Genetic basis of coaggregation receptor polysaccharide biosynthesis in Streptococcus sanguinis and related species

Interbacterial adhesion between streptococci and actinomyces promotes early dental plaque biofilm development. Recognition of coaggregation receptor polysaccharides (RPS) on strains of Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis by Actinomyces spp. type 2 fimbriae is the principal mechanism of these interactions. Previous studies of genetic loci for synthesis of RPS (rps) and RPS precursors (rml, galE1 and galE2) in S. gordonii 38 and S. oralis 34 revealed differences between these strains. To determine whether these differences are strain‐specific or species‐specific, we identified and compared loci for polysaccharide biosynthesis in additional strains of these species and in several strains of the previously unstudied species, S. sanguinis. Genes for synthesis of RPS precursors distinguished the rps loci of different streptococci. Hence, rml genes for synthesis of TDP‐L‐Rha were in rps loci of S. oralis strains but at other loci in S. gordonii and S. sanguinis. Genes for two distinct galactose epimerases were also distributed differently. Hence, galE1 for epimerization of UDP‐Glc and UDP‐Gal was in galactose operons of S. gordonii and S. sanguinis strains but surprisingly, this gene was not present in S. oralis. Moreover, galE2 for epimerization of both UDP‐Glc and UDP‐Gal and UDP‐GlcNAc and UDP‐GalNAc was at a different locus in each species, including rps operons of S. sanguinis. The findings provide insight into cell surface properties that distinguish different RPS‐producing streptococci and open an approach for identifying these bacteria based on the arrangement of genes for synthesis of polysaccharide precursors.     

Differential interactions of Streptococcus gordonii and Staphylococcus aureus with cultured osteoblasts

The impedance of normal osteoblast function by microorganisms is at least in part responsible for the failure of dental or orthopedic implants. Staphylococcus aureus is a major pathogen of bone, and exhibits high levels of adhesion and invasion of osteoblasts. In this article we show that the commensal oral bacterium Streptococcus gordonii also adheres to and is internalized by osteoblasts. Entry of S. gordonii cells had typical features of phagocytosis, similar to S. aureus, with membrane protrusions characterizing initial uptake, and closure of the osteoblast membrane leading to engulfment. The sensitivities of S. gordonii internalization to inhibitors cytochalasin D, colchicine and monensin indicated uptake through endocytosis, with requirement for actin accumulation. Internalization levels of S. gordonii were enhanced by expression of S. aureus fibronectin‐binding protein A (FnBPA) on the S. gordonii cell surface. Lysosomal‐associated membrane protein‐1 phagosomal membrane marker accumulated with intracellular S. aureus and S. gordonii FnBPA, indicating trafficking of bacteria into the late endosomal/lysosomal compartment. Streptococcus gordonii cells did not survive intracellularly for more than 12 h, unless expressing FnBPA, whereas S. aureus showed extended survival times (>48 h). Both S. aureus and S. gordonii DL‐1 elicited a rapid interleukin‐8 response by osteoblasts, whereas S. gordonii FnBPA was slower. Only S. aureus elicited an interleukin‐6 response. Hence, S. gordonii invades osteoblasts by a mechanism similar to that exhibited by S. aureus, and elicits a proinflammatory response that may promote bone resorption.     

Gingipain‐dependent augmentation by Porphyromonas gingivalis of phagocytosis of Tannerella forsythia

In the pathogenesis of periodontitis, Porphyromonas gingivalis plays a role as a keystone pathogen that manipulates host immune responses leading to dysbiotic oral microbial communities. Arg‐gingipains (RgpA and RgpB) and Lys‐gingipain (Kgp) are responsible for the majority of bacterial proteolytic activity and play essential roles in bacterial virulence. Therefore, gingipains are often considered as therapeutic targets. This study investigated the role of gingipains in the modulation by P. gingivalis of phagocytosis of Tannerella forsythia by macrophages. Phagocytosis of T. forsythia was significantly enhanced by coinfection with P. gingivalis in a multiplicity of infection‐dependent and gingipain‐dependent manner. Mutation of either Kgp or Rgp in the coinfecting P. gingivalis resulted in attenuated enhancement of T. forsythia phagocytosis. Inhibition of coaggregation between the two bacterial species reduced phagocytosis of T. forsythia in mixed infection, and this coaggregation was dependent on gingipains. Inhibition of gingipain protease activities in coinfecting P. gingivalis abated the coaggregation and the enhancement of T. forsythia phagocytosis. However, the direct effect of protease activities of gingipains on T. forsythia seemed to be minimal. Although most of the phagocytosed T. forsythia were cleared in infected macrophages, more T. forsythia remained in cells coinfected with gingipain‐expressing P. gingivalis than in cells coinfected with the gingipain‐null mutant or infected only with T. forsythia at 24 and 48 h post‐infection. Collectively, these results suggest that P. gingivalis, mainly via its gingipains, alters the clearance of T. forsythia, and provide some insights into the role of P. gingivalis as a keystone pathogen.     

