SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

In vivo expression of proteases and protease inhibitor,a serpin,by periodontal pathogens at teeth and implants

Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK‐proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri‐implant health and disease conditions. Biofilm and gingival crevicular fluid (GCF)/ peri‐implant sulcular fluid (PISF) samples were taken from healthy tooth and implant sites (n = 10), gingivitis and mucositis sites (n = 12), and periodontitis and peri‐implantitis sites (n = 10). Concentration of interleukin‐8 (IL‐8), IL‐1β and IL‐10 in GCF was determined by enzyme‐linked immunosorbent assay. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by quantitative PCR, whereas gene expression was estimated by quantitative RT‐PCR. The presence of P. gingivalis and T. forsythia, as well as the level of IL‐8 and IL‐1β, were associated with disease severity in the periodontal and peri‐implant tissues. In biofilm samples harboring T. forsythia, genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK‐protease genes and miropin. At the same time, detectable mRNA expression of individual genes ranged from 20.7% to 58.6% of samples (for forsylisin and miropsin‐1, respectively). In comparison with the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK‐proteases and especially miropin, might play a role in pathogenesis of both periodontal and peri‐implant diseases.     

Effects of different topical agents on enamel demineralization around orthodontic brackets: an in vivo and in vitro study

Background: The aim of this study was to evaluate the in vivo and in vitro effects of a casein phosphopeptide‐amorphous calcium phosphate (CPP‐ACP) and fluoride containing topical agents in reducing enamel demineralization around orthodontic brackets, and to compare this with a control group. Methods: Twenty‐one patients and 60 extracted premolars were divided into three groups: two experimental and one control. Tooth Mousse^® (CPP‐ACP gel; GC‐Corp, Tokyo, Japan) and Fluoridin N5^® (Fluoride gel; Voco‐GmbH, Cuxhaven, Germany) were applied to tooth surfaces around orthodontic brackets in the experimental groups. Teeth were extracted after 60 days to evaluate the in vivo effects of the testing materials. For the in vitro experiment, samples were cycled for 14 days through a daily procedure of demineralization. All teeth were sectioned and evaluated by superficial microhardness analysis. An indentation was made from two positions (occlusal‐cervical) and one depth (10 μm). Results: Comparisons of occlusal and cervical microhardness scores for all specimens showed no statistically significant side differences. A multiple comparison test showed that the use of CPP‐ACP and fluoride containing topical gels were more significantly efficient than the control group (p <0.001). No significant differences were detected between CPP‐ACP and the fluoride groups against demineralization. Conclusions: In vivo and in vitro evaluations indicated that CPP‐ACP and fluoride containing agents successfully inhibited caries around orthodontic brackets.     

Dentin tubule invasion by Enterococcus faecalis under stress conditions ex vivo

Enterococcus faecalis is the species most frequently isolated from failed endodontic treatments because it can survive under stress conditions imposed by root canal treatment. The objective of this study was to determine the ability of E. faecalis to invade dentine tubules under alkaline and energy‐starvation stress and to explore the potential mechanisms. Roots from single‐rooted human teeth were infected with E. faecalis under alkaline and energy‐starvation stress conditions. After 4 wk of culture, samples were processed to establish the tubule‐penetration distance. In addition, the hydrophobicity of E. faecalis cells under these conditions was analysed and the expression of genes involved in adhesion was quantified by real‐time quantitative PCR. Culture of E. faecalis under alkaline and energy‐starvation stress conditions resulted in a marked reduction of tubule‐penetration distance, a significant increase in hydrophobicity of the bacterial surface, and marked down‐regulation of most adhesin genes compared with E. faecalis cultured in tryptic soy broth. The results indicate that the dentine tubule invasion ability of E. faecalis was markedly decreased under alkaline and glucose‐starvation stress conditions, possibly because of the increase of hydrophobicity and down‐regulation of some adhesion genes.     

Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis-affected gingival tissues

Abe D, Kubota T, Morozumi T, Shimizu T, Nakasone N, Itagaki M, Yoshie H. Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis‐affected gingival tissues. J Periodont Res 2011; 46: 345–353. © 2011 John Wiley & Sons A/S Background and Objective: Gene expression is related to the pathogenesis of periodontitis and plays a crucial role in local tissue destruction and disease susceptibility. The aims of the present study were to identify the expression of specific genes and biological pathways in periodontitis‐affected gingival tissue using microarray and quantitative real‐time RT‐PCR analyses. Material and Methods: Healthy and periodontitis‐affected gingival tissues were taken from three patients with severe chronic periodontitis. Total RNAs from six gingival tissue samples were used for microarray analyses. Data‐mining analyses, such as comparisons, gene ontology and pathway analyses, were performed and biological pathways with a significant role in periodontitis were identified. In addition, quantitative real‐time RT‐PCR analysis was performed on samples obtained from 14 patients with chronic periodontitis and from 14 healthy individuals in order to confirm the results of the pathway analysis. Results: Comparison analyses found 15 up‐regulated and 13 down‐regulated genes (all of which showed a change of more than twofold in expression levels) in periodontitis‐affected gingival tissues. Pathway analysis identified 15 up‐regulated biological pathways, including leukocyte transendothelial migration, and five down‐regulated pathways, including cell communication. Quantitative real‐time RT‐PCR verified that five genes in the leukocyte transendothelial migration pathway were significantly up‐regulated, and four genes in the cell communication pathway were significantly down‐regulated, which was consistent with pathway analysis. Conclusion: We identified up‐regulated genes (ITGB‐2, MMP‐2, CXCL‐12, CXCR‐4 and Rac‐2) and down‐regulated genes (connexin, DSG‐1, DSC‐1 and nestin) in periodontitis‐affected gingival tissues; these genes may be related to the stimulation of leukocyte transendothelial migration and to the the impairment of cell‐to‐cell communication in periodontitis.     

Elevated MicroRNA‐128 in Periodontitis Mitigates Tumor Necrosis Factor‐α Response via p38 Signaling Pathway in Macrophages

Background: Periodontitis is a chronic inflammatory disease resulting from an inflammatory response to subgingival plaque bacteria, including Porphyromonas gingivalis. MicroRNA (miRNA) is a current focus in regulating the inflammatory processes. In this study, the inflammatory miRNA expression in gingival tissues of patients with periodontitis and of healthy individuals is compared, and its role in regulating the inflammatory response is examined. Methods: Gingival tissues from patients with periodontitis and healthy individuals were collected for miRNA microarray. THP‐1 and CA9‐22 cells were challenged with P. gingivalis, and miRNA expression was determined by real‐time polymerase chain reaction. Target genes for miRNA were predicted using TargetScanHuman database, and miRNA gene expressions were reviewed using public databases. For the functional study, THP‐1 cells were transfected with a miRNA‐128 mimic, and target gene expression was compared with THP‐1 cells challenged with P. gingivalis. For the tolerance test, THP‐1 cells transfected with miRNA‐128 mimic were treated with phorbol 12‐myristate 13‐acetate (PMA) or paraformaldehyde (PFA)‐fixed Escherichia coli. Tumor necrosis factor (TNF)‐α production was determined by enzyme‐linked immunosorbent assay, and mitogen‐activated protein kinase (MAPK) protein phosphorylation was determined by Western blot. Results: Gingival tissues from patients with periodontitis showed increased expression of miRNA‐128, miRNA‐34a, and miRNA‐381 and decreased expression of miRNA‐15b, miRNA‐211, miRNA‐372, and miRNA‐656. THP‐1 cells and CA9‐22 cells challenged with P. gingivalis showed increased miRNA‐128 expression. Among the predicted miRNA‐128 target genes, several genes that are involved in MAPK signaling pathway showed similar gene expression pattern between P. gingivalis challenge and miRNA‐128 mimic transfection. In THP‐1 cells transfected with miRNA‐128 mimic, TNF‐α production was lower, and phosphorylation of p38 was inhibited when challenged with PMA or PFA‐fixed E. coli. Conclusion: miRNA‐128 may be involved in mitigating the inflammatory response induced by P. gingivalis in periodontitis.     

