PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus

Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H₂O₂, whereas Dpr (an Fe²⁺-chelating protein)-dependent H₂O₂ protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H₂O₂-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl₂, S. oligofermentans increased survival after H₂O₂ stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn²⁺-mediated H₂O₂ resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn²⁺) unless it was supplemented with ≥2.5 μM MnCl₂ and caused 82 to 95% reduction of the cellular Mn²⁺ level, while mntABC overexpression increased cellular Mn²⁺ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn²⁺ transporter in S. oligofermentans. mntA mutation reduced the survival of H₂O₂-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H₂O₂-challenged survival 12-fold, indicating that MntABC-mediated Mn²⁺ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H₂O₂ pulsing upregulated mntABC, while H₂O₂-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H₂O₂ can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H₂O₂ stress defenses and may be distributed across all oral streptococci lacking catalase.     

Proteome Analysis Identifies the Dpr Protein of Streptococcus mutans as an Important Factor in the Presence of Early Streptococcal Colonizers of Tooth Surfaces

Oral streptococci are primary colonizers of tooth surfaces and Streptococcus mutans is the principal causative agent of dental caries in humans. A number of proteins are involved in the formation of monospecies biofilms by S. mutans. This study analyzed the protein expression profiles of S. mutans biofilms formed in the presence or absence of S. gordonii, a pioneer colonizer of the tooth surface, by two-dimensional gel electrophoresis (2-DE). After identifying S. mutans proteins by Mass spectrometric analysis, their expression in the presence of S. gordonii was analyzed. S. mutans was inoculated with or without S. gordonii DL1. The two species were compartmentalized using 0.2-μl Anopore membranes. The biofilms on polystyrene plates were harvested, and the solubilized proteins were separated by 2-DE. When S. mutans biofilms were formed in the presence of S. gordonii, the peroxide resistance protein Dpr of the former showed 4.3-fold increased expression compared to biofilms that developed in the absence of the pioneer colonizer. In addition, we performed a competition assay using S. mutans antioxidant protein mutants together with S. gordonii and other initial colonizers. Growth of the dpr-knockout S. mutans mutant was significantly inhibited by S. gordonii, as well as by S. sanguinis. Furthermore, a cell viability assay revealed that the viability of the dpr-defective mutant was significantly attenuated compared to the wild-type strain when co-cultured with S. gordonii. Therefore, these results suggest that Dpr might be one of the essential proteins for S. mutans survival on teeth in the presence of early colonizing oral streptococci.     

Catabolite control protein A controls hydrogen peroxide production and cell death in Streptococcus sanguinis

Streptococcus sanguinis is a commensal oral bacterium producing hydrogen peroxide (H₂O₂) that is dependent on pyruvate oxidase (Spx) activity. In addition to its well-known role in bacterial antagonism during interspecies competition, H₂O₂ causes cell death in about 10% of the S. sanguinis population. As a consequence of H₂O₂-induced cell death, largely intact chromosomal DNA is released into the environment. This extracellular DNA (eDNA) contributes to the self-aggregation phenotype under aerobic conditions. To further investigate the regulation of spx gene expression, we assessed the role of catabolite control protein A (CcpA) in spx expression control. We report here that CcpA represses spx expression. An isogenic ΔccpA mutant showed elevated spx expression, increased Spx abundance, and H₂O₂ production, whereas the wild type did not respond with altered spx expression in the presence of glucose and other carbohydrates. Since H₂O₂ is directly involved in the release of eDNA and bacterial cell death, the presented data suggest that CcpA is a central control element in this important developmental process in S. sanguinis.Initial development of dental biofilms is dominated by oral streptococci, which produce specific adhesins that interact with salivary proteins bathing the teeth and oral mucosa surfaces (29). Biofilm development is a highly competitive process, and different mechanisms are used by individual bacteria to compete with other initial colonizers (17). For example, Streptococcus gordonii binding to salivary components via the surface protein Hsa has been shown to provide a competitive measure during niche competition with Streptococcus sanguinis (30). The excretion of antimicrobial components by oral streptococci as a more aggressive mode of competition has been known for several decades. Bacteriocins produced by cariogenic Streptococcus mutans are effective in inhibiting the growth of several other oral streptococci (10). Conversely, competitive hydrogen peroxide (H₂O₂) production by commensal S. sanguinis and S. gordonii during aerobic growth inhibits S. mutans (18, 20). The enzyme responsible for competitive H₂O₂ production has been identified as pyruvate oxidase (Spx, also referred to as Pox) (5, 20). Isogenic Spx⁻ mutants of S. sanguinis and S. gordonii were unable to inhibit the growth of S. mutans in an in vitro competition assay (20). A similar effective role of pyruvate oxidase dependent H₂O₂ production has been shown in the Streptococcus pneumoniae-Staphylococcus aureus interference (38). Moreover, the inverse association between S. sanguinis and more cariogenic species has been shown in clinical studies, suggesting a protective effect of S. sanguinis colonization resulting in lower caries incidence (1, 3, 6, 43). Although molecular mechanisms of this inverse relationship are not well defined, H₂O₂ production might play a role. The initial colonization process during early biofilm formation occurs when oxygen tension is high enough to allow for respiration and H₂O₂ production (25). With the consequence that H₂O₂ susceptible species might be outcompeted. This has a profound consequence on the overall composition of the biofilm because the initial colonization process influences the spatial and temporal development of the dental biofilm (15). Detailed knowledge of the regulation of pyruvate oxidase-mediated H₂O₂ production could therefore provide important insights into dental biofilm ecology and eventually lead to new ways to promote biofilm development toward a healthy composition. Initial results have shown that the pyruvate oxidases of S. sanguinis and S. gordonii are differentially regulated by glucose, despite a high homology of the promoter region. S. gordonii is not able to inhibit the growth of S. mutans in the presence of glucose, while S. sanguinis inhibiting ability is not affected (20). Furthermore, it was shown that the pyruvate oxidase dependent production of H₂O₂ is correlated with bacterial cell death and the release of extracellular DNA (eDNA). eDNA is an important component of the extracellular matrix in biofilms and in the case of S. sanguinis confers cell-cell adhesion to a certain extent, thus providing evidence that H₂O₂ production not only increases competitiveness but also promotes biofilm development (19).In this report, the regulation of pyruvate oxidase gene expression was further investigated in S. sanguinis. Carbon catabolite control protein A (CcpA) plays a role in spx expression regulation, but the regulation is not influenced by glucose. Gene expression control was also verified on the protein level. Moreover, evidence of CcpA-dependent regulation of cell death is presented in the context of increased H₂O₂ production for a ΔccpA mutant background.     