Involvement of lipoprotein PpiA of Streptococcus gordonii in evasion of phagocytosis by macrophages

Streptococcus gordonii is a commensal gram‐positive bacterium that resides in the human oral cavity, and is one of the most common causes of infective endocarditis (IE). Bacterial surface molecules play an important role in establishing IE, and several S. gordonii proteins have been implicated in binding to host cells during the establishment of IE. In this study, we identified a putative lipoprotein, peptidyl‐prolyl cis/trans isomerase (PpiA), and clarified its role in evasion of phagocytosis by macrophages. Attenuation of the gene encoding prolipoprotein diacylglyceryl transferase (Lgt) altered the localization of PpiA from the cell surface to the culture supernatant, indicating that PpiA is lipid‐anchored in the cell membrane by Lgt. Both human and murine macrophages showed higher phagocytic activity towards ppiA and lgt mutants than the wild‐type, indicating that the presence of PpiA suppresses phagocytosis of S. gordonii. Human macrophages treated with dextran sulfate had significantly impaired phagocytosis of S. gordonii, suggesting that class A scavenger receptors in human macrophages are involved in the phagocytosis of S. gordonii. These results provide evidence that S. gordonii lipoprotein PpiA plays an important role in inhibiting phagocytic engulfment and in evasion of the host immune response.     

Role of lysine in interaction between surface protein peptides of Streptococcus gordonii and agglutinin peptide

Introduction: Streptococcus gordonii interacts with the salivary pellicle on the tooth surface and plays an important role in dental biofilm formation. Reports show that the analog Ssp peptide (A11K; alanine to lysine at position 11 in the arranged sequence, ¹DYQAKLAAYQAEL¹³) of SspA and SspB of S. gordonii increased binding to the salivary agglutinin (gp‐340/DMBT1) peptide (scavenger receptor cysteine‐rich domain 2: SRCRP2). To determine the role of lysine in the binding of the Ssp(A11K) peptide to SRCRP2, we investigated whether an additional substitution by lysine influenced the binding of Ssp(A11K) peptide to SRCRP2 using a BIAcore biosensor assay. Methods: Six analogs of the Ssp peptide with positive charges in surface positions on the structure were synthesized using substitution at various positions. Results: The binding activity of analog Ssp(A4K–A11K) peptide was significantly higher than the other Ssp analogs. The binding activity rose under low ionic strength conditions. The distance between positively charged amino acids in the Ssp(A4K–A11K) peptide between 4K and 11K was 1.24 ± 0.02 nm and was close to the distance (1.19 ± 0.00 nm) between Q and E, presenting a negative charged area, on SRCRP2 using chemical computing graphic analysis. The molecular angle connecting 1D–11K–4K in the Ssp(A4K–A11K) peptide secondary structure was smaller than the other peptide angles (1D–11K–XK). The Ssp(A4K–A11K) peptide showed higher inhibiting activity for Streptococcus mutans binding to saliva‐coated hydroxyapatite than the (A11K) peptide. Conclusion: The positioning of lysine is important for binding between Ssp peptide and SRCRP2, and the inhibiting effect on S. mutans binding to the tooth surface.     

A YadA‐like autotransporter,Hag1 in Veillonella atypica is a multivalent hemagglutinin involved in adherence to oral streptococci,Porphyromonas gingivalis,and human oral buccal cells

Dental biofilm development is a sequential process, and adherence between microbes and the salivary pellicle (adhesion) as well as among different microbes (co‐adhesion or coaggregation) plays a critical role in building a biofilm community. The Veillonella species are among the most predominant species in the oral cavity and coaggregate with many initial, early, middle, and late colonizers. Similar to oral fusobacteria, they are also considered bridging species in biofilm development. However, the mechanism of this ability has yet to be reported, due to the previous lack of a genetic transformation system in the entire genus. In this study, we used our recently discovered transformable Veillonella strain, Veillonella atypica OK5, to probe the mechanism of coaggregation between Veillonella species and other oral bacteria. By insertional inactivation of all eight putative hemagglutinin genes, we identified one gene, hag1, which is involved in V. atypica coaggregation with the initial colonizers Streptococcus gordonii, Streptococcus oralis and Streptococcus cristatus, and the periodontal pathogen Porphyromonas gingivalis. The hag1 mutant also abolished adherence to human buccal cells. Inhibition assays using various chemical or physiological treatments suggest different mechanisms being involved in coaggregation with different partners. The entire hag1 gene was sequenced and shown to be the largest known bacterial hemagglutinin gene.     

