Streptococcus mutans NADH oxidase lies at the intersection of overlapping regulons controlled by oxygen and NAD+ levels.

NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD(+). The critical nature of Nox is 2-fold: it serves to regenerate NAD(+), a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD(+) have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD(+) affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress.

BrpA is involved in regulation of cell envelope stress responses in Streptococcus mutans.

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by Streptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of brpA is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a Galleria mellonella (wax worm) model, BrpA deficiency was shown to diminish the virulence of S. mutans OMZ175, which, unlike S. mutans UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of S. mutans.

The Biology of Streptococcus mutans.

As a major etiological agent of human dental caries, Streptococcus mutans resides primarily in biofilms that form on the tooth surfaces, also known as dental plaque. In addition to caries, S. mutans is responsible for cases of infective endocarditis with a subset of strains being indirectly implicated with the onset of additional extraoral pathologies. During the past 4 decades, functional studies of S. mutans have focused on understanding the molecular mechanisms the organism employs to form robust biofilms on tooth surfaces, to rapidly metabolize a wide variety of carbohydrates obtained from the host diet, and to survive numerous (and frequent) environmental challenges encountered in oral biofilms. In these areas of research, S. mutans has served as a model organism for ground-breaking new discoveries that have, at times, challenged long-standing dogmas based on bacterial paradigms such as Escherichia coli and Bacillus subtilis. In addition to sections dedicated to carbohydrate metabolism, biofilm formation, and stress responses, this article discusses newer developments in S. mutans biology research, namely, how S. mutans interspecies and cross-kingdom interactions dictate the development and pathogenic potential of oral biofilms and how next-generation sequencing technologies have led to a much better understanding of the physiology and diversity of S. mutans as a species.

Biology of Oral Streptococci.

Bacteria belonging to the genus Streptococcus are the first inhabitants of the oral cavity, which can be acquired right after birth and thus play an important role in the assembly of the oral microbiota. In this article, we discuss the different oral environments inhabited by streptococci and the species that occupy each niche. Special attention is given to the taxonomy of Streptococcus, because this genus is now divided into eight distinct groups, and oral species are found in six of them. Oral streptococci produce an arsenal of adhesive molecules that allow them to efficiently colonize different tissues in the mouth. Also, they have a remarkable ability to metabolize carbohydrates via fermentation, thereby generating acids as byproducts. Excessive acidification of the oral environment by aciduric species such as Streptococcus mutans is directly associated with the development of dental caries. However, less acid-tolerant species such as Streptococcus salivarius and Streptococcus gordonii produce large amounts of alkali, displaying an important role in the acid-base physiology of the oral cavity. Another important characteristic of certain oral streptococci is their ability to generate hydrogen peroxide that can inhibit the growth of S. mutans. Thus, oral streptococci can also be beneficial to the host by producing molecules that are inhibitory to pathogenic species. Lastly, commensal and pathogenic streptococci residing in the oral cavity can eventually gain access to the bloodstream and cause systemic infections such as infective endocarditis.

Expression of BrpA in Streptococcus mutans is regulated by FNR-box mediated repression.

Our previous studies showed that brpA in Streptococcus mutans, which encodes a member of the LytR-CpsA-Psr family of proteins, can be co-transcribed with brpB upstream as a bicistronic operon, and the intergenic region also has strong promoter activity. To elucidate how brpA expression is regulated, the promoter regions were analyzed using polymerase chain reaction-based deletions and site-directed mutagenesis and a promoterless luciferase gene as a reporter. Allelic exchange mutagenesis was also used to examine genes encoding putative trans-acting factors, and the impact of such mutations on brpA expression was analyzed by reporter assays. Multiple elements in the short brpA promoter (nucleotide -1 to -344 relative to start cordon ATG) were shown to have a major impact on brpA expression, including an FNR-box, for a putative binding site of an FNR-type of transcriptional regulator. When compared with the intact brpA promoter, mutations of the highly conserved nucleotides in FNR-box from TTGATgtttAcCtt to TTACAgaaaGtTac resulted in 1362-fold increases of luciferase activity (P < .001), indicative of the FNR-box-mediated repression as a major mechanism in regulation of brpA expression. When luciferase reporter was fused to the upstream brpBA promoter (nucleotides -784 to -1144), luciferase activity was decreased by 4.5-fold (P < .001) in the brpA mutant, TW14D, and by 67.7-fold (P < .001) in the brpB mutant, JB409, compared with the wild-type, UA159. However, no such effects were observed when the reporter gene was fused to the short brpA promoter and its derivatives. These results also suggest that brpA expression in S. mutans is auto-regulated through the upstream brpBA promoter.

Streptococcus mutans: fructose transport, xylitol resistance, and virulence.