Effect of the quorum‐sensing luxS gene on biofilm formation by Enterococcus faecalis

Enterococcus faecalis is the species of bacterium most frequently isolated from the root canals of teeth that exhibit chronic apical periodontitis refractory to endodontic treatment. In this study, we evaluated the effect of the S‐ribosylhomocysteine lyase (luxS) quorum‐sensing gene on E. faecalis biofilm formation by constructing a knockout mutant. The biofilms formed by both E. faecalis and its luxS mutant strain were evaluated using the MTT method. Important parameters that influence biofilm formation, including cell‐surface hydrophobicity and the nutrient content of the growth medium, were also studied. Biofilm structures were observed using confocal laser scanning microscopy (CLSM), and expression of biofilm‐related genes was investigated using RT‐PCR. The results showed that the luxS gene can affect biofilm formation, whereas it does not affect the bacterial growth rate. Deletion of the luxS gene also increased cell‐surface hydrophobicity. Biofilm formation was accelerated by the addition of increasing concentrations of glucose. The CLSM images revealed that the luxS mutant strain tends to aggregate into distinct clusters and relatively dense structures, whereas the wild‐type strain appears confluent and more evenly distributed. All genes examined were up‐regulated in the biofilms formed by the luxS mutant strain. The quorum‐sensing luxS gene can affect E. faecalis biofilm formation.     

Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis

Hayashi Y, Matsunaga T, Yamamoto G, Nishii K, Usui M, Yamamoto M, Tachikawa T. Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis. J Periodont Res 2010; 45: 618–625. © 2010 John Wiley & Sons A/S Background and Objective: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. Material and Methods: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high‐quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real‐time PCR and immunohistochemistry. Results: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up‐regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50‐fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100‐fold higher than in the oral gingival epithelium by real‐time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. Conclusion: We developed a method for generating fresh‐frozen tissue sections suitable for extraction of good‐quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.     

Apoptotic gene expression by human periodontal ligament cells following cyclic stretch

Xu C, Hao Y, Wei B, Ma J, Li J, Huang Q, Zhang F. Apoptotic gene expression by human periodontal ligament cells following cyclic stretch. J Periodont Res 2011; 46: 742–748. © 2011 John Wiley & Sons A/S Background and Objective: Periodontal ligament cells play an important role in maintaining homeostasis of periodontal tissue upon mechanical force loading caused by mastication or orthodontic force. Previous studies revealed force‐driven periodontal ligament cell death via apoptosis, but the force‐sensing genes assigned to the apoptotic pathway have not been fully characterized. The present study aimed to identify force‐sensing genes implicated in the apoptotic pathway in periodontal ligament cells. Material and Methods: Human periodontal ligament cells were exposed to 20% stretch strain for 6 or 24 h, and the differential expression of 84 genes implicated in the apoptotic pathway were quantified by real‐time PCR array technology. Results: Ten and 11 genes showed upregulated expression after 6 and 24 h stretches, respectively, and there were two downregulated genes in response to both 6 and 24 h stretches. These genes included those encoding the tumor necrosis factor ligand family (TNFSF8), tumor necrosis factor receptor family (FAS, TNFRSF10B, TNFRSF11B, TNFRSF25 and CD27), the Bcl‐2 family (BAG3, BAK1, BCL2L11 and BCLAF1), the caspase family (CASP5 and CASP7), the inhibitor of apoptosis proteins family (BIRC3, BIRC6 and NAIP), the caspase recruitment domain family (RIPK2 and PYCARD) and the death domain family (DAPK1), as well as an oncogene (BRAF). Conclusion: This study identified several force‐sensing genes implicated in the apoptotic pathway in periodontal ligament cells and should facilitate future studies on force‐driven apoptosis by providing putative target genes.     