Identification of A New Gene Responsible for the Oxygen Tolerance in Aerobic Life of Streptococcus mutans

Alkyl hydroperoxide reductase in Streptococcus mutans consists of two components, Nox-1 and AhpC. Deletion of nox-1 and ahpC in a double mutant as well as the wild-type of Streptococcus mutans can form colonies in the presence of air to the same extent. The evidence suggested the presence of some other antioxidant system(s) independent of the Nox-1/AhpC system in the bacterium. Here we identified a new antioxidant gene (dpr) and the gene product (Dpr) which complements the defect of peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. The dpr-disruption mutant of S. mutans could form colonies anaerobically but not aerobically.     

Oxygen exposure increases resistance of Desulfovibrio vulgaris Hildenborough to killing by hydrogen peroxide

Inactivation of PerR by oxidative stress and a corresponding increase in expression of the perR regulon genes is part of the oxidative stress defense in a variety of anaerobic bacteria. Diluted anaerobic, nearly sulfide-free cultures of mutant and wild-type Desulfovibrio vulgaris (10⁵–10⁶ colony-forming units/ml) were treated with 0 to 2,500 μM H₂O₂ for only 5 min to prevent readjustment of gene expression. Survivors were then scored by plating. The wild type and perR mutant had 50% survival at 58 and 269 μM H₂O₂, respectively, indicating the latter to be 4.6-fold more resistant to killing by H₂O₂ under these conditions. Significantly increased resistance of the wild type (38-fold; 50% killing at 2188 μM H₂O₂) was observed if cells were pretreated with full air for 30 min, conditions that did not affect cell viability. The resistance of the perR mutant increased less (4.6-fold; 50% killing at 1230 μM H₂O₂), when similarly pretreated. Interestingly, no increased resistance of either was achieved by exposure with 10.6 μM H₂O₂ for 30 min, the highest concentration that could be used without killing the cells. Hence, in environments with low D. vulgaris biomass only the presence of external O₂ effectively activates the perR regulon. As a result, mutant strains lacking one of the perR regulon genes ahpC, dvu0772, rbr1 or rbr2 displayed decreased resistance to H₂O₂ stress only following pretreatment with air.     

