F-type proton-pumping ATPase mediates acid tolerance in Streptococcus mutans.

AIMS: Streptococcus mutans is highly sensitive to inhibitors of proton-pumping F-type ATPase (F-ATPase) under acidic conditions. Herein, we investigated the role of S. mutans F-ATPase in acid tolerance using a bacterium expressing the F-ATPase ß subunit at lower levels than the wild-type strain. METHODS AND RESULTS: We generated a mutant S. mutans expressing the catalytic ß subunit of F-ATPase at lower levels than the wild-type bacterium. The mutant cells exhibited a significantly slower growth rate at pH 5.30, whereas the rate was essentially the same as that of wild-type cells at pH 7.40. In addition, the colony-forming ability of the mutant was decreased at pH <4.30 but not at pH 7.40. Thus, the growth rate and survival of S. mutans expressing low levels of the ß subunit were reduced under acidic conditions. CONCLUSIONS: Together with our previous observations, this study indicates that F-ATPase is involved in the acid tolerance mechanism of S. mutans by secreting protons from the cytoplasm.

Immunoelectron Microscopic Analysis of Dipeptidyl-Peptidases and Dipeptide Transporter Involved in Nutrient Acquisition in Porphyromonas gingivalis.

Porphyromonas gingivalis is an asaccharolytic, Gram-negative, anaerobic bacterium representing a keystone pathogen in chronic periodontitis. The bacterium's energy production depends on the metabolism of amino acids, which are predominantly incorporated as dipeptides via the proton-dependent oligopeptide transporter (Pot). In this study, the localization of dipeptidyl-peptidases (DPPs) and Pot was investigated for the first time in P. gingivalis using immunoelectron microscopy with specific antibodies for the bacterial molecules and gold-conjugated secondary antibodies on ultrathin sections. High-temperature protein G and hemin-binding protein 35 were used as controls, and the cytoplasmic localization of the former and outer membrane localization of the latter were confirmed. P. gingivalis DPP4, DPP5, DPP7, and DPP11, which are considered sufficient for complete dipeptide production, were detected in the periplasmic space. In contrast, DPP3 was localized in the cytoplasmic space in accord with the absence of a signal sequence. The inner membrane localization of Pot was confirmed. Thus, spatial integration of the nutrient acquisition system exists in P. gingivalis, in which where dipeptides are produced in the periplasmic space by DPPs and readily transported across the inner membrane via Pot.

Expanded substrate specificity supported by P1' and P2' residues enables bacterial dipeptidyl-peptidase 7 to degrade bioactive peptides.

Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a pathogen related to chronic periodontitis, because its energy production is entirely dependent on the metabolism of amino acids predominantly incorporated as dipeptides. These dipeptides are produced by periplasmic dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11. Although the substrate specificities of these four DPPs cover most amino acids at the penultimate position from the N terminus (P1), no DPP is known to cleave penultimate Gly, Ser, Thr, or His. Here, we report an expanded substrate preference of bacterial DPP7 that covers those residues. MALDI-TOF mass spectrometry analysis demonstrated that DPP7 efficiently degraded incretins and other gastrointestinal peptides, which were successively cleaved at every second residue, including Ala, Gly, Ser, and Gln, as well as authentic hydrophobic residues. Intravenous injection of DPP7 into mice orally administered glucose caused declines in plasma glucagon-like peptide-1 and insulin, accompanied by increased blood glucose levels. A newly developed coupled enzyme reaction system that uses synthetic fluorogenic peptides revealed that the P1' and P2' residues of substrates significantly elevated kcat values, providing an expanded substrate preference. This activity enhancement was most effective toward the substrates with nonfavorable but nonrepulsive P1 residues in DPP7. Enhancement of kcat by prime-side residues was also observed in DPP11 but not DPP4 and DPP5. Based on this expanded substrate specificity, we demonstrate that a combination of DPPs enables proteolytic liberation of all types of N-terminal dipeptides and ensures P. gingivalis growth and pathogenicity.

A genomic sequence of the type II-A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system in Mycoplasma salivarium strain ATCC 29803.