Fos‐related activator‐1 is overexpressed in oral squamous cell carcinoma and associated with tumor lymph node metastasis

J Oral Pathol Med (2010) 39 : 470–476 Background: Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB cells. Then, expression microarray analysis showed that the gene encoding fos‐related activator‐1 (Fra‐1) was significantly upregulated in the cancerous HB cells compared with HIOECs. Methods: To confirm the expression of Fra‐1 at mRNA and protein levels by real‐time PCR and western blotting analysis in the carcinogenesis model of OSCC and CAL27 cells. To investigate Fra‐1 expression in clinical samples from 30 primary OSCC patients by immunohistochemistry. Results: Fra‐1 expression was increased both at mRNA and protein levels in this carcinogenesis model of OSCC and CAL27 cells. Nuclear and cytoplasmic Fra‐1 protein expressions both increased in the cancerous tissues compared with those in the paired adjacent non‐malignant epithelia (nuclear: P < 0.001, cytoplasmic: P = 0.003). A higher level of nuclear Fra‐1 expression was seen in the tumor samples of patients with lymph node metastasis than those without lymph node metastasis (5.07 ± 1.33 vs 3.81 ± 1.33, P = 0.023). Higher level of Fra‐1 expression was also found in the tumor invasive margin than tumor center. Conclusions: Fra‐1 is a positive gene of OSCC development and progression, Fra‐1 can be used as a potential therapeutic target gene and an additional marker for evaluation of lymph node metastasis.     

The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture

Saminathan A, Vinoth KJ, Wescott DC, Pinkerton MN, Milne TJ, Cao T, Meikle MC. The effect of cyclic mechanical strain on the expression of adhesion‐related genes by periodontal ligament cells in two‐dimensional culture. J Periodont Res 2012; 47: 212–221. © 2011 John Wiley & Sons A/S Background and Objective: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion‐related genes by human periodontal ligament cells. Material and Methods: Cultured periodontal ligament cells were subjected to a cyclic in‐plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6–24 h in a Flexercell FX‐4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase‐3 and ‐7 activity; and (iii) the expression of 84 genes encoding adhesion‐related molecules using real‐time RT‐PCR microarrays. Results: Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase‐3 and ‐7 activity at 6 and 12 h. Seventy‐three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α‐chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76‐fold induction of SPP1 at 12 h to a 2.49‐fold downregulation of COL11A1 at 24 h. Conclusion: The study has identified several mechanoresponsive adhesion‐related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis ‘executioner’ caspases.     

Porphyromonas gingivalis inhibits M2 activation of macrophages by suppressing α‐ketoglutarate production in mice

Reprograming of metabolic pathways is critical in governing the polarization of macrophages into classical proinflammatory M1 or alternative anti‐inflammatory M2 phenotypes in metabolic diseases, such as diabetes. Porphyromonas gingivalis, a keystone pathogen of periodontitis, causes an imbalance in M1/M2 activation, resulting in a hyperinflammatory environment that promotes the pathogenesis of periodontitis. However, whether P. gingivalis infection modulates metabolic pathways to alter macrophage polarization remains unclear. Bone‐marrow‐derived macrophages (BMDMs) were collected from 6‐week‐old female C57BL/6 mice and stimulated with P. gingivalis, P. gingivalis‐derived LPS or IL‐4. Relative gene expression and protein production were measured by quantitative real‐time PCR, RNA sequencing and western blotting. Colorimetric assays were also performed to assess the amounts of α‐ketoglutarate (α‐KG) and succinate. P. gingivalis or P. gingivalis‐derived LPS‐induced inflammatory responses enhanced M1 macrophages and suppressed M2 macrophages, even in the presence of IL‐4. P. gingivalis inhibited Idh1/2 and Gpt1/2 mRNA expression, and increased Akgdh mRNA expression, thus decreasing the ratio of α‐KG/succinate. Supplementation of cell‐permeable dimethyl‐α‐KG dramatically restored M2 activation during P. gingivalis infection. Our study suggests that P. gingivalis maintains a hyperinflammatory state by suppressing the production of α‐KG by M2 macrophages.     

