Porphyromonas gingivalis Mfa1 fimbria putatively binds to TLR2 and induces both IL-6 and IL-8 production in human bronchial epithelial cells.

Porphyromonas gingivalis (Pg) a major periodontal pathogen involved in periodontal disease development and progression. Moreover, Pg has two fimbriae surface proteins (FimA and Mfa1) that are genetically distinct and make-up the fimbrial shaft which in-turn form crucial attachment to oral bacteria and multiple host cells. However, unlike FimA, Mfa1 attachment to non-periodontal cells has not been fully elucidated. Considering Pg-associated periodontal disease contributes to pulmonary disease development, we investigated whether Mfa1 can functionally interact with human bronchial epithelial cells and, likewise, trigger a functional response. Initially, we simulated molecular docking and performed both luciferase and neutralization assays to confirm Mfa1-related functional interaction. Subsequently, we treated BEAS-2B cells with purified Mfa1 and performed cytokine quantification through real time-PCR and ELISA to establish Mfa1-related functional response. We found that both Mfa1-TLR2 and Mfa1-TLR4 docking is possible, however, only Mfa1-TLR2 showed a functional interaction. Additionally, we observed that both IL-8 and IL-6 gene expression and protein levels were induced confirming Mfa1-related functional response. Taken together, we propose that BEAS-2B human bronchial epithelial cells are able to recognize Pg Mfa1 and induce both IL-8 and IL-6 inflammatory responses.

Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells.

The effect of Mfa1 fimbriae of Porphyromonas gingivalis on the progression of bone resorption remains unclear, especially compared with another fimbriae, FimA. We investigated the effect of Mfa1 on osteoclastogenesis together with FimA. We also investigated the role of Toll-like receptors (TLRs) in Mfa1 recognition during osteoclast differentiation. Receptor activator of nuclear factor κß ligand (RANKL)-prestimulated RAW264 cells were used to examine the effects of purified Mfa1 fimbriae. The number of osteoclasts was examined by tartrate-resistant acid phosphate (TRAP) staining, osteoclast activation was investigated by bone resorption assays, and gene expression of differentiation markers was examined by quantitative real-time PCR. Transfection of Tlr2 and Tlr4 siRNAs into RAW264 cells was also employed and their role in Mfa1 recognition was investigated. Mfa1 effectively induced the formation of TRAP-positive multinucleated cells and activated osteoclasts. Mfa1 also increased gene expression of Acp5, Mmp9, and Ctsk in RANKL-prestimulated RAW264 cells compared with the control. The osteoclastogenesis induced by Mfa1 was significantly decreased in cells transfected with Tlr2 or Tlr4 siRNAs compared with control siRNA. Our results revealed the role of Mfa1 fimbriae in osteoclastogenesis that may contribute to the partial elucidation of the mechanisms of periodontal disease progression and the development of new therapeutic strategies.

Porphyromonas gingivalis Components/Secretions Synergistically Enhance Pneumonia Caused by Streptococcus pneumoniae in Mice.

Streptococcus pneumoniae is an important causative organism of respiratory tract infections. Although periodontal bacteria have been shown to influence respiratory infections such as aspiration pneumonia, the synergistic effect of S. pneumoniae and Porphyromonas gingivalis, a periodontopathic bacterium, on pneumococcal infections is unclear. To investigate whether P. gingivalis accelerates pneumococcal infections, we tested the effects of inoculating P. gingivalis culture supernatant (PgSup) into S. pneumoniae-infected mice. Mice were intratracheally injected with S. pneumoniae and PgSup to induce pneumonia, and lung histopathological sections and the absolute number and frequency of neutrophils and macrophages in the lung were analyzed. Proinflammatory cytokine/chemokine expression was examined by qPCR and ELISA. Inflammatory cell infiltration was observed in S. pneumoniae-infected mice and S. pnemoniae and PgSup mixed-infected mice, and mixed-infected mice showed more pronounced inflammation in lung. The ratios of monocytes/macrophages and neutrophils were not significantly different between the lungs of S. pneumoniae-infected mice and those of mixed-infected mice. PgSup synergistically increased TNF-α expression/production and IL-17 production compared with S. pneumoniae infection alone. We demonstrated that PgSup enhanced inflammation in pneumonia caused by S. pneumoniae, suggesting that virulence factors produced by P. gingivalis are involved in the exacerbation of respiratory tract infections such as aspiration pneumonia.

