Role of Streptococcus mutans surface proteins for biofilm formation.

Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans. An important virulence property of the bacterium is its ability to form biofilm known as dental plaque on tooth surfaces. In addition, this organism also produces glucosyltransferases, multiple glucan-binding proteins, protein antigen c, and collagen-binding protein, surface proteins that coordinate to produce dental plaque, thus inducing dental caries. Bacteria utilize quorum-sensing systems to modulate environmental stress responses. A major mechanism of response to signals is represented by the so called two-component signal transduction system, which enables bacteria to regulate their gene expression and coordinate activities in response to environmental stress. As for S. mutans, a signal peptide-mediated quorum-sensing system encoded by comCDE has been found to be a regulatory system that responds to cell density and certain environmental stresses by excreting a peptide signal molecule termed CSP (competence-stimulating peptide). One of its principal virulence factors is production of bacteriocins (peptide antibiotics) referred to as mutacins. Two-component signal transduction systems are commonly utilized by bacteria to regulate bacteriocin gene expression and are also related to biofilm formation by S. mutans.

Streptococcus mutans and biofilm formation

Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans [1] and one of its important virulence properties is an ability to form biofilm known as dental plaque on tooth surfaces [2]. The bacterium synthesizes adhesive glucan from sucrose by the action of glucosyltransferases (GTFs), then glucans mediate firm adherence of its cells to tooth surfaces [3]. S. mutans also produces multiple glucan-binding proteins (Gbp proteins), which are thought to promote adhesion [4]. Furthermore, the cell surface protein antigen c (PAc), a major surface protein of S. mutans, is correlated to its virulence in regard to development of dental caries, as it is known to participate in bacterial adherence to tooth surfaces via interaction with the salivary pellicle [5]. Together, these bacterial surface proteins coordinate to produce dental plaque, thus inducing dental caries.

Glucosyltransferases

S. mutans produces 3 types of GTFs (GTFB, GTFC, GTFD), whose cooperative action is essential for adherence of bacterial cells, with the highest level of sucrose-dependent cellular adhesion found at the ratio of 5:0.25:1 [6].

GTFB and GTFC, which mainly synthesize water-insoluble glucans rich in α-1,3-glucosidic linkages, are located on the cell surface, and encoded by the gtfB and gtfC genes, respectively [7], [8]. On the other hand, GTFD, which synthesizes water-soluble glucans rich in α-1,6-glucosidic linkages, has been detected in culture supernatant and known to be encoded by the gtfD gene [9]. Each enzyme is composed of 2 functional domains, an amino-terminal catalytic domain (CAT), which binds and hydrolyzes the substrate of sucrose, and a carboxyl-terminal glucan-binding domain (GBD), which functions as an acceptor for binding glucan and also plays an important role in determining the nature of the glucan synthesized by a GTF [10], [11], [12]. In a previous study of anti-caries activities of oolong tea, high-molecular-weight polyphenols were found to have site-specific actions, thus an oolong tea fraction rich in polymeric polyphenols reduced glucan synthesis in a noncompetitive manner by targeting the S. mutans glucan-binding domains of GTFB and GTFD in the solution phase [13].

Simultaneous synthesis of glucans by GTFB and GTFC is essential for establishment of a matrix that enhances the coherence of bacterial cells and adherence to tooth surfaces, allowing for formation of high density biofilm [14], [15], [16]. It has been shown that the presence of highly adherent and insoluble glucans in situ increases mechanical stability by binding bacterial cells together, as well as to an apatite surface (Fig. 1). In addition to interactions with specific Gbps expressed by S. mutans and other oral microorganisms, these polymers are critical for maintaining the 3-dimensional structure of biofilm (Fig. 1), thereby playing a role in modulating development of cariogenic biofilm [15], [16], [17].

Figure 1

Representative confocal images of bacterial cells (green) and glucans (red) within biofilm formed by S. mutans MT8148 on tooth enamel surface in presence of 0.5% (wt/vol) sucrose.

Glucan-binding proteins

Binding of S. mutans to glucans formed in situ is mediated by the presence of cell-associated GTF enzymes and non-GTF glucan-binding proteins (Gbps) [4]. This bacterial organism produces at least 4 glucan-binding proteins (Gbps); GbpA [18], GbpB [19], GbpC [20], and GbpD [21], which presumably promote its adhesion. GbpA, the first designated glucan-binding protein, contains carboxyl terminal repeats similar to the glucan-binding domain of GTF enzymes [21], [22]. This protein is involved in cellular adherence to tooth surfaces, and has been shown to contribute to the cariogenicity of S. mutans both in vitro and in vivo [23].

