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.

Oral and Gut Microbial Dysbiosis and Non-alcoholic Fatty Liver Disease: The Central Role of Porphyromonas gingivalis.

Gut microbiota play many important roles, such as the regulation of immunity and barrier function in the intestine, and are crucial for maintaining homeostasis in living organisms. The disruption in microbiota is called dysbiosis, which has been associated with various chronic inflammatory conditions, food allergies, colorectal cancer, etc. The gut microbiota is also affected by several other factors such as diet, antibiotics and other medications, or bacterial and viral infections. Moreover, there are some reports on the oral-gut-liver axis indicating that the disruption of oral microbiota affects the intestinal biota. Non-alcoholic fatty liver disease (NAFLD) is one of the systemic diseases caused due to the dysregulation of the oral-gut-liver axis. NAFLD is the most common liver disease reported in the developed countries. It includes liver damage ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. Recently, accumulating evidence supports an association between NAFLD and dysbiosis of oral and gut microbiota. Periodontopathic bacteria, especially Porphyromonas gingivalis, have been correlated with the pathogenesis and development of NAFLD based on the clinical and basic research, and immunology. P. gingivalis was detected in the liver, and lipopolysaccharide from this bacteria has been shown to be involved in the progression of NAFLD, thereby indicating a direct role of P. gingivalis in NAFLD. Moreover, P. gingivalis induces dysbiosis of gut microbiota, which promotes the progression of NAFLD, through disrupting both metabolic and immunologic pathways. Here, we review the roles of microbial dysbiosis in NAFLD. Focusing on P. gingivalis, we evaluate and summarize the most recent advances in our understanding of the relationship between oral-gut microbiome symbiosis and the pathogenesis and progression of non-alcoholic fatty liver disease, as well as discuss novel strategies targeting both P. gingivalis and microbial dysbiosis.

Epigallocatechin gallate stimulated histamine production and downregulated histamine H1 receptor in oral cancer cell lines expressing histidine decarboxylase.

OBJECTIVES: Increased histamine production and the overexpression of receptors (H1R∼H4R) has been reported in several tumors. The effects of TGFß1 and epigallocatechin gallate (EGCG) on histamine synthesizing enzymes (HDCs), and the histamine transporter systems and receptors were investigated in this study. METHODS: Four oral cancer cell lines (HSC2, HSC3, HSC4, and SAS) were treated with or without TGFß1 or EGCG for 24 h. The expression levels of HDC, SLC22A3, H1R∼H4R, and TAS2R14 were investigated by Western blotting. Histamine concentrations were determined using the enzyme immune assay. Bitter taste receptor (TAS2R14 and TAS2R39) mRNAs were investigated by RT-PCR. RESULTS: Varying expression levels of HDC, SLC22A3, H1R∼H4R, and TAS2R14 were observed in the four cell lines, where histamine concentrations were found to be ∼500 fmol/ml in cell culture media and induced 2-2.5 times higher amounts of histamine following EGCG treatment. TGFß1 increased HDC expression in three cell lines, SLC22A3 expression in three cell lines, H1R expression in two cell lines, H2R expression in three cell lines, H3R expression in three cell lines, and H4R expression in three cell lines. EGCG decreased HDC expression in all four cell lines, SLC22A3 expression in three expression, H1R expression in all four cell lines, H2R expression in two cell lines, H3R expression in three cell lines, and H4R expression in two cell lines. CONCLUSIONS: EGCG upregulated histamine production and decreased the expression level of H1R in the oral cancer cell lines. It might prove useful for cancer therapy during histamine regulation.

Uptake of Nanotitania by Gingival Epithelial Cells Promotes Inflammatory Response and Is Accelerated by Porphyromonas gingivalis Lipopolysaccharide: An In Vitro Study.

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonasgingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.

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.

Oral keratinocyte-derived exosomes regulate proliferation of fibroblasts and epithelial cells.

