Publications by authors named "Kazuyuki Ishihara"

101 Publications

Non-surgical treatment for periodontitis and peri-implantitis: longitudinal clinical and bacteriological findings-A case report with a 7-year follow-up evaluation.

SAGE Open Med Case Rep 2021 5;9:2050313X211029154. Epub 2021 Jul 5.

Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

The aim of this report is to show that periodontitis and peri-implantitis with horizontal bone resorption in a 68-year-old male patient were successfully treated by non-surgical treatment. Scaling with an ultrasonic device was performed for moderate periodontitis around the mandibular left first premolar and moderate peri-implantitis around the maxillary right molar implants. Root planing with a metal curette was performed for the periodontal site, and debridement with a plastic curette was performed for the peri-implant site. A month after treatment, probing depth decreased from 5 to 2 mm at the periodontal site and 8 to 3 mm at the peri-implant site. The investigation of bacterial composition by sequencing the 16S rRNA gene amplicons showed that the composition similarly changed at both sites, 5 years after treatment; the change reflected the typical recovery of periodontitis. The clinical condition was maintained for 7 years after treatment at both sites. This was a successful case of non-surgical treatment for peri-implantitis with horizontal bone resorption, promoting recovery of the microbiota from dysbiotic shift.
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http://dx.doi.org/10.1177/2050313X211029154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8261847PMC
July 2021

Systemic administration of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-Ig abrogates alveolar bone resorption in induced periodontitis through inhibition of osteoclast differentiation and activation: An experimental investigation.

J Periodontal Res 2021 Jun 15. Epub 2021 Jun 15.

Department of Periodontology, Tokyo Dental College, Tokyo, Japan.

Background/objectives: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical immunoregulatory molecule expressed on T cells. CTLA-4 also binds to the surfaces of monocytes and macrophages, precursors of osteoclasts. Research on rheumatoid arthritis demonstrated that CTLA-4 suppresses inflammation and bone resorption. However, its effects on alveolar bone have yet to be understood. The purpose of this study was to investigate the role and potential mechanism of CTLA-4 in bone resorption in periodontitis.

Materials And Methods: In vivo, the effects of systemic administration of CTLA-4 immunoglobulin fusion protein (CTLA-4-Ig) on alveolar bone resorption were investigated using a periodontitis mouse model. A total of 20 C57BL/6J mice were randomly assigned to two groups according to the administration modes. Periodontitis was induced by placing a ligature around the left maxillary second molar. The contralateral tooth was left un-ligated. In the CTLA-4-Ig (+) group, CTLA-4-Ig was administered by intraperitoneal injection at 1 and 3 days after ligature placement. Animals in the CTLA-4-Ig (-) group were given only phosphate-buffered saline each time. At 5 days after ligature placement, bone resorption was assessed by micro-computed tomography and histological examination, and the prevalence of osteoclast-like cells was assessed by tartrate-resistant acid phosphatase (TRAP) staining. In vitro, the effects of CTLA-4-Ig on osteoclasts were evaluated. Viability of RAW 264.7 cells treated with receptor activator of nuclear factor-κB ligand (RANKL) and CTLA-4-Ig was tested by WST-1 assay. Osteoclast-like cells were enumerated by TRAP staining, and osteoclast activity was evaluated by resorption pit assay. Gene expression levels of osteoclast differentiation markers (macrophage-colony stimulating factor receptor, carbonic anhydrase II, cathepsin K, and Trap) and protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, were assessed by quantitative real-time polymerase chain reaction. The effect of CTLA-4-Ig on the nuclear factor-κB (NF-κB) activation was assessed by enzyme-linked immunosorbent assay.

Results: In vivo, ligature-induced bone resorption and the numbers of osteoclast-like cells were significantly decreased by the administration of CTLA-4-Ig. In vitro, treatment with RANKL and CTLA-4-Ig had no significant effect on cell viability. CTLA-4-Ig significantly reduced the prevalence and activation of osteoclast-like cells and decreased the expressions of osteoclast differentiation markers, compared with the RANKL-treated control. CTLA-4-Ig significantly suppressed RANKL-induced phosphorylation of NF-κB p65 but increased PP2A expression.

