a statistically significant decrease in S. mutans population in the subgingival and supragingival plaque for HP samples between T1 and T2 no difference was found between T1 and T3 regardless of the location and the whitening product used (α = 0.05).

Conclusions

Although HP reduced S. mutans during treatment, the levels returned to baseline when assessed 30 days after the treatment.     

SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

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.     

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.     

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.     

Streptococcus mutans SpaP binds to RadD of Fusobacterium nucleatum ssp. polymorphum

Adhesin‐mediated bacterial interspecies interactions are important elements in oral biofilm formation. They often occur on a species‐specific level, which could determine health or disease association of a biofilm community. Among the key players involved in these processes are the ubiquitous fusobacteria that have been recognized for their ability to interact with numerous different binding partners. Fusobacterial interactions with Streptococcus mutans, an important oral cariogenic pathogen, have previously been described but most studies focused on binding to non‐mutans streptococci and specific cognate adhesin pairs remain to be identified. Here, we demonstrated differential binding of oral fusobacteria to S. mutans. Screening of existing mutant derivatives indicated SpaP as the major S. mutans adhesin specific for binding to Fusobacterium nucleatum ssp. polymorphum but none of the other oral fusobacteria tested. We inactivated RadD, a known adhesin of F. nucleatum ssp. nucleatum for interaction with a number of gram‐positive species, in F. nucleatum ssp. polymorphum and used a Lactococcus lactis heterologous SpaP expression system to demonstrate SpaP interaction with RadD of F. nucleatum ssp. polymorphum. This is a novel function for SpaP, which has mainly been characterized as an adhesin for binding to host proteins including salivary glycoproteins. In conclusion, we describe an additional role for SpaP as adhesin in interspecies adherence with RadD‐SpaP as the interacting adhesin pair for binding between S. mutans and F. nucleatum ssp. polymorphum. Furthermore, S. mutans attachment to oral fusobacteria appears to involve species‐ and subspecies‐dependent adhesin interactions.     

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.     

Molecular pathogenicity of Streptococcus anginosus

Streptococcus anginosus and the closely related species Streptococcus constellatus and Streptococcus intermedius, are primarily commensals of the mucosa. The true pathogenic potential of this group has been under‐recognized for a long time because of difficulties in correct species identification as well as the commensal nature of these species. In recent years, streptococci of the S. anginosus group have been increasingly found as relevant microbial pathogens in abscesses and blood cultures and they play a pathogenic role in cystic fibrosis. Several international studies have shown a surprisingly high frequency of infections caused by the S. anginosus group. Recent studies and a genome‐wide comparative analysis suggested the presence of multiple putative virulence factors that are well‐known from other streptococcal species. However, very little is known about the molecular basis of pathogenicity in these bacteria. This review summarizes our current knowledge of pathogenicity factors and their regulation in S. anginosus.     

Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans

Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Many of the proteins necessary for its colonization of the oral cavity and pathogenesis are exported to the cell surface or the extracellular matrix, a process that requires the assistance of the export machineries. Bioinformatic analysis revealed that the S. mutans genome contains a prsA gene, whose counterparts in other gram‐positive bacteria, including Bacillus and Lactococcus, encode functions involved in protein post‐export. In this study, we constructed a PrsA‐deficient derivative of S. mutans and demonstrated that the prsA mutant displayed an altered cell wall/membrane protein profile as well as cell‐surface‐related phenotypes, including auto‐aggregation, increased surface hydrophobicity and abnormal biofilm formation. Further analysis revealed that the disruption of the prsA gene resulted in reduced insoluble glucan production by cell surface localized glucosyltransferases, and mutacin as well as cell surface‐display of a heterologous expressed GFP fusion to the cell surface protein SpaP. Our study suggested that PrsA in S. mutans encodes functions similar to those identified in Bacillus, and so is likely to be involved in protein post‐export.     

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.     

Deficiency of PdxR in Streptococcus mutans affects vitamin B6 metabolism,acid tolerance response and biofilm formation

Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA‐binding N‐terminal domain and a C‐terminal domain highly homologous to the pyridoxal phosphate‐dependent aspartate aminotransferases. A pdxR‐deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR‐deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real‐time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B₆ biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR‐deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B₆. Further studies revealed that although S. mutans is known to require vitamin B₆ to grow in defined medium, B₆ vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B₆ metabolism, acid tolerance response and biofilm formation.     

d‐Alanine metabolism is essential for growth and biofilm formation of Streptococcus mutans

Part of the d ‐alanine (d ‐Ala) metabolic pathway in bacteria involves the conversion of l ‐alanine to d ‐Ala by alanine racemase and the formation of d ‐alanyl–d ‐alanine by d ‐alanine–d ‐alanine ligase, the product of which is involved in cell wall peptidoglycan synthesis. At present, drugs that target the metabolic pathway of d ‐Ala are already in clinical use – e.g. d ‐cycloserine (DCS) is used as an antibiotic against Mycobacterium tuberculosis. Streptococcus mutans is the main cariogenic bacterium in the oral cavity. Its d ‐Ala metabolism‐associated enzymes alanine racemase and d ‐alanine–d ‐alanine ligase are encoded by the genes smu.1834 and smu.599, respectively, which may be potential targets for inhibitors. In this study, the addition of DCS blocked the d ‐Ala metabolic pathway in S. mutans, leading to bacterial cell wall defects, significant inhibition of bacterial growth and biofilm formation, and reductions in extracellular polysaccharide production and bacterial adhesion. However, the exogenous addition of d ‐Ala could reverse the inhibitory effect of DCS. Through the means of drug regulation, our study demonstrated, for the first time, the importance of d ‐Ala metabolism in the survival and biofilm formation of S. mutans. If the growth of S. mutans can be specifically inhibited by designing drugs that target d ‐Ala metabolism, then this may serve as a potential new treatment for dental caries.     

Combined inhibitory effect of bovine immune whey and peroxidase-generated hypothiocyanite against glucose uptake by Streptococcus mutans

Immune whey product was obtained from Streptococcus mutans- and Streptococcus sobrinus-immunized cows. Hypothiocyanite (HOSCN/OSCN^-) was generated in VMG-buffer, pH 5.5 or 6.5, by bovine milk lactoperoxidase, KSCN and hydrogen peroxide. The glucose incorporation by late-log cells of S. mutans 10449, serotype c, was followed by measuring the uptake of ¹⁴C-glucose at 37°C. At pH 5.5 and 6.5 both immune whey product and HOSCN/OSCTST^- dosedependently inhibited glucose uptake. The inhibition by their combination was additive if bacterial cells were treated with HOSCN/OSCN^- before exposed to immune whey product. In contrast to immune whey product, the control product from sham-immunized cows increased the glucose uptake even when added simultaneously with HOSCN/OSCN^-. However, when bacterial cells were pretreated with HOSCN/OSCN^- an enhanced inhibitory effect was observed also with control product. The results indicate that colostral proteins from S. mutans- or S. sobrinus-immunized cows inhibit glucose uptake and that the effect is enhanced by pretreatment with lactoperoxidase-generated HOSCN/OSCN^-.     

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.     

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.