GbpA contributes to development of optimal plaque biofilm, which minimizes stress on the bacterial population [24], while it also has an important role in binding proteins and exopolysaccharides for construction of biofilm and maintenance of a balanced environment, while the structure of biofilm and its tolerance to various types of stress is affected by its absence [25]. A deficiency of GbpA results in loose binding to the EPS matrix, resulting in a weak non-uniform biofilm structure (Fig. 2). Thus, GbpA has important roles as a protein for formation of firm and stable biofilm.

Figure 2

Biofilm analysis using confocal laser scanning microscopy. (A) Representative three-dimensional images of biofilms formed by S. mutans MT8148 (a) and AD1 (b) in the presence of 0.5% sucrose. (B) x–z section and z-projection of biofilm formed by MT8148 (c) and AD1 (d). S. mutans was stained with SYTO®9 green fluorescent nucleic acid stain (green) and labeled-dextran (Alexa Fluor® 647; red) was used.

Alterations in biofilm structure cause harbored bacteria to be exposed to acid, making them susceptible to gene introduction, with the stress response proteins RecA, DnaK, and GroEL possibly related to that response, though the detailed mechanisms remain unclear [24], [25].

GbpB has been purified and shown to be immunologically distinct from other Gbps expressed by S. mutans and Streptococcus sobrinus [19]. It was also found to be homologous with peptidoglycan hydrolases of other Gram-positive microorganisms, while results of a comparative genomic analysis of the gbpB region suggested a functional relationship between genes involved in cell shape and cell wall maintenance [26], [27]. GbpB is considered to have some roles in the cariogenicity of S. mutans, as mucosal immunization has been found to induce protective immune responses against experimental dental caries [28], [29].

GbpC is a cell-surface-associated protein involved in dextran-induced aggregation and is expressed only under stress conditions [20]. Although the glucan-binding domain of GbpC has not been identified, it is homologous with the AgI/II family of proteins [20]. GbpC (and possibly GbpB) is a cell-wall bound protein that may function as a cell surface glucan receptor in S. mutans [4]. While each of the Gbps appears to have roles in sucrose-dependent adhesion and biofilm formation by S. mutans, loss of cell-surface-anchored GbpC has been shown to reduce the caries-inducing properties of the bacterium [17], [23]. It also plays an important role in sucrose-dependent adhesion by binding to soluble glucan synthesized by GTFD [30], indicating that GbpC is a cell-wall anchoring protein that possesses a specific binding domain for soluble glucans. Although GbpC does not contain repeat domains in regions involved in glucan-binding, the glucan-binding domain has been elucidated by bioinformatics analysis (Fig. 3) [31].

Figure 3

Results of NCBI Conserved Domains Database search of loop regions relative to the putative dextran-binding domain in GbpC. (A) Glucan-binding protein C: S. mutans. (B) Glucan binding protein C: S. macacae. (C) AgiII: S. mutans. (D) SpaA: S. sobrinus. (E) Probable cell-surface antigen I/II: S. intermedius. Asterisks indicate mid-points. Bold letters indicate core regions of potential dextran-binding domain sequences. Lower-case letters indicate no homology among strains.

GbpD was discovered and isolated using sequence analysis of the complete, annotated sequence of the UA159 strain of S. mutans [21]. After cloning and sequencing harbored proteins, it was identified as a secreted Gbp with lipase activity [21]. In addition, the amino acid sequence of gbpD is homologous to the carboxyl terminus regions of GTF and GbpA [21]. GbpD contributes to the cohesiveness of aggregates and adhesion to tooth surfaces, and is known to be mediated by glucans, particularly dextran, similar to GbpA [21].

Protein antigen c

The cell surface protein antigen c (PAc) is one of the major surface proteins of S. mutans [32] and known by a number of other names, including SpaP [33], antigen I/II [34] and B [35], P1 [36], and MSL-1 [37]. PAc is known to be correlated with virulence of the organism for development of dental caries and participates in bacterial adherence to teeth via interaction with the salivary pellicle [38], which is termed sucrose-independent adhesion. The gene for PAc of S. mutans serotype c has been cloned and sequenced [32], [33], and shown to include an N-terminal signal sequence, a region with a series of three 82-residue alanine-rich repeats (A-region) within the N-terminal third of the molecule, as well as a region with a series of three 39-residue proline-rich repeats (P-region) in the central portion of the molecule [32]. In addition, C-terminal sequences characteristic of wall- and membrane-spanning domains of streptococcal surface proteins have also been reported [39]. Furthermore, the A-region has a strong relationship with adhesion to tooth surfaces, while the P-region has a high affinity for PAc [40]. Several studies have reported an association of PAc with the virulence of S. mutans for infective endocarditis (IE) development. For example, PAc antibody titers were found to be elevated in a human subject with IE as compared with healthy subjects [41], whereas PAc was not related to endocarditis virulence in rat models in another study [42]. On the other hand, PAc has been shown to contribute to the interactions of S. mutans cells with fibronectin, collagen type I, and fibrinogen [43]. In addition, platelet aggregation occurring after pathogenic bacterial infection is considered to be one of the most important factors in the pathogenesis of IE. As for S. mutans, PAc is involved in human platelet aggregation, as it binds directly to platelets [44].