INTRODUCTION: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems are RNA-mediated adaptive immune systems that actagainst invading genetic elements such as phages or plasmids. CRISPR/Cas systems exist in nearly half of bacteria. Mycoplasma salivarium is a commensal species of the oropharynx. The American Type Culture Collection maintains five M. salivarium strains: ATCC 14277, 23064, 23557, 29803, and 33130. The genome sequence of ATCC 23064 revealed that it has an incomplete CRISPR/Cas system. However, the genome sequences of the remaining strains have not been analyzed. METHODS: We performed polymerase chain reaction-amplicon sequencing and de novo genome sequencing to evaluate the presence of the CRISPR/Cas system in four strains. RESULTS: Only ATCC 29803 possessed cas1, cas2, cas9, and csn2 genes, a CRISPR array, and tracrRNA. The sequences of most components were identical between the CRISPR/Cas systems of ATCC 29803 and ATCC 23064, whereas the spacer sequences and a region of the cas9 gene were different. Unlike the CRISPR/Cas system of ATCC 23064, the cas9 gene of ATCC 29803 was not disrupted by the presence of stop codons. CONCLUSION: ATCC 29803 possesses genomic components required to express the type II-A CRISPR/Cas system, which potentially functions as an RNA-guided endonuclease.

Tryptophanyl tRNA Synthetase from Human Macrophages Infected by Porphyromonas gingivalis Induces a Proinflammatory Response Associated with Atherosclerosis.

Porphyromonas gingivalis is the most common microorganism associated with adult periodontal disease, causing inflammation around the subgingival lesion. In this study, we investigated tryptophanyl tRNA synthase (WRS) production by THP-1 cells infected with P. gingivalis. Cytokine production, leukocyte adhesion molecules, and low-density lipoprotein receptor (LDLR) expressions in cultured cells were examined. WRS was detected in THP-1 cell culture supernatants stimulated with P. gingivalis from 1 to 24 h, and apparent production was observed after 4 h. No change in WRS mRNA expression was observed from 1 to 6 h in THP-1 cells, whereas its expression was significantly increased 12 h after stimulation with P. gingivalis. Lactate dehydrogenase (LDH) activity was observed from 4 to 24 h. The TNF-α, IL-6, IL-8, and CXCL2 levels of THP-1 cells were upregulated after treatment with recombinant WRS (rWRS) and were significantly reduced when THP-1 cells were treated with C29. The MCP-1, ICAM-1, and VCAM-1 levels in human umbilical vein endothelial cells were upregulated following treatment with rWRS, and TAK242 suppressed these effects. Additionally, unmodified LDLR, macrophage scavenger receptor A, and lectin-like oxidized LDLRs were upregulated in THP-1 cells treated with rWRS. These results suggest that WRS from macrophages infected with P. gingivalis is associated with atherosclerosis.

Associations of streptococci and fungi amounts in the oral cavity with nutritional and oral health status in institutionalized elders: a cross sectional study.

BACKGROUND: Disruption of the indigenous microbiota is likely related to frailty caused by undernutrition. However, the relationship between undernutrition and the oral microbiota, especially normal bacteria, is not obvious. The aim of this study was to elucidate the associations of nutritional and oral health conditions with prevalence of bacteria and fungi in the oral cavity of older individuals. METHODS: Forty-one institutionalized older individuals with an average age ± standard deviation of 84.6 ± 8.3 years were enrolled as participants. Body mass index (BMI) and oral health assessment tool (OHAT) scores were used to represent nutritional and oral health status. Amounts of total bacteria, streptococci, and fungi in oral specimens collected from the tongue dorsum were determined by quantitative polymerase chain reaction (PCR) assay results. This study followed the STROBE statement for reports of observational studies. RESULTS: There was a significant correlation between BMI and streptococcal amount (ρ = 0.526, p < 0.001). The undernutrition group (BMI < 20) showed a significantly lower average number of oral streptococci (p = 0.003). In logistic regression models, streptococcal amount was a significant variable accounting for "not undernutrition" [odds ratio 5.68, 95% confidential interval (CI) 1.64-19.7 (p = 0.06)]. On the other hand, participants with a poor oral health condition (OHAT ≥ 5) harbored significantly higher levels of fungi (p = 0.028). CONCLUSION: Oral streptococci were found to be associated with systemic nutritional condition and oral fungi with oral health condition. Thus, in order to understand the relationship of frailty with the oral microbiota in older individuals, it is necessary to examine oral indigenous bacteria as well as etiological microorganisms.