Competition and coexistence between Streptococcus mutans and Streptococcus sanguinis in the dental biofilm

The human mucosal surface is colonized by the indigenous microflora, which normally maintains an ecological balance among different species. Certain environmental or biological factors, however, may trigger disruption of this balance, leading to microbial diseases. In this study, we used two oral bacterial species, Streptococcus mutans and Streptococcus sanguinis (formerly S. sanguis), as a model to probe the possible mechanisms of competition/coexistence between different species which occupy the same ecological niche. We show that the two species engage in a multitude of antagonistic interactions temporally and spatially; occupation of a niche by one species precludes colonization by the other, while simultaneous colonization by both species results in coexistence. Environmental conditions, such as cell density, nutritional availability, and pH, play important roles in determining the outcome of these interactions. Genetic and biochemical analyses reveal that these interspecies interactions are possibly mediated through a well-regulated production of chemicals, such as bacteriocins (produced by S. mutans) and hydrogen peroxide (produced by S. sanguinis). Consistent with the phenotypic characteristics, production of bacteriocins and H₂O₂ are regulated by environmental conditions, as well as by juxtaposition of the two species. These sophisticated interspecies interactions could play an essential part in balancing competition/coexistence within multispecies microbial communities.     

A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans

The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H₂O₂ via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)–ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens.     

Antimicrobial effects of herbal extracts on Streptococcus mutans and normal oral streptococci

Streptococcus mutans is associated with dental caries. A cariogenic biofilm, in particular, has been studied extensively for its role in the formation of dental caries. Herbal extracts such as Cudrania tricuspidata, Sophora flavescens, Ginkgo biloba, and Betula Schmidtii have been used as a folk remedy for treating diseases. The purpose of this study was to evaluate and compare the antibacterial activity of herbal extracts against normal oral streptococci, planktonic and biofilm of S. mutans. Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius, Streptococcus sanguinis, and S. mutans were cultivated with brain heart infusion broth and susceptibility assay for the herbal extracts was performed according to the protocol of Clinical and Laboratory Standard Institute. Also, S. mutans biofilm was formed on a polystyrene 12-well plate and 8-well chamber glass slip using BHI broth containing 2% sucrose and 1% mannose after conditioning the plate and the glass slip with unstimulated saliva. The biofilm was treated with the herbal extracts in various concentrations and inoculated on Mitis-Salivarius bacitracin agar plate for enumeration of viable S. mutans by counting colony forming units. Planktonic S. mutans showed susceptibility to all of the extracts and S. mutans biofilm exhibited the highest level of sensitivity for the extracts of S. flavescens. The normal oral streptococci exhibited a weak susceptibility in comparison to S. mutans. S. oralis, however, was resistant to all of the extracts. In conclusion, the extract of S. flavescens may be a potential candidate for prevention and management of dental caries.     

Hydrogen Peroxide Contributes to the Epithelial Cell Death Induced by the Oral Mitis Group of Streptococci

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H₂O₂) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H₂O₂ may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H₂O₂-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H₂O₂, inhibited S. oralis cytotoxicity, and H₂O₂ alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H₂O₂ production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H₂O₂ induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H₂O₂ is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts.     

Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

Background

Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis.

Results

As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes α1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis.

Conclusions

These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.     

Identification of Anion Channels Responsible for Fluoride Resistance in Oral Streptococci

Recently, it has been reported that eriC and crcB are involved in bacterial fluoride resistance. However, the fluoride-resistance mechanism in oral streptococci remains unclear. BLAST studies showed that two types of eriCs (eriC1 and eriC2) and two types of crcBs (crcB1 and crcB2) are present across 18 oral streptococci, which were identified in ≥ 10% of 166 orally healthy subjects with ≥ 0.01% of the mean relative abundance. They were divided into three groups based on the distribution of these four genes: group I, only eriC1; group II, eriC1 and eriC2; and group III, eriC2, crcB1, and crcB2. Group I consisted of Streptococcus mutans, in which one of the two eriC1s predominantly affected fluoride resistance. Group II consisted of eight species, and eriC1 was responsible for fluoride resistance, but eriC2 was not, in Streptococcus anginosus as a representative species. Group III consisted of nine species, and both crcB1 and crcB2 were crucial for fluoride resistance, but eriC2 was not, in Streptococcus sanguinis as a representative species. Based on these results, either EriC1 or CrcBs play a role in fluoride resistance in oral streptococci. Complementation between S. mutans EriC1 and S. sanguinis CrcB1/CrcB2 was confirmed in both S. mutans and S. sanguinis. However, neither transfer of S. sanguinis CrcB1/CrcB2 into wild-type S. mutans nor S. mutans EriC1 into wild-type S. sanguinis increased the fluoride resistance of the wild-type strain. Co-existence of different F⁻ channels (EriC and CrcB) did not cause the additive effect on fluoride resistance in oral Streptococcus species.     