Comparison of disinfection effect between benzalkonium chloride and povidone iodine in nasotracheal intubation: a randomized trial.

BACKGROUND: Nasotracheal intubation can potentially result in microbial contamination from the upper respiratory tract to the lower respiratory tracts. However, an ideal nasotracheal disinfection method is yet to be determined. Therefore, we compared the disinfection effects between benzalkonium chloride and povidone iodine in nasotracheal intubation. METHODS: Overall, this study enrolled 53 patients aged 20-70 years who were classified into classes 1 and 2 as per American Society of Anesthesiologists-physical status and were scheduled to undergo general anesthesia with NTI. Patients who did not give consent (n = 2) and who has an allergy for BZK or PVI were excluded from the study. The patients were randomly divided into two groups on the basis of the disinfection method: BZK (n = 26, one patient was discontinued intervention) and PVI (n = 25). 50 patients were assessed finally. The subjects' nasal cavities were swabbed both before (A) and after disinfection (B), and the internal surface of the endotracheal tube was swabbed after extubation (C). The swabs were cultured on Brain heart infusion agar and Mannitol salt agar. The number of bacteria per swab was determined and the rates of change in bacterial count (B/A, C/B) were calculated. The growth inhibitory activity of the disinfectants on Staphylococcus aureus were also investigated in vitro. RESULTS: Although the initial disinfection effects (B/A) were inferior for benzalkonium chloride compared with those for povidone iodine, the effects were sustained for benzalkonium chloride (C/B). In the in vitro growth inhibitory assay against S. aureus, benzalkonium chloride showed higher inhibitory activity than povidone iodine. CONCLUSION: Although both disinfectants were inactivated or diffused/diluted over time, benzalkonium chloride maintained the threshold concentration and displayed antimicrobial effects longer than povidone iodine; therefore, benzalkonium chloride appeared to show a better sustained effect. Benzalkonium chloride can be used for creating a hygienic nasotracheal intubation environment with sustained sterilizing effects. TRIAL REGISTRATION: UMIN-CTR (Registration No. UMIN000029645 ). Registered 21 Oct 2017.

IL-15 and RANKL Play a Synergistically Important Role in Osteoclastogenesis.

Interleukin-15 (IL-15), a cytokine secreted by several cell types, has important physiological roles in the activity, proliferation, and viability of immune cells. It has both chemoattractant and proinflammatory properties, and may promote bone destruction. A previous study has shown that IL-15 alone exerts no effect on osteoclastogenesis. Therefore, the current study addressed the synergistic effect of IL-15 on osteoclast formation using RAW264.7 (RAW) cells by co-stimulation with receptor activator of nuclear factor (NF)-κB ligand (RANKL) that has a major role in osteoclastogenesis involving the pathogenesis of rheumatoid arthritis and periodontal disease. Co-stimulation of RAW cells by IL-15 and RANKL significantly increased the gene expression of osteoclast differentiation and osteoclastogenesis markers compared with stimulation by RANKL or IL-15 independently as evaluated by tartrate-resistant acid phosphate-positive cell numbers, the fusion index, a pit formation assay with Alizarin red staining (calcification estimation), and quantitative polymerase chain reaction. Phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase, p38 mitogen-activated protein kinase, and NF-κB was significantly increased by RANKL and IL-15 (P < 0.05) compared with RANKL alone. In addition, these differentiation activities induced by RANKL and IL-15 were comparatively suppressed by inhibition of ERK, suggesting that this synergistic effect on osteoclastogenesis is mainly mediated by ERK. Taken together, our results demonstrate that IL-15 and RANKL induce osteoclastogenesis synergistically, and IL-15 might play a novel and major role in destructive inflammatory bone diseases. J. Cell. Biochem. 118: 739-747, 2017. © 2016 Wiley Periodicals, Inc.