Potential role for Streptococcus gordonii‐derived hydrogen peroxide in heme acquisition by Porphyromonas gingivalis

Streptococcus gordonii, an accessory pathogen and early colonizer of plaque, co‐aggregates with many oral species including Porphyromonas gingivalis. It causes α‐hemolysis on blood agar, a process mediated by H₂O₂ and thought to involve concomitant oxidation of hemoglobin (Hb). Porphyromonas gingivalis has a growth requirement for heme, which is acquired mainly from Hb. The paradigm for Hb heme acquisition involves the initial oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb), followed by heme release and extraction through the actions of K‐gingipain protease and/or the HmuY hemophore‐like protein. The ability of S. gordonii to mediate Hb oxidation may potentially aid heme capture during co‐aggregation with P. gingivalis. Hemoglobin derived from zones of S. gordonii α‐hemolysis was found to be metHb. Generation of metHb from oxyHb by S. gordonii cells was inhibited by catalase, and correlated with levels of cellular H₂O₂ production. Generation of metHb by S. gordonii occurred through the higher Hb oxidation state of ferrylhemoglobin. Heme complexation by the P. gingivalis HmuY was employed as a measure of the ease of heme capture from metHb. HmuY was able to extract iron(III)protoporphyrin IX from metHb derived from zones of S. gordonii α‐hemolysis and from metHb generated by the action of S. gordonii cells on isolated oxyHb. The rate of HmuY‐Fe(III)heme complex formation from S. gordonii‐mediated metHb was greater than from an equivalent concentration of auto‐oxidized metHb. It is concluded that S. gordonii may potentially aid heme acquisition by P. gingivalis by facilitating metHb formation in the presence of oxyHb.     

Interactions between Streptococcus oralis,Actinomyces oris,and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual‐species biofilms, or three‐species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non‐fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.     

Erythritol alters microstructure and metabolomic profiles of biofilm composed of Streptococcus gordonii and Porphyromonas gingivalis

The effects of sugar alcohols such as erythritol, xylitol, and sorbitol on periodontopathic biofilm are poorly understood, though they have often been reported to be non‐cariogenic sweeteners. In the present study, we evaluated the efficacy of sugar alcohols for inhibiting periodontopathic biofilm formation using a heterotypic biofilm model composed of an oral inhabitant Streptococcus gordonii and a periodontal pathogen Porphyromonas gingivalis. Confocal microscopic observations showed that the most effective reagent to reduce P. gingivalis accumulation onto an S. gordonii substratum was erythritol, as compared with xylitol and sorbitol. In addition, erythritol moderately suppressed S. gordonii monotypic biofilm formation. To examine the inhibitory effects of erythritol, we analyzed the metabolomic profiles of erythritol‐treated P. gingivalis and S. gordonii cells. Metabolome analyses using capillary electrophoresis time‐of‐flight mass spectrometry revealed that a number of nucleic intermediates and constituents of the extracellular matrix, such as nucleotide sugars, were decreased by erythritol in a dose‐dependent manner. Next, comparative analyses of metabolites of erythritol‐ and sorbitol‐treated cells were performed using both organisms to determine the erythritol‐specific effects. In P. gingivalis, all detected dipeptides, including Glu‐Glu, Ser‐Glu, Tyr‐Glu, Ala‐Ala and Thr‐Asp, were significantly decreased by erythritol, whereas they tended to be increased by sorbitol. Meanwhile, sorbitol promoted trehalose 6‐phosphate accumulation in S. gordonii cells. These results suggest that erythritol has inhibitory effects on dual species biofilm development via several pathways, including suppression of growth resulting from DNA and RNA depletion, attenuated extracellular matrix production, and alterations of dipeptide acquisition and amino acid metabolism.     

Inhibition of Streptococcus mutans adherence and biofilm formation using analogues of the SspB peptide

Objective

Streptococcus gordonii is a pioneer colonizer of the enamel salivary pellicle that forms biofilm on the tooth surfaces. Recent reports show the surface protein analogue peptide {400 (T) of SspB 390-402 is substituted to K forming SspB (390-T400K-402)} from S. gordonii interacts strongly with salivary receptors to cariogenic bacteria, Streptococcus mutans. To characterize the analogue peptide biological activities, we investigated its binding and inhibiting effects, and the role of its amino acid moities.