Extracellular vesicles (EVs), including exosomes, are small membrane-bound particles released by cells. From a therapeutic point of view, EVs can often convey similar biological function as their parent cell. Grafts originating from oral mucosa have frequently been used in regenerative medicine, and we have previously described the use of oral cell sheets to prevent stricture formation of the esophagus. Further, we recently found that exosomes derived from these cell sheets have pro-regenerative effect on skin wound healing. Here, we have isolated exosomes from conditioned media from oral keratinocyte ("OKEx") and dermal fibroblast ("FEx") cultures. The exosomes were probed for classical EV-markers by western blot (CD9, annexin V and Flotillin-1), FEx were positive for all markers while OKEx were positive only for CD9. Tunable resistive pulse sensing indicated a mean size of around 110 nm and transmission electron microscopy showed a spherical morphology, for both groups. After fluorescent labelling, we studied the uptake of exosomes co-cultured with fibroblasts or keratinocytes. Signal from OKEx could be detected after 90 min, and signal could be detected in all groups after 16 h. Finally we studied the exosomes' modulation of cell proliferation. Both groups suppressed proliferation of healthy keratinocyte and fibroblasts, at some doses to similar levels as dexamethasone (a drug commonly used to prevent stricture formation). In contrast, the exosomes also suppressed the proliferation of the carcinoma cell line TR146, while dexamethasone had no effect. In conclusion, we believe that exosome-signaling might be one of the mode-of-actions of cell sheet-therapy for stricture prevention.

Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing.

The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer.

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.

Porphyromonas gingivalis is highly sensitive to inhibitors of a proton-pumping ATPase.

Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane.

Laminins 411 and 421 differentially promote tumor cell migration via α6ß1 integrin and MCAM (CD146).

α4-laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by blood and lymphatic vessels and some tumor cells. Laminin-411 promotes migration of leukocytes and endothelial cells, but the effect of this laminin and laminin-421 on tumor cells is poorly understood. In the present study, we demonstrate that laminin-411 and, to a greater extent, laminin-421 significantly promote migration of tumor cells originated from melanomas, gliomas and different carcinomas via α6ß1 integrin. In solid-phase binding assays, both laminins similarly bound α6ß1 integrin but only laminin-421, among several laminin isoforms, readily bound MCAM (CD146), a cell-surface adhesion molecule strongly associated with tumor progression. Accordingly, a function-blocking mAb to MCAM inhibited tumor cell migration on laminin-421 but not on laminins 411 or 521. In tumor tissues, melanoma cells co-expressed MCAM, laminin α4, ß1, ß2 and γ1 chains, and integrin α6 and ß1 chains. The present data highlight the novel role of α4-laminins in tumor cell migration and identify laminin-421 as a primary ligand for MCAM and a putative mediator of tumor invasion and metastasis.

Monoclonal antibodies to human laminin α4 chain globular domain inhibit tumor cell adhesion and migration on laminins 411 and 421, and binding of α6ß1 integrin and MCAM to α4-laminins.

α4-Laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by vascular and lymphatic endothelial cells, leukocytes and other normal cell types. These laminins are recognized by α6ß1 and α6ß4 integrins and MCAM (CD146), and promote adhesion and migration of the cells. α4-Laminins are also expressed and secreted by some tumor cells and strongly promote tumor cell migration. Moreover, the abluminal side of blood and/or lymphatic vessels and the nerve perineurium, common tracks of tumor cell dissemination, express α4-laminins, and these laminin isoforms, when expressed in the stroma, may contribute to tumor invasion. In the present study, we examined ten mAbs to human laminin α4 chain for their reactivity with the isolated laminin α4 globular domain, their ability to inhibit tumor cell adhesion and migration on laminins 411 and 421, and their effect on the binding of α6ß1 integrin and MCAM to both α4-laminins. Most of the mAbs reacted with the laminin α4 globular domain, but only two, mAbs FC10 and 084, significantly inhibited tumor cell adhesion and migration on laminin-411. When used in combination, these antibodies practically abolished the cell adhesion and migration on laminin-411 and significantly reduced the cellular responses on laminin-421. Accordingly, mAbs FC10 and 084 significantly inhibited the binding of purified α6ß1 integrin and MCAM to laminins 411 and 421. These results indicate that mAbs to the laminin α4 globular domain are able to inhibit tumor cell adhesion and migration on laminins 411 and 421, and that α6ß1 integrin and MCAM bind α4-laminins at very close sites on the globular domain. These reagents contribute to a better understanding of the biology of α4-laminins and may have a therapeutic potential in malignant and inflammatory diseases.

A novel monoclonal antibody to human laminin α5 chain strongly inhibits integrin-mediated cell adhesion and migration on laminins 511 and 521.

Laminins, a large family of αßγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5ß1γ1) and 521 (α5ß2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3ß1/α6ß1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3ß1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMß1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.