Comparative analysis of motility and other properties of Treponema denticola strains.

The periodontitis-associated pathogen Treponema denticola is a spirochetal bacterium that swims by rotating its cell body like a corkscrew using periplasmic flagella. We compared physiologic and pathogenic properties, including motility, in four strains of T. denticola. Phase-contrast microscopy showed differential motility between the strains; ATCC 35404 showed the highest motility, followed by ATCC 33521, and the remaining two strains (ATCC 35405 and ATCC 33520) showed the lowest motility. Transmission electron microscopy showed that the low motility strains exhibited extracellular flagellar protrusions resulting from elongated flagella. Treponemal flagellar filaments are composed of three flagellins of FlaB1, FlaB2 and FlaB3. FlaB1 expression was comparable between the strains, whereas FlaB2 expression was lowest in ATCC 35404. FlaB3 expression varied among strains, with ATCC 35405, ATCC 33520, ATCC 33521, and ATCC 35404 showing the highest to lowest expression levels, respectively. Additionally, the low motility strains showed faster electrophoretic mobility of FlaB3, suggesting that posttranslational modifications of these proteins may have varied, because the amino acid sequences of FlaB3 were identical between the strains. These results suggest that inappropriate expression of FlaB2 and FlaB3 caused the unusual elongation of flagella that resulted in decreased motility. Furthermore, the low motility strains grew to higher bacterial density, and showed greater chymotrypsin-like protease activity, and more bacterial cells associated with gingival epithelial cells in comparison with the high motility strains. There may be a relationship between motility and these properties, but the genetic factors underlying this association remain unclear.

Production of 4-hydroxybutyrate from succinate semialdehyde in butyrate biosynthesis in Porphyromonas gingivalis.

BACKGROUND: Despite evidence demonstrating the importance of butyrate-producing bacteria in host health and disease, the characterization of enzymes responsible for butyrate production has not been fully elucidated in the periodontopathogen, Porphyromonas gingivalis. METHODS: LC-MS/MS and colorimetric analyses were employed to enzymatically characterize recombinant PGN_0724 in P. gingivalis as a succinate semialdehyde reductase. The concentration of short chain fatty acids in the culture supernatant of the wild-type bacteria and a mutant strain lacking the PGN_0724 gene were quantified using GC-MS. RESULTS: Incubation of recombinant PGN_0724 with succinate semialdehyde and NADH resulted in the production of 4-hydroxybutyrate as well as consumption of succinate semialdehyde. Double reciprocal plots showed that the reaction catalyzed by the PGN_0724 protein was associated with a ternary complex mechanism. The growth speed and final turbidity of the mutant strain were much lower than those of the wild-type cells. The capacity of the mutant strain to produce butyrate, isobutyrate, and isovalerate was 30%, 15%, and 45%, respectively, of that of the wild-type strain, while the mutant strain produced approximately 3.9-fold more propionate than the wild type. CONCLUSIONS: The pathway responsible for butyrate production is important for the growth of P. gingivalis and appears to be associated with production of the other short chain fatty acids. GENERAL SIGNIFICANCE: The aim of this study was to delineate the mechanisms involved in the production of 4-hydroxybutyrate, which is an intermediate in the biosynthetic pathway for production of butyrate, which is a virulence factor in P. gingivalis.

Acyl-CoA reductase PGN_0723 utilizes succinyl-CoA to generate succinate semialdehyde in a butyrate-producing pathway of Porphyromonas gingivalis.