Streptococcus mutans, the primary etiological agent of human dental caries, possesses at least two fructose phosphotransferase systems (PTSs), encoded by fruI and fruCD. fruI is also responsible for xylitol transport. We hypothesized that fructose and xylitol transport systems do not affect virulence. Thus, colonization and cariogenicity of fruI(-) and fruCD(-) single and double mutants, their WT (UA159), and xylitol resistance (X(r)) of S. mutans were studied in rats fed a high-sucrose diet. A sucrose phosphorylase (gtfA(-)) mutant and a reference strain (NCTC-10449S) were additional controls. Recoveries of fruI mutant from the teeth were decreased, unlike those for the other strains. The fruCD mutation was associated with a slight loss of cariogenicity on enamel, whereas mutation of fruI was associated with a loss of cariogenicity in dentin. These results also suggest why xylitol inhibition of caries is paradoxically associated with spontaneous emergence of so-called X(r) S. mutans in habitual human xylitol users.

Transcription factor Rex in regulation of pathophysiology in oral pathogens.

The NAD(+) and NADH-sensing transcriptional regulator Rex is widely conserved across gram-positive bacteria. Rex monitors cellular redox poise and controls the expression of genes/operons involved in diverse pathways including alternative fermentation, oxidative stress responses, and biofilm formation. The oral cavity undergoes frequent and drastic fluctuations in nutrient availability, pH, temperature, oxygen tension, saliva, and shear forces. The oral streptococci are major colonizers of oral mucosa and tooth surfaces and include commensals as well as opportunistic pathogens, including the primary etiological agent of dental caries, Streptococcus mutans. Current understanding of the Rex regulon in oral bacteria is mostly based on studies in S. mutans and endodontic pathogen Enterococcus faecalis. Indeed, other oral bacteria encode homologs of the Rex protein and much is to be gleaned from more in-depth studies. Our current understanding has Rex positioned at the interface of oxygen and energy metabolism. In biofilms, heterogeneous oxygen tension influences the ratio of intracellular NADH and NAD(+) , which is finely tuned through glycolysis and fermentation. In S. mutans, Rex regulates the expression of glycolytic enzyme NAD(+) -dependent glyceraldehyde 3-phosphate dehydrogenase, and NADH-dependent fermentation enzymes/complexes lactate dehydrogenase, pyruvate dehydrogenase, alcohol-acetaldehyde dehydrogenase, and fumarate reductase. In addition, Rex controls the expression of NADH oxidase, a major enzyme used to eliminate oxidative stress and regenerate NAD(+) . Here, we summarize recent studies carried out on the Rex regulators in S. mutans and E. faecalis. This research has important implications for understanding how Rex monitors redox balance and optimizes fermentation pathways for survival and subsequent pathogenicity.

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.

Characterization of two operons that encode components of fructose-specific enzyme II of the sugar:phosphotransferase system of Streptococcus mutans.

Three genes, designated as fruC, fruD and fruI, were predicted to encode polypeptides homologous to fructose-specific enzyme II (II(Fru)) of the phosphoenolpyruvate-dependent sugar:phosphotransferase system, and were cloned from Streptococcus mutans, the primary etiological agent of human dental caries. The fruC and fruD genes encoded domains BC and domain A of II(Fru), respectively. The fruI gene encoded IICBA(Fru). Northern hybridization and slot blot analysis showed that expression of fruI was inducible by sucrose and fructose, while fruCD were expressed constitutively and at much lower levels. Inactivation of either fruI or fruCD alone, or of both fruCD and fruI, had no major impact on growth on fructose at a concentration of 0.5% (w/v). However, when the strains were grown with 0.2% fructose as the sole carbohydrate source, a significant decrease in the growth rate was seen with the fruCD/fruI double mutants. Assays of sugar:phosphotransferase activity showed that the fruCD/fruI double mutants had roughly 30% of the capacity of the wild-type strain to transport fructose via the phosphoenolpyruvate-dependent sugar:phosphotransferase system. Xylitol toxicity assays indicated that the inducible fructose permease was responsible for xylitol transport.

Antibacterial dental composites with chlorhexidine and mesoporous silica.

