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.     

Environment and Colonisation Sequence Are Key Parameters Driving Cooperation and Competition between Pseudomonas aeruginosa Cystic Fibrosis Strains and Oral Commensal Streptococci

Cystic fibrosis (CF) patient airways harbour diverse microbial consortia that, in addition to the recognized principal pathogen Pseudomonas aeruginosa, include other bacteria commonly regarded as commensals. The latter include the oral (viridans) streptococci, which recent evidence indicates play an active role during infection of this environmentally diverse niche. As the interactions between inhabitants of the CF airway can potentially alter disease progression, it is important to identify key cooperators/competitors and environmental influences if therapeutic intervention is to be improved and pulmonary decline arrested. Importantly, we recently showed that virulence of the P. aeruginosa Liverpool Epidemic Strain (LES) could be potentiated by the Anginosus-group of streptococci (AGS). In the present study we explored the relationships between other viridans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus sanguinis) and the LES and observed that co-culture outcome was dependent upon inoculation sequence and environment. All four streptococcal species were shown to potentiate LES virulence factor production in co-culture biofilms. However, in the case of S. oralis interactions were environmentally determined; in air cooperation within a high cell density co-culture biofilm occurred together with stimulation of LES virulence factor production, while in an atmosphere containing added CO₂ this species became a competitor antagonising LES growth through hydrogen peroxide (H₂O₂) production, significantly altering biofilm population dynamics and appearance. Streptococcus mitis, S. gordonii and S. sanguinis were also capable of H2O2 mediated inhibition of P. aeruginosa growth, but this was only visible when inoculated as a primary coloniser prior to introduction of the LES. Therefore, these observations, which are made in conditions relevant to the biology of CF disease pathogenesis, show that the pathogenic and colonisation potential of P. aeruginosa isolates can be modulated positively and negatively by the presence of oral commensal streptococci.     

dpr and sod in Streptococcus mutans Are Involved in Coexistence with S. sanguinis,and PerR Is Associated with Resistance to H2O2

Large numbers of bacteria coexist in the oral cavity. Streptococcus sanguinis, one of the major bacteria in dental plaque, produces hydrogen peroxide (H₂O₂), which interferes with the growth of other bacteria. Streptococcus mutans, a cariogenic bacterium, can coexist with S. sanguinis in dental plaque, but to do so, it needs a means of detoxifying the H₂O₂ produced by S. sanguinis. In this study, we investigated the association of three oxidative stress factors, Dpr, superoxide dismutase (SOD), and AhpCF, with the resistance of S. sanguinis to H₂O₂. The knockout of dpr and sod significantly increased susceptibility to H₂O₂, while the knockout of ahpCF had no apparent effect on susceptibility. In particular, dpr inactivation resulted in hypersensitivity to H₂O₂. Next, we sought to identify the factor(s) involved in the regulation of these oxidative stress genes and found that PerR negatively regulated dpr expression. The knockout of perR caused increased dpr expression levels, resulting in low-level susceptibility to H₂O₂ compared with the wild type. Furthermore, we evaluated the roles of perR, dpr, and sod when S. mutans was cocultured with S. sanguinis. Culturing of the dpr or sod mutant with S. sanguinis showed a significant decrease in the S. mutans population ratio compared with the wild type, while the perR mutant increased the ratio. Our results suggest that dpr and sod in S. mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H₂O₂ in regulating the expression of Dpr.     

Antimicrobial Effects of Peptides from Human Beta-Defensin-3 on Planktonic and Biofilm States of Streptococci

Human beta-defensin-3 (hBD3) acts as a first line of defense against both Gram-positive and Gram-negative bacteria infection. Streptococci are the significant cause for oral biofilm associated diseases. We synthesized three fragments (hBD3-1, hBD3-2, hBD3-3) from the hBD3 and evaluated the antibacterial efficacy on oral streptococci. All of the three fragments from hBD3 had good estimated solubility and hBD3-3 had a higher net positive charge than others. Structure analysis showed that the three fragments shared stable β-sheet structure, but tyrosine were not found in hBD3-2 and hBD3-3 by using Raman and circular dichroism spectroscopy. The inhibition ability of the peptides was examined on the bioactivity of Streptococcus oralis (S.oralis), Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii) by minimal inhibitory concentration, minimum bactericidal concentration and anti-biofilm formation test. Three fragments had antimicrobial activity on planktonic state of streptococci, and S. oralis had much more sensitive to the three peptides. Results of antibiofilm experiment showed that streptococci biofilm formation was more sensitive to hBD3-3. Confocal laser scanning microscopy and scanning electron microscopy showed the decrease of biomass and bacterial morphology destruction, which indicated that the antimicrobial mechanism of hBD3-3 might involve an electrostatic charge-based impact on membrane permeability. In conclusion, hBD3-3 possessed the potential capacity for depressing the growth of bacteria, especially first colonizers during the development of oral biofilm. Powerful, endogenous antimicrobial peptide provides the potential to interfere with biofilm by disorganizing early biofilm formation and thereby inhibiting biofilm-associated diseases.     

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.     