The molecular basis of butyrate production in Porphyromonas gingivalis has not been fully elucidated, even though butyrate, a short chain fatty acid (SCFA), can exert both beneficial and harmful effects on a mammalian host. A database search showed that the amino acid sequence of PGN_0723 protein was 50.6% identical with CoA-dependent succinate semialdehyde dehydrogenase (SSADH) in Clostridium kluyveri. By contrast, the protein has limited identity (19.1%) with CoA-independent SSADH in Escherichia coli. Compared with the wild type, growth speed, and final turbidity were lower in the PGN_0723 deletion strain that was constructed by replacing the PGN_0723 gene with an erythromycin resistance cassette. Gas chromatography mass spectrometry revealed the supernatant concentrations of the SCFAs butyrate, isobutyrate, and isovalerate, but not propionate, in the PGN_0723 deletion strain were also lower than those in the wild type. The wild-type phenotype was restored in a complemented strain. We cloned the PGN_0723 gene, purified the recombinant protein, and computationally constructed its three-dimensional model. A colorimetric assay and liquid chromatography-tandem mass spectrometry analysis demonstrated that the recombinant PGN_0723 produces succinate semialdehyde, which is an intermediate in the P. gingivalis butyrate synthesis pathway, not from succinate but from succinyl-CoA in the presence of NAD(P)H via a ping-pong bi-bi mechanism. Asn110Ala and Cys239Ala mutations resulted in a significant loss of the CoA-dependent PGN_0723 enzymatic activity. The study provides new insights into butyrate production, which constitutes a virulence factor in P. gingivalis.

Separation of novel phosphoproteins of Porphyromonas gingivalis using phosphate-affinity chromatography.

Phosphorylation of serine, threonine and tyrosine is a central mechanism for regulating the structure and function of proteins in both eukaryotes and prokaryotes. However, the action of phosphorylated proteins present in Porphyromonas gingivalis, a major periodontopathogen, is not fully understood. Here, six novel phosphoproteins that possess metabolic activities were identified, namely PGN_0004, PGN_0375, PGN_0500, PGN_0724, PGN_0733 and PGN_0880, having been separated by phosphate-affinity chromatography. The identified proteins were detectable by immunoblotting specific to phosphorylated Ser (P-Ser), P-Thr, and/or P-Tyr. These results imply that novel phosphorylated proteins might play an important role for regulation of metabolism in P. gingivalis.

Serum Amyloid A Promotes E-Selectin Expression via Toll-Like Receptor 2 in Human Aortic Endothelial Cells.

Periodontitis is a chronic inflammatory disease that affects the periodontium. Recent studies suggest an association between periodontal and cardiovascular diseases. However, the detailed molecular mechanism is unknown. A previous study has demonstrated that experimental periodontitis induces serum amyloid A (SAA) in the liver and peripheral blood of ApoE-deficient mice as an atherosclerosis model. SAA is an acute-phase protein that affects systemic inflammation. The aim of this study is to investigate the atherosclerosis-onset mechanism using human aortic endothelial cells (HAECs) stimulated by SAA in vitro. Atherosclerosis PCR array and qPCR analyses showed upregulation of adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin in HAECs upon SAA stimulation. In addition, the results demonstrated that Toll-like receptor, TLR2, could serve as an important receptor of SAA in HAECs. Furthermore, small interfering RNA (siRNA) against TLR2 inhibited the upregulation of adhesion molecules in HAECs stimulated by SAA. Our results suggest that SAA stimulates the expression of adhesion molecules via TLR2. SAA could be an important molecule for atherosclerosis induced by periodontal disease.

Characterization of wheat germ agglutinin lectin-reactive glycosylated OmpA-like proteins derived from Porphyromonas gingivalis.

Glycosylation is one of the common posttranslational modifications in eukaryotes. Recently, glycosylated proteins have also been identified in prokaryotes. A few glycosylated proteins, including gingipains, have been identified in Porphyromonas gingivalis, a major pathogen associated with chronic periodontitis. However, no other glycosylated proteins have been found. The present study identified glycoproteins in P. gingivalis cell lysates by lectin blotting. Whole-cell lysates reacted with concanavalin A (ConA), Lens culinaris agglutinin (LCA), Phaseolus vulgaris erythroagglutinin (PHA-E4), and wheat germ agglutinin (WGA), suggesting the presence of mannose-, N-acetylgalactosamine-, or N-acetylglucosamine (GlcNAc)-modified proteins. Next, glycoproteins were isolated by ConA-, LCA-, PHA-E4-, or WGA-conjugated lectin affinity chromatography although specific proteins were enriched only by the WGA column. Mass spectrometry analysis showed that an OmpA-like, heterotrimeric complex formed by Pgm6 and Pgm7 (Pgm6/7) was the major glycoprotein isolated from P. gingivalis. Deglycosylation experiments and Western blotting with a specific antibody indicated that Pgm6/7 was modified with O-GlcNAc. When whole-cell lysates from P. gingivalis mutant strains with deletions of Pgm6 and Pgm7 were applied to a WGA column, homotrimeric Pgm7, but not Pgm6, was isolated. Heterotrimeric Pgm6/7 had the strongest affinity for fibronectin of all the extracellular proteins tested, whereas homotrimeric Pgm7 showed reduced binding activity. These findings suggest that the heterotrimeric structure is important for the biological activity of glycosylated WGA-binding OmpA-like proteins in P. gingivalis.