Conclusion: These results suggest that CTLA-4-Ig abrogates bone resorption in induced periodontitis, possibly via inhibition of osteoclast differentiation and activation. The regulation of the NF-κB pathway and PP2A expression may be one mechanism by which CTLA-4-Ig suppresses osteoclast behavior.
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http://dx.doi.org/10.1111/jre.12909DOI Listing
June 2021

Taxonomic and Gene Category Analyses of Subgingival Plaques from a Group of Japanese Individuals with and without Periodontitis.

Int J Mol Sci 2021 May 18;22(10). Epub 2021 May 18.

Department of Microbiology, Tokyo Dental College, Chiyoda-ku, Tokyo 101-0061, Japan.

Periodontitis is an inflammation of tooth-supporting tissues, which is caused by bacteria in the subgingival plaque (biofilm) and the host immune response. Traditionally, subgingival pathogens have been investigated using methods such as culturing, DNA probes, or PCR. The development of next-generation sequencing made it possible to investigate the whole microbiome in the subgingival plaque. Previous studies have implicated dysbiosis of the subgingival microbiome in the etiology of periodontitis. However, details are still lacking. In this study, we conducted a metagenomic analysis of subgingival plaque samples from a group of Japanese individuals with and without periodontitis. In the taxonomic composition analysis, genus and demonstrated significantly different compositions between healthy sites and sites with periodontal pockets. The results from the relative abundance of functional gene categories, carbohydrate metabolism, glycan biosynthesis and metabolism, amino acid metabolism, replication and repair showed significant differences between healthy sites and sites with periodontal pockets. These results provide important insights into the shift in the taxonomic and functional gene category abundance caused by dysbiosis, which occurs during the progression of periodontal disease.
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http://dx.doi.org/10.3390/ijms22105298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157553PMC
May 2021

Patient-specific establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering.

J Periodontal Res 2021 May 31. Epub 2021 May 31.

Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Background And Objective: Dysbiosis, a loss of balance in the microbiota, is a potential factor of peri-implantitis. However, compositional change of the peri-implant microbiota soon after implant uncovering is still unknown. In this study, bacterial composition in the peri-implant sulcus was examined to understand the establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering.

Methods: Microbiota samples were collected at weeks 1, 2, 4, and 6 after stage-two surgery. Bacterial DNA was isolated from the samples, and a 16S rRNA gene library was constructed. Sequence reads were obtained using a high-throughput sequencing platform and were taxonomically assigned at the phylum and genus levels.

Results: Alpha diversity indices, which did not include taxonomic information, were at similar levels throughout the four time points. At 1 and 2 weeks, the bacterial composition was similar among patients with the predominance of Firmicutes and Proteobacteria. However, the composition was diverse at 4 and 6 weeks and significantly dissimilar to the composition at 1 week.

Conclusions: At 1 week, the peri-implant microbiota was already formed with alpha diversity as high as that at the later time points. However, the bacterial composition was not highly dissimilar among patients at 1 week. The composition changed over the passage of several weeks and was specific for each patient.
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http://dx.doi.org/10.1111/jre.12898DOI Listing
May 2021

Role of Hyalin-like Protein in Gliding and Biofilm Formation by Capnocytophaga Ochracea.

Bull Tokyo Dent Coll 2021 Jun 14;62(2):89-98. Epub 2021 May 14.

Department of Microbiology, Tokyo Dental College.

Capnocytophaga ochracea possesses a type-IX secretion system that exports proteins which have a gliding motility-associated C-terminal (CTD) domain. This system is found in several species of the Bacteroidetes phylum. Hyalin, a large protein encoded by Coch_0033 in C. ochracea ATCC 27872, has a CTD domain and is posited to be involved in quorum sensing according to the database of the Kyoto Encyclopedia of Genes and Genomes. This suggests that it plays a role in biofilm formation via interbacterial communication. The aim of this study was to investigate the potential role of the hyalin-like protein coded by the Coch_0033 gene in gliding and biofilm formation of C. ochracea. A hyalin-like protein-deficient mutant strain of C. ochracea, designated mutant WR-1, was constructed through insertion of the ermF-ermAM cassette into the target gene. The spreading feature at the edge of the colony was lost in the mutant strain. Crystal violet and confocal laser scanning microscopy revealed no difference between the quantity of biofilm organized by the mutant and that organized by the wild-type strain. These data suggest that the hyalin-like protein encoded by the Coch_0033 gene is indeed involved in C. ochracea gliding activity.
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http://dx.doi.org/10.2209/tdcpublication.2020-0051DOI Listing
June 2021

Inhibitory Effect of Resveratrol on Candida albicans Biofilm Formation.