Abiotrophia defectiva DnaK Promotes Fibronectin-Mediated Adherence to HUVECs and Induces a Proinflammatory Response.

Abiotrophia defectiva is a nutritionally variant streptococci that is found in the oral cavity, and it is an etiologic agent of infective endocarditis. We have previously reported the binding activity of A. defectiva to fibronectin and to human umbilical vein endothelial cells (HUVECs). However, the contribution of some adhesion factors on the binding properties has not been well delineated. In this study, we identified DnaK, a chaperon protein, as being one of the binding molecules of A. defectiva to fibronectin. Recombinant DnaK (rDnaK) bound immobilized fibronectin in a concentration-dependent manner, and anti-DnaK antiserum reduced the binding activity of A. defectiva with both fibronectin and HUVECs. Furthermore, DnaK were observed on the cell surfaces via immune-electroscopic analysis with anti-DnaK antiserum. Expression of IL-8, CCL2, ICAM-1, and VCAM-1 was upregulated with the A. defectiva rDnaK treatment in HUVECs. Furthermore, TNF-α secretion of THP-1 macrophages was also upregulated with the rDnaK. We observed these upregulations in rDnaK treated with polymyxin B, but not in the heat-treated rDnaK. The findings show that A. defectiva DnaK functions not only as an adhesin to HUVECs via the binding to fibronectin but also as a proinflammatory agent in the pathogenicity to cause infective endocarditis.

Effect of Butyric Acid in the Proliferation and Migration of Junctional Epithelium in the Progression of Periodontitis: An In Vitro Study.

PURPOSE: To elucidate the effects of butyric acid (BA), a metabolite of bacteria involved in periodontitis, and a possible enhancer of the junctional epithelial cells. METHODS: A murine junctional epithelial cell line, JE-1, was used to assess the effects of sodium butyrate (NaB) as BA. Cell proliferation, migration and attachment were analyzed. Additionally, gene and promoter expression analysis was performed, i.e., cap analysis of gene expression (CAGE) and gene ontology (GO) term enrichment analysis. RESULTS: NaB affected junctional epithelial cell proliferation, migration and attachment. A high concentration of NaB caused cell death and a low concentration tended to promote migration and adhesion. CAGE analysis revealed 75 upregulated and 96 downregulated genes in the cells after 0.2 mM NaB stimulation for 3 h. Regarding GO term enrichment, the genes upregulated >4-fold participated predominantly in cell migration and proliferation. The results of this study suggest that BA produced from periodontopathic bacteria is involved in periodontal tissue destruction at high concentrations. Furthermore, at low concentrations, BA potentially participates in periodontal disease progression by increasing proliferation, migration and attachment of the junctional epithelium and thereby increasing epithelial down-growth.

Preferential dipeptide incorporation of Porphyromonas gingivalis mediated by proton-dependent oligopeptide transporter (Pot).