Identification of Critical Amino Acid Residues of a Two-Component Sensor Protein for Signal Sensing in Porphyromonas gingivalis Fimbriation via Random Mutant Library Construction.

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli-Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure-function relationships of proteins of interest.

The AtoC family response regulator upregulates an operon encoding putative outer membrane proteins sorted by type IX secretion system in Porphyromonas gingivalis.

OBJECTIVES: Porphyromonas gingivalis, a keystone periodontopathogen, has multiple two-component systems that are thought to modulate virulence. In this study, we focused on PGN_0775 response regulator (RR), an AtoC homolog, and attempted to identify the target gene that it regulates in P. gingivalis. METHODS: Comparative proteomic analyses comprising two-dimensional electrophoresis and peptide mass fingerprinting were applied to total protein samples from parent (WT) and atoC gene knockout (KO) strains to screen for affected protein spots. Fluctuations in the expression of corresponding genes were further confirmed using relative quantitative real-time polymerase chain reaction (RQPCR). RESULTS: Five protein spots with fluctuating expression levels were identified in pgn_0775 KO strains along with their masses and physiological features, which contained two hypothetical proteins with higher expression levels in the WT than in the KO strains. RQPCR analysis confirmed that mRNA levels were consistently decreased in KO and recovered in pgn_0775-complemented KO strains. The two hypothetical proteins appeared to be the products of an operon that comprises four genes encoding three hypothetical but putative type IX secretion system sorting domain-containing proteins and an N-terminal region of the C25 cysteine peptidase. CONCLUSIONS: The AtoC RR homolog in P. gingivalis upregulates the expression of the operon encoding potentially antigenic proteins retained on the cell surface; thus, it could be a promising target for P. gingivalis-specific antivirulence therapy.

Structural and antigenic characterization of a novel genotype of Mfa1 fimbriae in Porphyromonas gingivalis.

Background: Mfa1 fimbriae of the periodontal pathogen Porphyromonas gingivalis are responsible for biofilm formation and comprise five proteins: Mfa1-5. Two major genotypes, mfa170 and mfa153, encode major fimbrillin. The mfa170 genotype is further divided into the mfa170A and mfa170B subtypes. The properties of the novel mfa170B remain unclear. Methods: Fimbriae were purified from P. gingivalis strains JI-1 (mfa170A), 1439 (mfa170B), and Ando (mfa153), and their components and their structures were analyzed. Protein expression and variability in the antigenic specificity of fimbrillins were compared using Coomassie staining and western blotting using polyclonal antibodies against Mfa170A, Mfa170B, and Mfa153 proteins. Cell surface expression levels of fimbriae were analyzed by filtration enzyme-linked immunosorbent assays. Results: The composition and structures of the purified Mfa1 fimbriae of 1439 was similar to that of JI-1. However, each Mfa1 protein of differential subtype/genotype was specifically detected by western blotting. Mfa170B fimbriae were expressed in several strains such as 1439, JKG9, B42, 1436, and Kyudai-3. Differential protein expression and antigenic heterogeneities were detected in Mfa2-5 between strains. Conclusion: Mfa1 fimbriae from the mfa170A and mfa170B genotypes indicated an antigenic difference suggesting the mfa170B, is to be utilized for the novel classification of P. gingivalis.

Accessory fimbrial subunits and PPAD are necessary for TLR2 activation by Porphyromonas gingivalis.