Bull Tokyo Dent Coll 2021 Mar 15;62(1):1-6. Epub 2021 Feb 15.

Department of Microbiology, Tokyo Dental College.

Candida albicans is the primary candidiasis-causing fungal pathogen in humans, and one of its most important virulence factors is the ability to form biofilms. Moreover, these biofilms are often resistant to antifungal agents, so there is a need to develop alternative elimination strategies and therapeutic agents for such infections. The antifungal activity of resveratrol, a phytoalexin polyphenolic compound, impairs the morphological transition of C. albicans under various hypha-inducing conditions and inhibits growth of the yeast-form and mycelia. The purpose of this study was to investigate the effect of resveratrol against C. albicans biofilm formation. The developmental, sustained, and mature stages of biofilm formation were affected or inhibited by resveratrol. Exposure to resveratrol at the developmental stage inhibited growth of C. albicans in a dose-dependent manner. A >30% reduction was observed in sustained biofilm growth in the presence of 200 μg/ml resveratrol in comparison with in its absence. In terms of disruption of matured biofilm, 6.25-100 μg/ml resveratrol significantly reduced cell viability of C. albicans compared with in a control sample (p<0.05). The present results indicate that resveratrol has the potential to serve as an anti-Candida treatment and preventive tool which functions by inhibiting existing or under-forming C. albicans biofilms.
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http://dx.doi.org/10.2209/tdcpublication.2020-0023DOI Listing
March 2021

Investigation of the potential regulator proteins associated with the expression of major surface protein and dentilisin in .

J Oral Microbiol 2020 Oct 11;12(1):1829404. Epub 2020 Oct 11.

Department of Microbiology, Tokyo Dental College, Tokyo, Japan.

is involved in 'chronic' periodontitis pathogenesis. The mechanism underlying the regulation of the expression of its virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine specific protease (dentilisin) is yet to be clarified. We determined the gene expression profiles of Msp- and dentilisin-deficient mutants of to identify the regulation network of gene expression concomitant with the inactivation of these virulence genes. Gene expression profiles of ATCC 35405 (wild type), dentilisin-deficient mutant K1, and deficient mutant DMSP3 were determined using DNA microarray analysis and quantitative real-time reverse transcription PCR (qRT-PCR). Msp and dentilisin protein levels were determined by immunoblotting and proteolytic activity assays. In addition to several differentially expressed genes, dentilisin expression was reduced in DMSP3; expression was significantly reduced in K1 (p < 0.05), both at the gene and protein levels. To identify the regulatory system involved, the expression levels of the potential regulators whose expression showed changes in the mutants were evaluated using qRT-PCR. Transcriptional regulators TDE_0127 and TDE_0814 were upregulated in K1, and the potential repressor, TDE_0344, was elevated in DMSP3. Dentilisin and Msp expression were interrelated, and gene expression regulators, such as TDE_0127, may be involved in their regulation.
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http://dx.doi.org/10.1080/20002297.2020.1829404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586716PMC
October 2020

Characterization of the Treponema denticola Virulence Factor Dentilisin.

Methods Mol Biol 2021 ;2210:173-184

Department of Microbiology, Tokyo Dental College, Tokyo, Japan.

Treponema denticola is a potent periodontal pathogen that forms a red complex with Porphyromonas gingivalis and Tannerella forsythia. It has many virulence factors, yet there are only a few reports detailing these factors. Among them, dentilisin is a well-documented surface protease. Dentilisin is reported to be involved in nutrient uptake, bacterial coaggregation, complement activation, evasion of the host immune system, inhibition of the hemostasis system, and cell invasion as a result of its action, in addition to its original proteolysis function. Therefore, characterization of dentilisin, and clarifying the relationship between T. denticola and the onset of periodontal disease will be important to better understanding this disease. In this chapter, we explain the methods for analysis of dentilisin activity and pathogenicity.
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http://dx.doi.org/10.1007/978-1-0716-0939-2_17DOI Listing
March 2021

Investigation of the antimicrobial activity of Bilberry (Vaccinium myrtillus L.) extract against periodontopathic bacteria.