Methods

We measured binding activity of analogue peptides to salivary components using the BIAcore assay; assayed inhibition activities of peptides for bacterial binding and growth on saliva-coated hydroxyapatite beads (s-HA); and describe the peptides interfering with biofilm formation of S. mutans on polystyrene surfaces.

Results

The SspB (390-T400K-402 and -401) peptides significantly bound with salivary components and inhibited the binding of S. mutans and S. gordonii to s-HA without bactericidal activity; but did not inhibit binding of Streptococcus mitis, a beneficial commensal. Further, the lack of D and E-L at position 390 and 401-402 in the peptide, and substituted peptide SspB (D390H- or D390K-T400K-402) did not bind to salivary components or inhibit binding of S. mutans. The SspB (390-T400K-402) peptide inhibited biofilm formation on salivary components-coated polystyrene surfaces in absence of conditioned planktonic cells.

Conclusions

We found constructing the peptide to include positions 390(D), 400(K) and 401(E), two surface positive and negative connective charges, and at least 12 amino acids are required to bind salivary components and inhibit the binding of S. mutans and S. gordonii.     

Disruption of heterotypic community development by Porphyromonas gingivalis with small molecule inhibitors

Porphyromonas gingivalis is one of the main etiological organisms in periodontal disease. On oral surfaces P. gingivalis is a component of multispecies biofilm communities and can modify the pathogenic potential of the community as a whole. Accumulation of P. gingivalis in communities is facilitated by interspecies binding and communication with the antecedent colonizer Streptococcus gordonii. In this study we screened a library of small molecules to identify structures that could serve as lead compounds for the development of inhibitors of P. gingivalis community development. Three small molecules were identified that effectively inhibited accumulation of P. gingivalis on a substratum of S. gordonii. The structures of the small molecules are derived from the marine alkaloids oroidin and bromoageliferin and contain a 2‐aminoimidazole or 2‐aminobenzimidazole moiety. The most active compounds reduced expression of mfa1 and fimA in P. gingivalis, genes encoding the minor and major fimbrial subunits, respectively. These fimbrial adhesins are necessary for P. gingivalis co‐adhesion with S. gordonii. These results demonstrate the potential for a small molecular inhibitor‐based approach to the prevention of diseases associated with P. gingivalis.     

Streptococcal peptides that signal Enterococcus faecalis cells carrying the pheromone‐responsive conjugative plasmid pAM373

Pheromone‐mediated conjugative transfer of enterococcal plasmids can contribute to the dissemination of genes involved in antibiotic resistance, fitness, and virulence among co‐residents of mixed microbial communities. We have previously shown that intergeneric signaling by the Streptococcus gordonii strain Challis heptapeptide s.g.cAM373 (SVFILAA) induces an aggregation substance‐mediated mating response and facilitates plasmid transfer from Enterococcus faecalis cells carrying the pheromone‐responsive plasmid pAM373 to both pheromone‐producing and non‐pheromone‐producing oral streptococcal recipients. To further investigate the streptococcal pheromone‐like peptides, s.g.cAM373‐like sequences were identified in the signal sequences of streptococcal CamG lipoproteins and their abilities to induce a mating response in E. faecalis/pAM373 cells were examined. Synthetic heptamers with the consensus sequence (A/S)‐(I/V)‐F‐I‐L‐(A/V/T)‐(S/A) induced AS‐mediated clumping. The conserved pheromone ABC transporter encoded by S. gordonii genome loci SGO_RS02660 and SGO_RS02665 was identified and confirmed to be required for s.g.cAM373 activity. Functional assays of culture supernatants from representative oral and blood isolates of S. gordonii showed that in addition to strains encoding s.g.cAM373, strain SK120, encoding the newly identified pheromone s.g.cAM373‐V (SVFILVA), was able to induce enterococcal clumping, whereas strains SK6, SK8, SK9, and SK86 which encoded s.g.cAM373‐T (SVFILTA) did not elicit a detectable mating response. Absence of pheromone activity in supernatants of heterologous hosts encoding its CamG precursor suggested that s.g.cAM373‐T was not effectively processed and/or transported. Overall, these studies demonstrated the distribution of active pheromone peptides among strains of S. gordonii, and support a potential role for enterococcal–streptococcal communication in contributing to genetic plasticity in the oral metagenome.     