Porphyromonas gingivalis is an oral pathogen that promotes dysbiosis by quenching the bactericidal activity of the host immune system while maintaining chronic inflammation, leading to periodontitis. This involves the secretion of virulence factors such as P. gingivalis peptidyl arginine deiminase (PPAD), which converts the C-terminal Arg residues of bacterial and host-derived proteins and peptides into citrulline. We have previously shown that PPAD activity and major fimbriae (containing FimA) are necessary for P. gingivalis to activate Toll-like receptor 2 (TLR2). TLR2 is an important component of the innate immune system and plays a predominant role in the recognition of P. gingivalis by host cells. Here, we extend those findings to show that P. gingivalis strains deficient for PPAD and fimbriae induced almost identical transcriptional profiles in infected primary human gingival fibroblasts (PHGFs), but these differed substantially from the transcriptome elicited by the wild-type ATCC 33277 strain. Apparently, PPAD-modified fimbriae trigger the host cell response to P. gingivalis, as confirmed by showing that the proinflammatory host cell response mediated by TLR2 is dependent on PPAD activity and the presence of fimbriae, with type I fimbriae as the most potent TLR2 activators. We also found that PPAD-modified accessory fimbrial subunits (FimC, FimD, and FimE) alone or in combination are TLR2 ligands in a reporter cell line. Although FimA polymerization to form the fimbrial shaft was not required for TLR2 activation, the secretion and proteolytic maturation of FimA were necessary for signaling by accessory Fim proteins. This was supported by showing that the proinflammatory activation of PHGFs is dependent on PPAD and accessory fimbrial subunits. We conclude that accessory fimbrial subunits are modified by PPAD and stimulate the response to P. gingivalis infection in a TLR2-dependent manner.

Effects of cleaning sports mouthguards with ethylene-vinyl acetate on oral bacteria.

Background: Sports mouthguards, worn in the oral cavity to prevent sports injuries, are constantly exposed to various microorganisms that cause oral infections. Hence, the optimal cleaning methods for sports mouthguards have been thoroughly examined. In this study, we evaluated the efficiency of cleaning effects with a mouthguard cleaner (MC) on microbial biofilm formation in sports mouthguards in vitro and in vivo. Methods: We evaluated the cleaning effects of the discs produced by ethylene-vinyl acetate (EVA) on bacterial biofilms formed by the commensal bacterium Streptococcus oralis, the cariogenic bacterium Streptococcus mutans, and the opportunistic pathogen Staphylococcus aureus in vitro. EVA discs with biofilm were subjected to sterile distilled water (CTRL) and ultrasonic washing (UW), followed by treatment with MC and sodium hypochlorite (NaClO) as positive controls. Thereafter, the viable bacterial cell counts were determined. The bacteria adhering to the sheets before and after the treatment were observed under an electron microscope. The degree of cleanliness and measurement of viable microbial cell counts for total bacteria, Streptococci and Candida, opportunistic fungi, were evaluated on the used experimental sports mouthguards with and without UW and MC treatment in vivo. Results: The number of bacterial cells significantly decreased against all the tested biofilm bacteria upon treatment with MC, compared with CTRL and UW. Electron microscopy analysis revealed the biofilm formation by all bacteria on the EVA discs before cleaning. We observed fewer bacteria on the EVA discs treated with MC than those treated with CTRL and UW. Furthermore, the degree of cleanliness of the used experimental sports mouthguards cleaned using MC was significantly higher than that of the CTRL-treated mouthguards. Moreover, the viable microbial cell counts on the used experimental sports mouthguard were considerably lower than those on the CTRL ones. Conclusion: The cleaning effect of MC against oral bacteria was more effective than that of UW. MC treatment might have a potential future application as a cleaning method for sports mouthguards to protect athletes from oral infection.

Porphyromonas gingivalis FimA and Mfa1 fimbriae: Current insights on localization, function, biogenesis, and genotype.