J Oral Biosci 2020 06 11;62(2):169-174. Epub 2020 Feb 11.

Department of Microbiology, Tokyo Dental College, 2-9-18, Kandamisaki-cho, Chiyoda-ku, Tokyo, Japan.

Objective: The purpose of this study was to isolate the active antibacterial compounds from Bilberry (Vaccinium myrtillus L.) against periodontopathic bacteria.

Methods: The acetone soluble fraction of Bilberry was extracted from the oil layer by oil/water separation. The extract was then purified by a silica gel open column chromatography. The minimum inhibitory concentration (MIC) of the total extract or purified fractions against bacteria was measured at each step.

Results: The MIC of the total extract against Porphyromonas gingivalis was 500 μg/mL. The fraction exhibiting antibacterial activity against P. gingivalis was called NU4-TDC, and its MICs against P. gingivalis, Fusobacterium nucleatum, and Prevotella intermedia were 26.0 ± 7.8 μg/mL, 59.0 ± 10.4 μg/mL, and 45.1 ± 16.5 μg/mL, respectively. The MIC against Streptococcus mutans was >62.5 μg/mL.

Conclusion: Bilberry contains antibacterial components against periodontopathic bacteria, such as P. gingivalis, F. nucleatum, and P. intermedia.
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http://dx.doi.org/10.1016/j.job.2020.01.009DOI Listing
June 2020

Synergistic biofilm formation by Parvimonas micra and Fusobacterium nucleatum.

Anaerobe 2020 Apr 12;62:102100. Epub 2019 Sep 12.

Oral Health Science Center, 2-1-14 Kanda-Misakicho, Chiyoda-ku, Tokyo, 101-0061, Japan; Department of Microbiology, 2-1-14 Kanda-Misakicho, Chiyoda-ku, Tokyo, 101-0061, Japan. Electronic address:

Parvimonas micra is frequently isolated from lesions of apical periodontitis and is a major disease-related pathogen. One of the main causes of apical periodontitis is extraradicular biofilm. In this study, we investigated polymicrobial biofilm formation by P. micra and species associated with apical periodontitis. The coaggregation activity of P. micra with partner strains was investigated by visual assays. Synergistic biofilm formation was evaluated by cocultures of P. micra and partner strains. Growth of planktonic cells was measured by evaluating the absorbance at OD, and biofilm formation was examined by staining with crystal violet. The effects of soluble components on synergistic biofilm formation and planktonic cell growth were examined after coculture of P. micra and other strains separated with a 0.4-μm pore-size porous membrane. P. micra coaggregated with Fusobacterium nucleatum, Porphyromonas gingivalis, or Capnoctyophaga ochracea. P. micra showed no coaggregation with Staphylococcus aureus, S. epidermidis, or Prevotella intermedia. In mixed cultures, biofilm formation by P. micra and F. nucleatum was greater than that by P. micra and P. gingivalis or C. ochracea. In separated cocultures, planktonic cell growth of P. micra was enhanced by each of the three species. Biofilm formation by P. micra was enhanced by F. nucleatum or C. ochracea; however, no significant enhancement was observed with P. gingivalis. These data indicated that P. micra and F. nucleatum had synergistic effects in biofilm formation and that these effects may be important for colonization by these two species in apical periodontitis lesions.
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http://dx.doi.org/10.1016/j.anaerobe.2019.102100DOI Listing
April 2020

Inhibitory Effects of Lingonberry Extract on Oral Streptococcal Biofilm Formation and Bioactivity.

Bull Tokyo Dent Coll 2019 Feb 31;60(1):1-9. Epub 2019 Jan 31.

Department of Microbiology, Tokyo Dental College.