Porphyromonas gingivalis Lipopolysaccharide Upregulates Insulin Secretion From Pancreatic β Cell Line MIN6

Background: A close association between periodontitis and diabetes has been demonstrated in human cross‐sectional studies, but an exact relationship between periodontitis and prediabetes has not been established. Previous studies using animal model systems consistently have shown that hyperinsulinemia occurs in animals with periodontitis compared to animals with healthy periodontium (while maintaining normoglycemia). Because bacterial lipopolysaccharide (LPS) plays an important role in the pathogenesis of periodontitis, we hypothesized that LPS may stimulate insulin secretion through a direct effect on β cell function. To test this hypothesis, pancreatic β cell line MIN6 cells were used to determine the effect of Porphyromonas gingivalis (Pg) LPS on insulin secretion. Furthermore, expression of genes altered by Pg LPS in innate immunity and insulin‐signaling pathways was determined. Methods: MIN6 cells were grown in medium with glucose concentration of normoglycemia (5.5 mM). Pg LPS was added to each well at final concentrations of 50, 200, and 500 ng/mL. Insulin secretion was measured using enzyme‐linked immunosorbent assay. Gene expression levels altered by Pg LPS were determined by polymerase chain reaction (PCR) array for mouse innate and adaptive immunity response and mouse insulin‐signaling pathways, and results were confirmed for specific genes of interest by quantitative PCR. Results: Pg LPS stimulated insulin secretion in the normoglycemic condition by ≈1.5‐ to 3.0‐fold depending on the concentration of LPS. Pg LPS treatment altered the expression of several genes involved in innate and adaptive immune response and insulin‐signaling pathway. Pg LPS upregulated the expression of the immune response–related genes cluster of differentiation 8a (Cd8a), Cd14, and intercellular adhesion molecule‐1 (Icam1) by about two‐fold. LPS also increased the expression of two insulin signaling–related genes, glucose‐6‐phosphatase catalytic subunit (G6pc) and insulin‐like 3 (Insl3), by three‐ to four‐fold. Conclusions: We have demonstrated for the first time that Pg LPS stimulates insulin secretion by pancreatic β cell line MIN cells. Pg LPS may have significant implications on the development of β cell compensation and insulin resistance in prediabetes in individuals with periodontitis.     

Deletion of csn2 gene affects acid tolerance and exopolysaccharide synthesis in Streptococcus mutans

Csn2 is an important protein of the CRISPR‐Cas system. The physiological function of this protein and its regulatory role in Streptococcus mutans, as the primary causative agent of human dental caries, is still unclear. In this study, we investigated whether csn2 deletion would affect S. mutans physiology and virulence gene expression. We used microscopic imaging, acid killing assays, pH drop, biofilm formation, and exopolysaccharide (EPS) production tests to determine whether csn2 deletion influenced S. mutans colony morphology, acid tolerance/production, and glucan formation abilities. Comparisons were made between quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR) data from the UA159 and csn2 deletion strain to determine the impact of csn2 knockout on S. mutans gene expression. The results showed that deletion of S. mutans csn2 changed its colony morphotype and made it more sensitive to acid. The expression levels of aciduricity genes, including leuA, leuB, leuC, and leuD, were significantly down‐regulated. Acid adaptation restored the aciduricity of csn2 mutant and enhanced the ability to synthesize EPS. The expression levels of EPS synthesis‐related genes, including gtfC and gtfD, were significantly up‐regulated after acid adaptation. In summary, deletion of S. mutans csn2 exerted multiple effects on the virulence traits of this pathogen, including acid tolerance and EPS formation, and that these alterations could partially be attributed to changes in gene expression upon loss of csn2. Understanding the function of csn2 in S. mutans might lead to novel strategies to prevent or treat imbalances in oral microbiota that may favor diseases.