In general, the periodontal pathogen Porphyromonas gingivalis expresses distinct FimA and Mfa1 fimbriae. Each of these consists of five FimA-E and five Mfa1-5 proteins encoded by the fim and mfa gene clusters, respectively. The main shaft portion comprises FimA and Mfa1, whereas FimB and Mfa2 are localized on the basal portion and function as anchors and elongation terminators. FimC-E and Mfa3-5 participate in the assembly of an accessory protein complex on the tips of each fimbria. Hence, they serve as ligands for the receptors on host cells and other oral bacterial species. The crystal structures of FimA and Mfa1 fimbrial proteins were recently elucidated and new insights into the localization, function, and biogenesis of these proteins have been reported. Several studies indicated a correlation between P. gingivalis pathogenicity and the fimA genotype but not the mfa1 genotype. We recently revealed polymorphisms of all genes in the fim and mfa gene clusters. Intriguingly, mfa5 occurred in numerous different forms and underwent duplication. Detailed structural and functional knowledge of the fimbrial proteins in the context of the entire filament could facilitate the development of innovative therapeutic strategies for structure-based drug design.

Construction of a Gene-Deletion Mutant in Tannerella forsythia.

Tannerella forsythia, a gram-negative anaerobic bacterium, is one of the most important pathogens in periodontal disease. However, it has been difficult to construct a gene-deletion mutant in this organism, which may serve as a useful tool in microbiological research. We reported a highly efficient method to construct a gene-deletion mutant of T. forsythia in 2007, and it was accomplished by preparing competent cells from a colony grown on an agar medium instead of a broth culture. Here, we describe the same method with some improvements.

Clinical classification of tooth position in the alveolar bone housing with periodontal defects.

A new classification of tooth position in the alveolar bone housing, which indicates the width of alveolar bone for buccolingual direction, with bone defects caused by periodontal disease is proposed. This classification highlights the importance of tooth position in the alveolar bone housing in terms of the progression of the regenerative process and the factors that may affect the prognosis of compromised teeth after regenerative surgery. Tooth positions were divided into two groups: (i) The whole tooth is centrally positioned in the existing alveolar bone housing (Grade I) and (ii) A part of the tooth is exposed out of the existing alveolar bone housing (Grade II). Grade II is further divided into two subgroups according to situations encountered in clinical practice. The following subclasses are suggested: Subgroup A, where the alveolar bone housing is broader than the tooth, and Subgroup B, where the alveolar bone housing is narrower than the tooth. These subgroups represent a discrepancy between tooth size and alveolar bone dimensions in the buccolingual orientation. This classification could be useful for planning the correct regenerative treatment for each type of the tooth position in the alveolar bone housing with periodontal defects.

Diversity analysis of genes encoding Mfa1 fimbrial components in Porphyromonas gingivalis strains.

Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is associated with the development of periodontal disease. The genetic diversity in virulence factors, such as adhesive fimbriae, among its strains affects the bacterial pathogenicity. P. gingivalis generally expresses two distinct types of fimbriae, FimA and Mfa1. Although the genetic diversity of fimA, encoding the major FimA fimbrilin protein, has been characterized, the genes encoding the Mfa1 fimbrial components, including the Mfa1 to Mfa5 proteins, have not been fully studied. We, therefore, analyzed their genotypes in 12 uncharacterized and 62 known strains of P. gingivalis (74 strains in total). The mfa1 genotype was primarily classified into two genotypes, 53 and 70. Additionally, we found that genotype 70 could be further divided into two subtypes (70A and 70B). The diversity of mfa2 to mfa4 was consistent with the mfa1 genotype, although no subtype in genotype 70 was observed. Protein structure modeling showed high homology between the genotypes in Mfa1 to Mfa4. The mfa5 gene was classified into five genotypes (A to E) independent of other genotypes. Moreover, genotype A was further divided into two subtypes (A1 and A2). Surprisingly, some strains had two mfa5 genes, and the 2nd mfa5 exclusively occurred in genotype E. The Mfa5 protein in all genotypes showed a homologous C-terminal half, including the conserved C-terminal domain recognized by the type IX secretion system. Furthermore, the von Willebrand factor domain at the N-terminal was detected only in genotypes A to C. The mfa1 genotypes partially correlated with the ragA and ragB genotypes (located immediately downstream of the mfa gene cluster) but not with the fimA genotypes.