One of the leading causes for the failure of dental composite restorations is secondary caries. Effectively inhibiting cariogenic biofilms and reducing secondary caries could extend the service life of composite restorations. Dental composites releasing antibacterial agents such as chlorhexidine (CHX) have shown biofilm-inhibitory efficacy, but they usually have poor physical and mechanical properties. Herein, we present a study of a new method to encapsulate and release CHX from dental composite using mesoporous silica nanoparticles (MSNs). SBA-15 MSNs were synthesized according to a reported procedure. CHX (62.9 wt%) was encapsulated into dried MSN from 0.3 M CHX ethanol solution. The dental composites containing 0% (control), 3%, 5%, and 6.3% CHX or the same amounts of CHX entrapped in MSN (denoted as CHX@MSN) were fabricated with methacrylate monomers and silanized glass fillers (CHX or CHX@MSN + glass filler particle = 70 wt%). The monomer mixture consisted of bisphenol A glycidyl methacrylate (BisGMA), hexanediol dimethacrylate (HDDMA), ethoxylated bisphenol A dimethacrylate (EBPADMA), and urethane dimethacrylates (UEDMA) at a weight ratio of 40:30:20:10. The composites were tested for CHX release and recharge, flexural strength and modulus (at 24 hr and 1 mo), surface roughness, in vitro wear, and antibacterial activity against Streptococcus mutans and Lactobacillus casei (in both planktonic growth and biofilm formation). The results showed that the composites with CHX@MSN largely retained mechanical properties and smooth surfaces and showed controlled release of CHX over a long time. In contrast, the composites with directly mixed CHX showed reduced mechanical properties, rough surfaces, and burst release of CHX in a short time. The composites with CHX either directly mixed or in MSN showed strong inhibition to S. mutans and L. casei. This research has demonstrated the successful application of MSNs as a novel nanotechnology in dental materials to inhibit oral biofilm without sacrificing materials' mechanical properties and surface integrity.

Transcriptome analysis of LuxS-deficient Streptococcus mutans grown in biofilms.

We previously reported that LuxS in Streptococcus mutans is involved in stress tolerance and biofilm formation. In this study, flowcells and confocal laser scanning microscopy were used to further examine the effects of LuxS-deficiency on biofilm formation. Similar to the wild-type strain (UA159), a strain deficient in LuxS (TW26D) bound efficiently to the flowcells and formed microcolonies 4 h after inoculation. Unlike UA159, which accumulated and formed compact, evenly distributed biofilms after 28 h, TW26D showed only loose, sporadic, thin biofilms. DNA microarray analysis revealed alterations in transcription of more than 60 genes in TW26D biofilms by at least 1.5-fold (P < 0.001). Among the upregulated genes were those for sugar-specific enzymes II of the phosphotransferase (PTS) system and the atp operon, which codes for the proton-pumping F-ATPase. Of the downregulated genes, several encode proteins with putative functions in DNA repair. Mutation of selected genes caused severe defects in the ability of the mutants to tolerate low pH and oxidative stress. These results provide additional proof that LuxS-deficiency causes global alterations in the expression of genes central to biofilm formation and virulence of S. mutans, including those involved in energy metabolism, DNA repair and stress tolerance.

Construction of a new integration vector for use in Streptococcus mutans.

A new integration vector, pBGK, was constructed for delivery of heterologous DNA into the chromosome of Streptococcus mutans. A kanamycin resistance element (OmegaKm), which is flanked by transcriptional and translational terminators and which is selectable in both Escherichia coli and streptococci, was inserted into a 2.4-kb EcoRI fragment carrying the S. mutans gtfA gene. A unique SmaI site flanking OmegaKm is available for cloning of promoter:reporter gene fusions or foreign genes, which can then be integrated into the S. mutans chromosome by allelic exchange with the gtfA gene. The vector was used to analyze the activity of an S. mutans promoter by fusing it to a chloramphenicol acetyltransferase gene. The reporter fusions could readily be cloned into the vector at a unique SmaI site and the vector and passenger DNA were stable in E. coli. DNAs flanked by gtfA sequees integrated efficiently into the chromosome of S. mutans and were stably maintained in the absence of selective pressure. Expression levels of the reporter gene were consistent regardless of orientation or repeated subculturing.

Regulation of expression of the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and carbon catabolite repression.

The polymers of fructose, levan and inulin, as well as sucrose and raffinose, are substrates for the product of the fruA gene of Streptococcus mutans GS-5. The purpose of this study was to characterize the DNA immediately flanking fruA, to explore the regulation of expression of fruA by the carbohydrate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3' to fruA was an open reading frame (ORF) with similarity to beta-fructosidases which was cotranscribed with fruA. A transcriptional initiation site, located an appropriate distance from an extended -10-like promoter, was mapped at 165 bp 5' to the fruA structural gene. By the use of computer algorithms, two overlapping, stable stem-loop sequences with the potential to function as rho-independent terminators were found in the 5' untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by functioning as negative cis elements in gram-positive bacteria, were located close to the promoter. The levels of production of fruA mRNA and FruA were elevated in cells growing on levan, inulin, or sucrose as the sole carbohydrate source, and repression was observed when cells were grown on readily metabolizable hexoses. Deletion derivatives containing fusions of fruA promoter regions, lacking sequences 5' or 3' to the promoter, and a promoterless chloramphenicol acetyltransferase gene were used (i) to demonstrate the functionality of the promoter mapped by primer extension, (ii) to demonstrate that CCR of the fru operon requires the CRE that is located 3' to the promoter region, and (iii) to provide preliminary evidence that supports the involvement of an antitermination mechanism in fruA induction.