Multiple dipeptidyl-peptidases (DPPs) are present in the periplasmic space of Porphyromonas gingivalis, an asaccharolytic periodontopathic bacterium. Dipeptides produced by DPPs are presumed to be transported into the bacterial cells and metabolized to generate energy and cellular components. The present study aimed to identify a transporter responsible for dipeptide uptake in the bacterium. A real-time metabolic analysis demonstrated that P. gingivalis preferentially incorporated Gly-Xaa dipeptides, and then, single amino acids, tripeptides and longer oligopeptides to lesser extents. Heterologous expression of the P. gingivalis serine/threonine transporter (SstT; PGN_1460), oligopeptide transporter (Opt; PGN_1518) and proton-dependent oligopeptide transporter (Pot; PGN_0135) genes demonstrated that Escherichia coli expressing Pot exclusively incorporated Gly-Gly, while SstT managed Ser uptake and Opt was responsible for Gly-Gly-Gly uptake. Dipeptide uptake was significantly decreased in a P. gingivalis Δpot strain and further suppressed in a Δpot-Δopt double-deficient strain. In addition, the growth of the Δpot strain was markedly attenuated and the Δpot-Δopt strain scarcely grew, whereas the ΔsstT strain grew well almost like wild type. Consequently, these results demonstrate that predominant uptake of dipeptide in P. gingivalis is mostly managed by Pot. We thus propose that Pot is a potential therapeutic target of periodontal disease and P. gingivalis-related systemic diseases.

Tyrosine tRNA synthetase as a novel extracellular immunomodulatory protein in Streptococcus anginosus.

Streptococcus anginosus is frequently detected in patients with infective endocarditis, abscesses or oral cancer. Although S. anginosus is considered the causative pathogen of these diseases, the pathogenic mechanisms of the bacterium have remained unclear. Previously, we suggested that an extracellular antigen from S. anginosus (SAA) serves as a pathogenic factor by inducing nitric oxide production in murine macrophages. In the present study, we identified SAA using LC-MS/MS and assessed the biological activities of His-tagged recombinant SAA in murine macrophages. SAA was identified as a tyrosine tRNA synthetase (SaTyrRS) that was isolated from the extracellular fraction of S. anginosus but not from other oral streptococci. In addition, inducible nitric oxide synthase and TNF-α mRNA expression was induced in recombinant SaTyrRS-stimulated murine macrophages. However, their mRNA expression was not induced in macrophages stimulated with truncated or heat-inactivated recombinant SaTyrRS, and the activation motif was identified as Arg264-Thr270. Consequently, these results indicated that SaTyrRS could be a novel and specific immunomodulatory protein in S. anginosus.

Abiotrophia defectiva adhere to saliva-coated hydroxyapatite beads via interactions between salivary proline-rich-proteins and bacterial glyceraldehyde-3-phosphate dehydrogenase.

Abiotrophia defectiva is a species of nutritionally variant streptococci that is found in human saliva and dental plaques and that has been associated with infective endocarditis. In our previous study, it was found that A. defectiva could bind specifically to saliva-coated hydroxyapatite beads (SHA). This study identified a cell surface component of A. defectiva that promotes adherence to SHA beads. The binding of A. defectiva to SHA was reduced in the presence of antibodies against human proline-rich protein (PRP); these results suggested that PRP may be a critical component mediating interactions between A. defectiva and the salivary pellicle. Two-dimensional gel electrophoresis of whole A. defectiva cells followed by Far-Western blotting was conducted by probing with synthetic peptides analogous to the binding region of PRP known as PRP-C. The results indicate that an A. defectiva protein of 37 kDa interacts with PRP-C. The results of amino-terminal sequencing of the adhesive A. defectiva protein revealed significant similarity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Recombinant GAPDH bound to immobilized PRP-C in a dose-dependent manner and binding of A. defectiva to SHA or to PRP was reduced in the presence of anti-GAPDH antiserum. Western blotting or electron immunomicroscopic observations with anti-GAPDH antiserum revealed that this protein was expressed in both cytosolic and cell wall fractions. These results suggest that A. defectiva could specifically bind to PRP via interactions with cell surface GAPDH; the findings suggest a mechanism underlying A. defectiva-mediated adherence to saliva-coated tooth surfaces.

Effects of butyric acid, a bacterial metabolite, on the migration of ameloblastoma mediated by laminin 332.