Phenolic compounds in fruits such as cranberries have been shown to promote a number of biological activities. The purpose of this study was to investigate the effects of polyphenolic compound-containing lingonberry extract on oral streptococci and compare them with the known anti-cariogenic activity of cranberries. Water-soluble and polyphenol-rich fractions (Fractions I and II, respectively) were isolated from cranberries and lingonberries. The effects of those fractions on the biofilm formation ability and bioactivity of Streptococcus mutans MT8148R, Streptococcus sobrinus 6715, and Streptococcus sanguinis ATCC 10556 were then evaluated. Cranberry or lingonberry Fraction II (at 0.5-1 mg/ml) significantly reduced biofilm formation by S. mutans, S. sobrinus, and S. sanguinis. In contrast, cranberry or lingonberry Fraction I (at 0.5-2 mg/ml) increased biofilm formation by S. mutans and S. sobrinus, but not by S. sanguinis. Fractions I and II (at 1-2 mg/ml) also reduced the bioactivity of S. mutans, while Fraction II (at 0.5 mg/ml) enhanced the bioactivity of all tested strains. The results revealed that lingonberries contained a larger amount of polyphenol than cranberries and that they showed almost the same level of activity against the biofilm formation ability and bioactivity of oral streptococci. This indicates that polyphenol-rich lingonberry fraction offers a promising natural food derivative for prevention of dental caries.
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http://dx.doi.org/10.2209/tdcpublication.2018-0007DOI Listing
February 2019

Dysbiosis of oral microbiota in palmoplantar pustulosis patients.

J Dermatol Sci 2019 01 2;93(1):67-69. Epub 2019 Jan 2.

Department of Dermatology, Tokyo Dental College Ichikawa General Hospital, Ichikawa, Chiba, Japan.

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http://dx.doi.org/10.1016/j.jdermsci.2018.12.003DOI Listing
January 2019

Treponema denticola Induces Epithelial Barrier Dysfunction in Polarized Epithelial Cells.

Bull Tokyo Dent Coll 2018 Nov 18;59(4):265-275. Epub 2018 Oct 18.

Department of Microbiology, Tokyo Dental College.

Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 10 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=10), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.
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http://dx.doi.org/10.2209/tdcpublication.2017-0052DOI Listing
November 2018

Characterization of a novel potential peptide import system in Treponema denticola.

Microb Pathog 2018 Oct 1;123:467-472. Epub 2018 Aug 1.

Oral Health Science Center, Tokyo Dental College, 2-9-18 Kanda-Misakicho, Chiyoda-ku, Tokyo 101-0061, Japan; Department of Microbiology, Tokyo Dental College, 2-1-14 Kanda-Misakicho, Chiyoda-ku, Tokyo 101-0061, Japan. Electronic address:

Treponema denticola is a major etiologic agent of chronic periodontitis. On the outer sheath of T. denticola, several proteins, such as the major outer sheath protein and dentilisin were detected, and among them, a 95 kDa protein which has not yet been characterized. The aim of this study was to characterize the function of this 95 kDa protein containing gene cluster. A gene encoding this 95 kDa protein (TDE_1072) of T. denticola was inactivated by homologous recombination. We compared growth curves between the TDE_1072 mutant and wild-type strains as well as differences in gene expression by DNA microarray analysis. Differential expression of genes identified by microarray analysis was confirmed by quantitative reverse transcription-polymerase chain reaction. The proteins encoded by TDE_1072, TDE_1073, TDE_1074, TDE_1075, and TDE_1076 shared respective similarities to the substrate-binding domain (DppA) of an ABC-type dipeptide/oligopeptide/nickel transport system, and to the permease components (DppB and DppC) and ATPase components (DppD and DppF) of an ABC-type dipeptide/oligopeptide/nickel transport system. Inactivation of dppA attenuated the growth of T. denticola and dppA-dppF were co-transcribed. In contrast, expression of oppB-oppF was up-regulated in the mutant. Our findings indicate that TDE_1072 may be a potential periplasmic solute binding protein encoded by dppA that is involved in the organization of a peptide uptake system with dppB-dppF.
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http://dx.doi.org/10.1016/j.micpath.2018.07.045DOI Listing
October 2018

Treponema denticola transcriptional profiles in serum-restricted conditions.

FEMS Microbiol Lett 2018 08;365(16)

Department of Microbiology, Tokyo Dental College, 2-1-14 Kanda-Misakicho, Chiyoda-ku, Tokyo 101-0061, Japan.