Collagen-binding protein

Recently, an approximately 120-kDa Cnm protein related to the collagen-binding activity of S. mutans was identified, and its encoding gene was cloned and sequenced [45]. This protein consists of a collagen-binding domain, a putative B-repeat domain, and a cell-wall-anchored LPXTG motif, such as PAc. The distribution frequency of strains with the cnm gene among oral isolates has been estimated to be in the range of 10–20% and cnm-positive strains are known to possess high collagen-binding properties [45], [46], [47], [48]. These strains have been predominantly identified in serotype f and k strains, though those are considered to be minor serotypes in the oral cavity [46], [49]. The Cnm protein has also been shown to possess binding activity to type I collagen [45], a major organic component of dentin, which is regarded as advantageous for binding to exposed dentin [50]. Recently, Cnm was reported to be involved in adherence to and invasion of human coronary artery endothelial cells, indicating its possible contributions to cardiovascular infections and pathology [51]. In addition, Cnm of S. mutans was shown to related to deterioration caused by cerebral hemorrhage [52]. Another collagen-binding protein, Cbm, has been predominantly identified in serotype k strains [53] and also proposed to be a potential important factor for inducing infective endocarditis [54].

Important factors related to biofilm formation by S. mutans

Biofilm formation is initiated by interactions between planktonic bacteria and an oral surface in response to appropriate environmental signals [55], [56], [57], [58], [59], [60], [61]. S. mutans metabolizes carbohydrates to adhere to and form biofilm on tooth surfaces, thus allowing the pathogen to tolerate rapid and frequent environmental fluctuations such as nutrient availability, aerobic-to-anaerobic transitions, and pH changes [62], [63] In addition, in response to physical and chemical signals, bacteria regulate diverse physiological processes in a cell density-dependent manner, known as quorum sensing [64], which they utilize to modulate environmental stress responses. Major participants in signal response are the so-called two-component signal transduction systems (TCSTSs), which enable bacteria to regulate their gene expression and coordinate activities in response to environmental stress [65], [66].

Two-component signal transduction systems (TCSTSs) of

Several TCSTSs have been reported present in S. mutans. Initially, a signal peptide-mediated quorum-sensing system encoded by comCDE was found to function as a regulatory system that responds to cell density and certain environmental stresses by excreting a peptide signal molecule termed CSP (competence-stimulating peptide) that is encoded by the comC gene [67], [68], [69], [70]. comCDE genes are located in the same locus and function together for generating and responding to CSP [67]. While comC encodes the precursor CSP, comDE genes encode a TCSTS comprised of a membrane-bound histidine kinase (ComD) and its cognate response regulator (ComE) [67]. When the genetic components of the S. mutans quorum-sensing system were initially identified, it was noted that genetic transformability was 10- to 600-fold higher in cells derived from biofilms as compared with those obtained from planktonic cultures [67]. Hence, in addition to competence development, this system is also involved in biofilm formation. To examine this assumption, mutants deficient in comC, comD, and comE were constructed and assayed for their ability to initiate biofilm formation. All of the mutants formed biofilms, though they lacked architectural integrity as compared to wild-type biofilm, while the comD- and comE-deficient mutant biofilms also had reduced biomass [68]. By interfering with this cell-cell signaling mechanism, biofilm formation by S. mutans, which utilizes quorum-sensing to control virulence, could potentially be attenuated. Another system termed HK/RR11 is involved in the relationship of S. mutans to acid tolerance [71]. This system consists of a membrane-associated histidine kinase (HK) protein, which senses a specific stimulus, as well as a cytoplasmic response regulator (RR) protein, which enables bacterial cells to respond to stimulus via regulation of gene expression [72]. Environmental signals are sensed by HK, resulting in autophosphorylation at a specific histidine residue, thus creating a high-energy phosphoryl group that is subsequently transferred to a specific aspartate residue within the N-terminal half of the cognate RR protein. Phosphorylation induces a conformational change in the regulatory domain resulting in activation of the RR protein, which then regulates gene expression by functioning as a DNA-binding transcriptional regulator that activates or represses genes whose products are specifically utilized to respond to the given input signal [73]. In addition, deletion of hk11 or rr11 has been reported to result in biofilm with a sponge-like architecture and composed of cells organized in very long chains [74]. Furthermore, inactivation of either hk11 or rr11 has been shown to lead to an abnormal biofilm phenotype, similar to that formed by a comC mutant, thus HK11 is suspected to be the second receptor for competence-stimulating peptide (CSP) [68]. On the other hand, an hk11-deficient mutant also showed reduced bacteriocin production, deficiency in transformability, and diminished ability to tolerate stress [75]. A major mechanism of signal transduction known to be widespread in bacteria is represented by TCSTSs, which enable bacteria to regulate their gene expression and coordinate activities in response to environmental stimuli [65], [66], [72]. In addition, the CiaR/H system has been characterized and found to play a coordinating role with the ComCDE quorum-sensing system for regulating genetic competence and stress response [76]. The ciaH and ciaR genes include a histidine kinase sensor protein and its cognate response regulator, as well as hk11/rr11, while activation of either ciaR or ciaH resulted in reduced biofilm biomass, whereas absence of ciaH alters sucrose-dependent biofilm formation [78]. In addition, CiaH signal transduction may be linked with the surface-anchored serine-protease HtrA, which is connected with CiaH on the stress response pathway [76]. That report also showed that deletion of CiaH resulted in biofilm with reduced biomass and very short chains, suggesting its role in regulating cell growth and/or cell division.