Mitis group streptococci express variable pilus islet 2 pili

Background

Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguinis are members of the Mitis group of streptococci and agents of oral biofilm, dental plaque and infective endocarditis, disease processes that involve bacteria-bacteria and bacteria-host interactions. Their close relative, the human pathogen S. pneumoniae uses pilus-islet 2 (PI-2)-encoded pili to facilitate adhesion to eukaryotic cells.

Methodology/Principal Findings

PI-2 pilus-encoding genetic islets were identified in S. oralis, S. mitis, and S. sanguinis, but were absent from other isolates of these species. The PI-2 islets resembled the genetic organization of the PI-2 islet of S. pneumoniae, but differed in the genes encoding the structural pilus proteins PitA and PitB. Two and three variants of pitA (a pseudogene in S. pneumoniae) and pitB, respectively, were identified that showed ≈20% difference in nucleotide as well as corresponding protein sequence. Species-independent combinations of pitA and pitB variants indicated prior intra- and interspecies horizontal gene transfer events. Polyclonal antisera developed against PitA and PitB of S. oralis type strain ATCC35037 revealed that PI-2 pili in oral streptococci were composed of PitA and PitB. Electronmicrographs showed pilus structures radiating >700 nm from the bacterial surface in the wild type strain, but not in an isogenic PI-2 deletion mutant. Anti-PitB-antiserum only reacted with pili containing the same PitB variant, whereas anti-PitA antiserum was cross-reactive with the other PitA variant. Electronic multilocus sequence analysis revealed that all PI-2-encoding oral streptococci were closely-related and cluster with non-PI-2-encoding S. oralis strains.

Conclusions/Significance

This is the first identification of PI-2 pili in Mitis group oral streptococci. The findings provide a striking example of intra- and interspecies horizontal gene transfer. The PI-2 pilus diversity provides a possible key to link strain-specific bacterial interactions and/or tissue tropisms with pathogenic traits in the Mitis group streptococci.     

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.     

Mechanisms underlying interactions between two abundant oral commensal bacteria

Complex polymicrobial biofilm communities are abundant in nature particularly in the human oral cavity where their composition and fitness can affect health. While the study of these communities during disease is essential and prevalent, little is known about interactions within the healthy plaque community. Here we describe interactions between two of the most abundant species in this healthy microbiome, Haemophilus parainfluenzae and Streptococcus mitis. We discovered that H. parainfluenzae typically exists adjacent to mitis group streptococci in vivo with which it is also positively correlated based on microbiome data. By comparing in vitro coculture data to ex vivo microscopy we revealed that this co-occurrence is density dependent and further influenced by H₂O₂ production. We discovered that H. parainfluenzae utilizes a more redundant, multifactorial response to H₂O₂ than related microorganisms and that this system’s integrity enhances streptococcal fitness. Our results indicate that mitis group streptococci are likely the in vivo source of NAD for H. parainfluenzae and also evoke patterns of carbon utilization in vitro for H. parainfluenzae similar to those observed in vivo. Our findings describe mechanistic interactions between two of the most abundant and prevalent members of healthy supragingival plaque that contribute to their in vivo survival.Subject terms: Microbiome, Bacterial physiology, Bacteriology, Biofilms, Microbial ecology     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

Genotypic Heterogeneity of Streptococcus oralis and Distinct Aciduric Subpopulations in Human Dental Plaque

The genotypic heterogeneity of Streptococcus oralis isolated from the oral cavity was investigated using repetitive extragenic palindromic PCR. Unrelated subjects harbored unique genotypes, with numerous genotypes being isolated from an individual. S. oralis is the predominant aciduric bacterium isolated from noncarious tooth sites. Genotypic comparison of the aciduric populations isolated at pH 5.2 with those isolated from mitis-salivarius agar (MSA) (pH 7.0) indicated that the aciduric populations were genotypically distinct in the majority of subjects (χ² = 13.09; P = 0.0031). Neither the aciduric nor the MSA-isolated strains were stable, with no strains isolated at baseline being isolated 4 or 12 weeks later in the majority of subjects. The basis of this instability is unknown but is similar to that reported for Streptococcus mitis. Examination of S. oralis strains isolated from cohabiting couples demonstrated that in three of five couples, genotypically identical strains were isolated from both partners and this was confirmed by using Salmonella enteritidis repetitive element PCR and enterobacterial PCR typing. These data provide further evidence of the physiological and genotypic heterogeneity of non-mutans streptococci. The demonstration of distinct aciduric populations of S. oralis implies that the role of these and other non-mutans streptococci in the caries process requires reevaluation.     