Ameloblastoma is a benign tumor that develops in the jawbone. Occasionally, however, it may become malignant and metastasize to other tissues. Although it has been suggested that various cytokines and several adhesion factors may play a role in its malignant transformation, the details have not been elucidated. In this context, it has been reported that butyric acid produced by periodontopathic bacteria causes progression of malignant tumors occurring in the mouth via podoplanin. However, the influence of butyric acid on ameloblastoma has not been clarified. In the present study, therefore, the expression of various cytokines and adhesion factors in ameloblastoma upon stimulation with butyric acid or cytokines was investigated using real-time reverse-transcription polymerase chain reaction. Three cell lines (HAM1, HAM2 and HAM3) established from the same ameloblastoma were used in the experiments. It was found that the expression of mRNAs for epidermal growth factor (EGF) and transforming growth factor beta 1 (TGFß1) was increased in HAM2 and HAM3, respectively, upon stimulation with butyric acid. In addition, stimulation with EGF and TGFß1 led to an increase in the expression of laminin ß-3 mRNA in the respective cell lines. These results suggest that butyric acid may be involved in ameloblastoma exacerbation through the expression of laminin 332 (LM332) via EGF and TGFß1 produced by ameloblastoma itself.

Establishment and use of a three-dimensional ameloblastoma culture model to study the effects of butyric acid on the transcription of growth factors and laminin ß3.

OBJECTIVE: This study aimed to establish a three-dimensional (3D) culture method for ameloblastoma cell lines and to use the model to investigate the effect of butyric acid (BA), a periodontopathic bacterial metabolite, on the malignant transformation of ameloblastoma. DESIGN: Three ameloblastoma cell lines (HAM1, HAM2, and HAM3) established from the same tumor were used in this study. A 3D culture model was established in low absorption dishes and was incubated for 48 h. The effects of BA on the transcription of growth factors and LMß3 were examined by real-time reverse transcription PCR. Various BA concentrations (0.02, 0.2, 2, and 20 mM) were used to stimulate the cell cultures for 6 and 12 h. RESULTS: A 3D culture model was established. Gene expression levels of epithelial growth factor (EGF), transforming growth factor beta 1 (TGFß1), and laminin ß3 (LMß3) were higher in 3D than in 2D cultures. Cell morphology in 3D cultures did not change, while the transcription levels of EGF, TGFß1, and LMß3 were upregulated by BA in all cell lines. CONCLUSION: The 3D culture model is more responsive to BA than the 2D culture model, and there is a possibility that the malignancy and progression of ameloblastoma via laminin 332 (LM332) is mediated by BA.

Characterization and pathogenicity of fibronectin binding protein FbpI of Streptococcus intermedius.

Streptococcus intermedius is a causative agent of brain or liver abscesses. S. intermedius produces intermedilysin that plays a pivotal role in pathogenicity. We identified other pathogenic factors and described a fibronectin binding protein (FBP) homolog of S. intermedius (FbpI) that mediated bacterial adhesion to epithelial cells and virulence for mice. The amino acid sequence of FbpI is similar to that of atypical FBPs, which do not possess a conventional secretion signal and an anchoring motif. A full-length recombinant FbpI (rFbpI) bound to immobilized fibronectin in a dose-dependent manner. The fibronectin binding activity of an N-terminal construct of rFbpI comprising the translation initiation methionine of the open reading frame to lysine 265 (rFbpI-N) bound immobilized fibronectin to a much lesser extent compared with rFbpI. A construct comprising the C-terminal domain (alanine 266 to methionine 549; rFbpI-C) bound immobilized fibronectin equivalently to rFbpI. Adherence of the isogenic mutant ΔfbpI to cultured epithelial cells and immobilized fibronectin was significantly lower than that of the wild-type strain. Abscess formation of ΔfbpI reduced in a mouse infection model compared with that in the wild-type. Thus, FbpI may play a role in bacterial adhesion to host cells and represent a critical pathogenic factor of S. intermedius.

Effects of denture adhesives on growth and morphological transformation of Candida albicans.