Role of an autoinducer-2-like molecule from Veillonella tobetsuensis in Streptococcus gordonii biofilm formation

Objectives

Dental plaque is a multispecies biofilm that causes dental caries and periodontal diseases. In particular, Streptococcus spp. and Veillonella spp. have been identified as early colonizers of oral biofilms. Coaggregation of bacterial species and/or autoinducers using the quorum sensing system has been suggested to plan an important role in biofilm formation. In our previous study, we showed that Veillonella tobetsuensis clearly promoted the development of Streptococcus gordonii biofilms. To clarify the mechanism, coaggregation between S. gordonii and V. tobetsuensis and the effect of an autoinducer-like molecule were investigated in this study.

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

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

Toll‐like receptor 2‐mediated modulation of growth and functions of regulatory T cells by oral streptococci

This study was designed to determine whether oral streptococci modulate the growth and functions of regulatory T cells. Heat‐killed cells of wild‐type strains of Streptococcus gordonii and Streptococcus mutans induced the Toll‐like receptor 2 (TLR2) ‐mediated nuclear factor‐κB (NF‐κB) activation, but their lipoprotein‐deficient strains did not. Stimulation with these streptococci resulted in a significant increase in the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in splenocytes derived from both TLR2^+/+ and TLR2^?/? mice, but the level of increase in TLR2^+/+ splenocytes was stronger than that in TLR2^?/? splenocytes. Both strains of S. gordonii enhanced the proliferation of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells isolated from TLR2^+/+ mice at the same level as those from TLR2^?/? mice in an interleukin‐2‐independent manner. However, wild‐type and lipoprotein‐deficient strains of both streptococci did not enhance the suppressive activity of the isolated regulatory T cells in vitro, but rather inhibited it. TLR ligands also inhibited the suppressive activity of the regulatory T cells. Inhibition of the suppressive activity was recovered by the addition of anti‐IL‐6 antibody. Pretreatment of antigen‐presenting cells with the NF‐κB inhibitor BAY11‐7082 enhanced the suppressive activity of the regulatory T cells. These results suggested that interleukin‐6 produced by antigen‐presenting cells inhibits the suppressive activity of the regulatory T cells. Wild‐type strain, but not lipoprotein‐deficient strain, of S. gordonii reduced the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in the acute infection model, whereas both strains of S. gordonii increased it in the chronic infection model mice. Hence, this study suggests that oral streptococci are capable of modulating the growth and functions of regulatory T cells in vitro and in vivo.     

Treponema denticola improves adhesive capacities of Porphyromonas gingivalis

Porphyromonas gingivalis, an important etiological agent of periodontal disease, is frequently found associated with Treponema denticola, an anaerobic spirochete, in pathogenic biofilms. However, interactions between these two bacteria are not well understood at the molecular level. In this study, we seek to link the influence of T. denticola on the expression of P. gingivalis proteases with its capacities to adhere and to form biofilms. The P. gingivalis genes encoding Arg‐gingipain A (RgpA), Lys‐gingipain (Kgp), and hemagglutinin A (HagA) were more strongly expressed after incubation with T. denticola compared with P. gingivalis alone. The amounts of the three resulting proteins, all of which contain hemagglutinin adhesion domains, were increased in culture supernatants. Moreover, incubation of P. gingivalis with T. denticola promoted static and dynamic biofilm formation, primarily via a time‐dependent enhancement of P. gingivalis adhesion capacities on bacterial partners such as Streptococcus gordonii. Adhesion of P. gingivalis to human cells was also increased. These results showed that interactions of P. gingivalis with other bacterial species, such as T. denticola, induce increased adhesive capacities on various substrata by hemagglutinin adhesion domain‐containing proteins.     

CcpA regulates biofilm formation and competence in Streptococcus gordonii

Streptococcus gordonii is an important member of the oral biofilm community. As an oral commensal streptococcus, S. gordonii is considered beneficial in promoting biofilm homeostasis. CcpA is known as the central regulator of carbon catabolite repression in Gram‐positive bacteria and is also involved in the control of virulence gene expression. To further establish the role of CcpA as central regulator in S. gordonii, the effect of CcpA on biofilm formation and natural competence of S. gordonii was investigated. These phenotypic traits have been suggested to be important to oral streptococci in coping with environmental stress. Here we demonstrate that a CcpA mutant was severely impaired in its biofilm‐forming ability, showed a defect in extracellular polysaccharide production and reduced competence. The data suggest that CcpA is involved in the regulation of biofilm formation and competence development in S. gordonii.