Staphylococcus aureus PerR Is a Hypersensitive Hydrogen Peroxide Sensor using Iron-mediated Histidine Oxidation

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerR_BS protein, represses target genes when bound to either Mn²⁺ or Fe²⁺ as corepressor, but only the Fe²⁺-bound form responds to H₂O₂. The orthologous protein in the human pathogen Staphylococcus aureus, PerR_SA, plays important roles in H₂O₂ resistance and virulence. However, PerR_SA is reported to only respond to Mn²⁺ as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H₂O₂ sensing. Here we demonstrate that PerR_SA uses either Fe²⁺ or Mn²⁺ as corepressor, and that, like PerR_BS, the Fe²⁺-bound form of PerR_SA senses physiological levels of H₂O₂ by iron-mediated histidine oxidation. Moreover, we show that PerR_SA is poised to sense very low levels of endogenous H₂O₂, which normally cannot be sensed by B. subtilis PerR_BS. This hypersensitivity of PerR_SA accounts for the apparent lack of Fe²⁺-dependent repressor activity and consequent Mn²⁺-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerR_SA is directly correlated with virulence, whereas it is inversely correlated with H₂O₂ resistance, suggesting that PerR_SA may be an attractive target for the control of S. aureus pathogenesis.     

Complementation of the Fo c Subunit of Escherichia coli with That of Streptococcus mutans and Properties of the Hybrid FoF1 ATP Synthase

The c subunit of Streptococcus mutans ATP synthase (F_oF₁) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid F_oF₁ is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F₁ bound to the hybrid F_o with the S. mutans c subunit showed N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli F_oF₁. Thus, the S. mutans c subunit assembled into a functional F_o together with the E. coli a and b subunits, forming a normal F₁ binding site. Although the H⁺ pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H⁺ transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H⁺ transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid F_o with the S. mutans c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans.     

Reduced Glutathione Mediates Resistance to H2S Toxicity in Oral Streptococci

Periodontal disease is associated with changes in the composition of the oral microflora, where health-associated oral streptococci decrease while Gram-negative anaerobes predominate in disease. A key feature of periodontal disease-associated anaerobes is their ability to produce hydrogen sulfide (H₂S) abundantly as a by-product of anaerobic metabolism. So far, H₂S has been reported to be either cytoprotective or cytotoxic by modulating bacterial antioxidant defense systems. Although oral anaerobes produce large amounts of H₂S, the potential effects of H₂S on oral streptococci are currently unknown. The aim of this study was to determine the effects of H₂S on the survival and biofilm formation of oral streptococci. The growth and biofilm formation of Streptococcus mitis and Streptococcus oralis were inhibited by H₂S. However, H₂S did not significantly affect the growth of Streptococcus gordonii or Streptococcus sanguinis. The differential susceptibility of oral streptococci to H₂S was attributed to differences in the intracellular concentrations of reduced glutathione (GSH). In the absence of GSH, H₂S elicited its toxicity through an iron-dependent mechanism. Collectively, our results showed that H₂S exerts antimicrobial effects on certain oral streptococci, potentially contributing to the decrease in health-associated plaque microflora.     

Sphingosine-1-phosphate (S1P) mediates darkness-induced stomatal closure through raising cytosol pH and hydrogen peroxide (H2O2) levels in guard cells in Vicia faba

The role and signaling of sphingosine-1-phosphate (S1P) during darkness-induced stomatal closure were examined in Vicia faba. Darkness substantially raised S1P and hydrogen peroxide (H₂O₂) levels and closed stomata. These darkness effects were significantly suppressed by DL-threo-dihydrosphingosine (DL-threo-DHS) and N,N-dimethylsphingosine (DMS), two inhibitors of long-chain base kinases. Exogenous S1P led to stomatal closure and H₂O₂ production, and the effects of S1P were largely prevented by the H₂O₂ modulators ascorbic acid, catalase, and diphenyleneiodonium. These results indicated that S1P mediated darkness-induced stomatal closure by triggering H₂O₂ production. In addition, DL-threo-DHS and DMS significantly suppressed both darkness-induced cytosolic alkalization in guard cells and stomatal closure. Exogenous S1P caused cytosolic alkalization and stomatal closure, which could be largely abolished by butyric acid. These results demonstrated that S1P synthesis was necessary for cytosolic alkalization during stomatal closure caused by darkness. Furthermore, together with the data described above, inhibition of darkness-induced H₂O₂ production by butyric acid revealed that S1P synthesis-induced cytosolic alkalization was a prerequisite for H₂O₂ production during stomatal closure caused by darkness, a conclusion supported by the facts that the pH increase caused by exogenous S1P had a shorter lag and peaked faster than H₂O₂ levels and that butyric acid prevented exogenous S1P-induced H₂O₂ production. Altogether, our data suggested that darkness induced S1P synthesis, causing cytosolic alkalization and subsequent H₂O₂ production, finally leading to stomatal closure.