PURPOSE: We investigated the effects of denture adhesives (cream (Cr), powder (Po), and cushion (Cu)) on growth and adhesive-related morphological transformation of Candida albicans. For this purpose, the numbers of adherent C. albicans, hyphae-specific gene expressions, and the SEM images were examined. METHODS: Acrylic resin blocks were prepared as controls (Co). Cr, Po, and Cu were thinly spread on the surface of the resin block.C. albicans suspension was seeded on the specimens and incubated at 4 °C for 2 h. The numbers of C. albicans adhering to each specimen at each incubation time period (1, 2, 3, 6, 12, and 24 h) were quantified using real-time RT-PCR. The hyphae-specific genes expressions were examined. The surface of each specimen was observed under the SEM to detect the transformation to the hyphal form. RESULTS: The initial adhesion rates in all groups were not statistically significant. The numbers of C. albicans adhering increased with time in all groups, and those adhering to the Cr, Po, and Cu were significantly greater than that adhering to the Co. In the Cr and Po, the hyphal-specific genes expressions were higher after incubation for 6 h. The transformation to the hyphal form was identified in the Cr and Po after incubation for 6 and 12 h. CONCLUSIONS: The denture adhesives used in this study accelerated the growth of C. albicans. Moreover, the early transformation to the hyphal form on the Cr- and Po-type adhesives was observed, suggesting that we should carefully use Cr- and Po-type adhesives.

Contribution of different adherent properties of Granulicatella adiacens and Abiotrophia defectiva to their associations with oral colonization and the risk of infective endocarditis.

Granulicatella adiacens (G. adiacens) and Abiotrophia defectiva (A. defectiva) colonize the oral cavity and form part of the normal flora in the intestinal and genitourinary tracts. As reported previously, the frequency of isolation of G. adiacens from the oral cavity was much higher than that of A. defectiva. However, it has been reported that compared with G. adiacens, A. defectiva was isolated at considerably higher frequencies from the blood of patients with infective endocarditis (IE). Hence, in this study, the in vitro interaction of G. adiacens and A. defectiva strains with host surfaces and biofilm formation was examined to assess whether their different adhesive properties contribute to their associations with oral colonization and IE, respectively. G. adiacens exhibited an increased binding ability to saliva-coated hydroxyapatite beads than A. defectiva following the addition of CaCl2. Furthermore, biofilm formation was observed only for G. adiacens with the use of a polystyrene tube and scanning electron microscopy analysis. Conversely, A. defectiva displayed significantly greater adherence to human umbilical vein endothelial cells and immobilized fibronectin than G. adiacens. These findings suggest that differences in binding properties to host components imply specific binding mechanisms in G. adiacens and A. defectiva, which might mediate selective colonization in the oral cavity or are associated with the pathogenicity of endocarditis.

Proton-pumping F-ATPase plays an important role in Streptococcus mutans under acidic conditions.

Streptococcus mutans, a bacterium mainly inhabiting the tooth surface, is a major pathogen of dental caries. The bacterium metabolizes sugars to produce acids, resulting in an acidic microenvironment in the dental plaque. Hence, S. mutans should possess a mechanism for surviving under acidic conditions. In the current study, we report the effects of inhibitors of Escherichia coli proton-pumping F-type ATPase (F-ATPase) on the activity of S. mutans enzyme, and the growth and survival of S. mutans under acidic conditions. Piceatannol, curcumin, and demethoxycurcumin strongly reduced the ATPase activity of S. mutans F-ATPase. Interestingly, these compounds inhibited the growth of S. mutans at pH 5.3 but not at pH 7.3. They also significantly reduced the colony-forming ability of S. mutans after incubation at pH 4.3, while showing essentially no effect at pH 7.3. These observations indicate that S. mutans is highly sensitive to F-ATPase inhibitors under acidic conditions and that F-ATPase plays an important role in acid tolerance of this bacterium.

Distribution of dipeptidyl peptidase (DPP) 4, DPP5, DPP7 and DPP11 in human oral microbiota-potent biomarkers indicating presence of periodontopathic bacteria.