Distribution of Streptococcus troglodytae and Streptococcus dentirousetti in chimpanzee oral cavities

The aim of this study was to analyze the distribution and phenotypic properties of the indigenous streptococci in chimpanzee (Pan troglodytes) oral cavities. Eleven chimpanzees (aged from 9 to 44 years, mean ± SD, 26.9 ± 12.6 years) in the Primate Research Institute of Kyoto University were enrolled in this research and brushing bacterial samples collected from them. Streptococci were isolated from the oral cavities of all chimpanzees. The isolates (n = 46) were identified as thirteen species by 16S rRNA genes analysis. The predominant species was Streptococcus sanguinis of mitis streptococci from five chimpanzees (45%). Mutans streptococci were isolated from six chimpanzees (55%). The predominant species in the mutans streptococci were Streptococcus troglodytae from four chimpanzees (36%), this species having been proposed as a novel species by us, and Streptococcus dentirousetti from three chimpanzees (27%). Streptococcus mutans was isolated from one chimpanzee (9%). However, Streptococcus sobrinus, Streptococcus macacae and Streptococcus downei, which are indigenous to human and monkey (Macaca fasciclaris) oral habitats, were not isolated. Of the mutans streptococci, S. troglodytae, S. dentirousetti, and S. mutans possessed strong adherence activity to glass surface.     

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.     

Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway

Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine–threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine–threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine–threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells.     

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.     

Effect of Acidic pH on Expression of Surface-Associated Proteins of Streptococcus oralis

Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.     

Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg²⁺ and Ca²⁺ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

Characterization of Hydrogen Peroxide-Induced DNA Release by Streptococcus sanguinis and Streptococcus gordonii

Extracellular DNA (eDNA) is produced by several bacterial species and appears to contribute to biofilm development and cell-cell adhesion. We present data showing that the oral commensals Streptococcus sanguinis and Streptococcus gordonii release DNA in a process induced by pyruvate oxidase-dependent production of hydrogen peroxide (H₂O₂). Surprisingly, S. sanguinis and S. gordonii cell integrity appears unaffected by conditions that cause autolysis in other eDNA-producing bacteria. Exogenous H₂O₂ causes release of DNA from S. sanguinis and S. gordonii but does not result in obvious lysis of cells. Under DNA-releasing conditions, cell walls appear functionally intact and ribosomes are retained over time. During DNA release, intracellular RNA and ATP are not coreleased. Hence, the release mechanism appears to be highly specific for DNA. Release of DNA without detectable autolysis is suggested to be an adaptation to the competitive oral biofilm environment, where autolysis could create open spaces for competitors to invade. Since eDNA promotes cell-to-cell adhesion, release appears to support oral biofilm formation and facilitates exchange of genetic material among competent strains.The release of bacterial DNA into the environment is of recent interest since this polymer is now recognized to stabilize cell-to-cell adherence and biofilm architecture (1, 35, 37). Treatment of extracellular DNA (eDNA) with DNase results in reduced intercellular stickiness, consistent with an adhesive function for eDNA. Furthermore, eDNA from Neisseria meningitis appears to have sufficient structural integrity to transform competent strains (11), indicating chromosomal origin. Since the abundance of eDNA is influenced by growth conditions, DNA release can also be regulated (40).DNA release is typically a consequence of cell lysis. Linked to DNA release, genetic transformation is the natural ability of competent bacterial species to take up DNA from the environment (13, 34, 42). During competence development, Streptococcus pneumoniae DNA is released by lysis of a subpopulation of cells (30, 42). Cell lysis and DNA release are controlled in a cell density-dependent signal transduction process. The S. pneumoniae comX regulon, carrying late competence genes, also includes the murein hydrolase genes lytA and cbpD (19, 42). Murein hydrolases digest structural components of the peptidoglycan, contributing to remodeling, recycling, and daughter cell separation. Furthermore, murein hydrolases trigger autolytic cell wall digestion, leading to release of DNA and other cellular content into the environment (36). The autolysis of bacterial cells as part of a regulated death program seems to be an important source for eDNA in diverse species, including Staphylococcus aureus (4, 36, 37), Staphylococcus epidermidis (35), Enterococcus faecalis (44), and Pseudomonas aeruginosa (1). In these species, the eDNA contributes to biofilm formation as a component of the extracellular biofilm matrix (35, 37, 44).Unlike for cell lysis-dependent release, the oral streptococci appear to induce eDNA release by a novel mechanism. In dual-species cultures, the oral commensals Streptococcus sanguinis and Streptococcus gordonii release eDNA in a manner dependent on pyruvate oxidase (Pox) generation of hydrogen peroxide (H₂O₂) under the control of ambient oxygen (23). In this report, we now provide direct evidence of selective H₂O₂-induced eDNA release by these oral commensal streptococci.     

Repressed Respiration of Oral Streptococci Grown in Biofilms

The respiratory activities of oral streptococci grown in biofilms were found to be markedly repressed compared with those of cells from aerobic culture, or for Streptococcus mutans GS-5, even for those grown in static culture. Respiration rates generally reflected levels of NADH oxidase activities in cell extracts. Superoxide dismutase levels were somewhat reduced in biofilm cells. However, sensitivities to oxidative damage caused by H₂O₂, t-butylhydroperoxide, or 8-hydroxyquinoline were not greatly different for cells from suspension cultures and those from either intact or dispersed biofilms. The capacities of S. sanguis and S. gordonii to produce H₂O₂ also were markedly repressed by biofilm growth, and presumably this repression would affect the ecology of dental plaque by reducing oxidative stresses under crowded conditions. Received: 23 April 2001 / Accepted: 2 August 2001