The VicRK system, which shares a high similarity to CovSR of Streptococcus pyogenes, has been found to regulate sucrose-dependent biofilm formation by S. mutans [77], [79]. In addition, VicR and CovR directly regulate a panel of genes implicated in the synthesis of and interaction with extracellular polysaccharides [79], [80], [81]. CovR negatively regulates expression of the gtfB and gtfC genes by directly binding to the promoter region [80]. Also, vicK-deficient mutants showed defects regarding separation of daughter cells and in sucrose-dependent biofilm formation [79]. On the other hand, the ScnRK system in S. mutans has been shown to regulate hydrogen resistance and macrophage killing, as a study found that ScnRK contributes to hydrogen peroxide stress tolerance and enhances the resistance of S. mutans to killing by macrophages [82]. In addition, ScnRK, which has also been termed HK/RR3, was reported to have a modest effect on acid tolerance, while an apparent ortholog of this system in Streptococcus pyogenes has been found to influence the expression of bacteriocin [83], [84]. Although each of these systems is related to biofilm formation, the only mechanism elucidated up to this point is gene expression of surface proteins. Additional studies are needed to clarify the related mechanisms.

Bacteriocin immunity proteins

One of the principal virulence factors of S. mutans is production of bacteriocins (peptide antibiotics), referred to as mutacins [85]. Although these peptide molecules are not required for growth, they may help the microorganisms that produce them to compete for limited nutrients in their environment [86]. TCSTs are commonly utilized by bacteria to regulate bacteriocin gene expression [87], [88], while several CSP-induced genes have stress-related roles, or participate in synthesis and transport of bacteriocin-like peptides [69], [89]. Two types of bacteriocins have been characterized, lantibiotics and non-lantibiotics. The former are lanthionine-containing small-peptide antibiotics that tend to show a wide spectrum of activities against Gram-positive bacteria, including non-producing strains of S. mutans, while non-lantibiotic bacteriocins, with one or two peptides, have thus far been shown to be primarily active against closely related species [90], [91]. Furthermore, the non-lantibiotic group of bacteriocins contains peptides that do not require modification for their biological activities [92], whereas those in the lantibiotic group possess peptides that require posttranslational modification for their antimicrobial activities [93], [94]. In S. mutans, the ComCDE TCSTS plays a direct role in regulation of a variety of mostly non-lantibiotic bacteriocins. Phosphorylated ComE then activates gene expression by its target bacteriocin promoters, which results in greatly increased bacteriocin production [69], [70], [89], [95], [96].

Producer organisms usually encode specific immunity proteins to protect themselves from the deleterious effects of their own lantibiotics, with immunity protein encoding genes often present within the same lantibiotic biosynthesis operon [97], [98], [99]. Generally, bacteriocin immunity protein (Bip) is an integral membrane proteins that confer protection against certain classes of antimicrobial agents and often enhance stress tolerance [97]. Inactivation of bip genes, which encode Bip, has effects on sensitivity to a variety of antimicrobial agents. Thus, the antimicrobial sensitivity of a bacterium can be modulated by a putative Bip expressed by the organism. These observations have implications regarding the evolution of bip protein genes as well as for potential new chemotherapeutic strategies.

Conflict of interest

The authors have no conflicts of interest to declare in regard to this study.