Dipeptidyl peptidase (DPP) 4, DPP5, DPP7 and DPP11, expressed in the periplasmic space, are crucial for energy production for Porphyromonas gingivalis, an asaccharolytic bacterium that causes periodontal disease. Bacterial DPP4 seems to be involved in regulation of blood glucose level via degradation of incretins. The present study aimed to identify four dpp orthologs in oral microbiota by database searches, and their enzymatic activities in periodontopathic and cariogenic bacteria, as well as oral specimens were determined. Search in the databases suggested that 43 species of 772 taxa possess dpp4 and other dpp genes. Most species are in the genera Bacteroides, Capnocytophaga, Porphyromonas, Prevotella and Tannerella, indicating a limited distribution of dpp orthologs in anaerobic periodontopathic rods. In accordance with those results, activities of all four DPPs were demonstrated in P. gingivalis, Porphyromonas endodontalis and Tannerella forsythia, while they were negligible in Treponema denticola, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Furthermore, DPP activities were also detected in subgingival dental plaque at different intensities among individual specimens, while DPP4 activity presumably derived from human entity was solely predominant in saliva samples. These findings demonstrated that DPP activities in dental plaque serve as potent biomarkers to indicate the presence of periodontopathic bacteria.

The fibronectin-binding protein homologue Fbp62 of Streptococcus anginosus is a potent virulence factor.

Streptococcus anginosus appears to be able to adhere to cultured epithelial cells or fibronectin and this may be associated with bacterial pathogenicity. In the present study, the molecular characteristics and virulence of the fibronectin-binding protein (FBP), Fbp62, of S. anginosus were investigated in animal models to determine the role of the molecule in bacterial infection. fbp62 encodes a 549 amino acid residue with an apparent molecular mass of 62.8 kDa that lacks a membrane anchor motif and a leader peptide, suggesting that fbp62 codes for an atypical FBP. It has been observed that the S. anginosus Fbp62 is very similar to the FbpA of Streptococcus gordonii, PavA of Streptococcus pneumoniae, SmFnB of Streptococcus mutans and Fbp54 of Streptococcus pyogenes. Recombinant Fbp62 prepared from pGEX-4T-2 was found to bind to fibronectin in a dose-dependent manner and competitively inhibit the binding of S. anginosus to fibronectin. Furthermore, anti-Fbp62 antiserum abrogated the binding of S. anginosus to fibronectin. Adhesion of the isogenic mutant, Δfbp62, constructed from S. anginosus NCTC 10713 (wild-type, WT) by homologous recombination to HEp-2 cells and DOK cells was significantly weaker than that of S. anginosus WT. In addition, Δfbp62's lethality and ability to form abscesses were weaker in a mouse model of infection than in the WT strain. Taken together, these results suggest that Fbp62 is an important pathogenic factor of S. anginosus.

Aciduricity and acid tolerance mechanisms of Streptococcus anginosus.

Although Streptococcus anginosus constitutes a proportion of the normal flora of the gastrointestinal and genital tracts, and the oral cavity, it has been reported that S. anginosus infection could be closely associated with abscesses at various body sites, infective endocarditis, and upper gastrointestinal cancers. The colonization in an acidic environment due to the aciduricity of S. anginosus could be the etiology of the systemic infection of the bacteria. To elucidate the aciduricity and acid tolerance mechanisms of the microbe, we examined the viability and growth of S. anginosus under acidic conditions. The viabilities of S. anginosus NCTC 10713 and Streptococcus mutans ATCC 25175 at pH 4.0 showed as being markedly higher than those of Streptococcus sanguinis ATCC 10556, Streptococcus gordonii ATCC 10558, and Streptococcus mitis ATCC 49456; however, the viability was partially inhibited by dicyclohexylcarbodiimide, an H+-ATPase inhibitor, suggesting that H+-ATPase could play a role in the viability of S. anginosus under acidic conditions. In addition, S. anginosus NCTC 10713 could grow at pH 5.0 and showed a marked arginine deiminase (ADI) activity, unlike its ΔarcA mutant, deficient in the gene encoding ADI, and other streptococcal species, which indicated that ADI could also be associated with aciduricity. These results suggest that S. anginosus has significant aciduric properties, which can be attributed to these enzyme activities.