Treponema denticola is a major pathogen in periodontal disease and is frequently isolated from the lesions of patients with chronic periodontitis. Treponema denticola utilizes serum components as nutrient sources so as to colonize and proliferate in the gingival crevice. However, the mechanisms of serum utilization remain unclear. Therefore, the aim of the present study was to identify T. denticola serum utilization genes. Precultured T. denticola cells were suspended in a tryptone-yeast extract-gelatin-volatile fatty acids medium containing 0, 1% and 10% serum, respectively, and incubated anaerobically for 17 h. Total RNA was isolated, and T. denticola gene expression was compared by microarray and reverse transcription-polymerase chain reaction. In serum-depleted conditions, the expression levels of a potential hydroxylamine reductase, several ABC transporters, and phosphoenolpyruvate synthase were increased, while those of genes encoding methyl-accepting chemotaxis proteins and a transcriptional regulator were decreased. These results suggest that T. denticola may uptake serum components mainly through the action of ABC transporters. In particular, the decrease in the dmcA expression level with decreasing serum concentration suggests its involvement in chemotaxis toward serum-rich environments.
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http://dx.doi.org/10.1093/femsle/fny171DOI Listing
August 2018

Identification of a specific domain of Porphyromonas gingivalis Hgp44 responsible for adhesion to Treponema denticola.

Pathog Dis 2018 07;76(5)

Department of Microbiology, Tokyo Dental College, Tokyo 101-0061, Japan.

Interaction between two periodontal pathogens, Porphyromonas gingivalis and Treponema denticola, contributes to plaque biofilm formation. Porphyromonas gingivalis forms aggregates with T. denticola through its adhesion/hemagglutinin domain (Hgp44). In this study, we investigated the specific domain of P. gingivalis Hgp44 responsible for adhesion to T. denticola using expression vectors harboring P. gingivalis Hgp44 DNA sequences encoding amino acid residues 1-419. Six plasmids harboring fragments in this region were generated by PCR amplification and self-ligation, and recombinant proteins r-Hgp44 (residues 1-419), r-Hgp441 (residues 1-124), r-Hgp442 (1-199), r-Hgp443 (1-316), r-Hgp444 (199-419), r-Hgp445 (124-198) and r-Hgp446 (199-316) were produced, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. r-Hgp44, r-Hgp443 and r-Hgp446 showed greater adhesion to T. denticola sonicates than the control, as determined by enzyme-linked immunosorbent assay. r-Hgp446 reduced the coaggregation of P. gingivalis and T. denticola. Scanning electron and confocal laser scanning microscopy analyses revealed that r-Hgp446 reduced dual-species biofilm formation. Our results indicate that residues 199-316 of P. gingivalis Hgp44 are mainly responsible for adhesion to T. denticola; inhibiting this domain could potentially disrupt periodontopathic biofilm formation and maturation.
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http://dx.doi.org/10.1093/femspd/fty047DOI Listing
July 2018

Effect of Porphyromonas gingivalis infection in the placenta and umbilical cord in pregnant mice with low birth weight.

Acta Odontol Scand 2018 Aug 15;76(6):433-441. Epub 2018 Jan 15.

a Department of Periodontology, Graduate School of Medical and Dental Sciences , Tokyo Medical and Dental University , Tokyo , Japan.

Objective: Growing evidence indicates an association between periodontitis and delivery outcome; however, the mechanism is unclear. This study aimed to investigate the influence of Porphyromonas gingivalis (Pg) infection on delivery outcome in mice.

Materials And Methods: Bacteremia was induced in pregnant Slc:ICR mice (8 weeks old) by intravenous injection of Pg. Mice were randomly divided into a control group (CO), and those receiving Pg injection at gestational day 1 (GD1), gestational day 15 (GD15) or every day (ED). Delivery outcome, Pg infection, and gene expression in the placenta and umbilical cord were evaluated.

Results: Birth weight was lower in the ED and GD15 groups than in the CO group. A remarkable increase in anti-Pg IgG antibody was observed in the ED and GD1 groups, although Pg was not detected in the placenta or umbilical cord. mRNA expression of Tnfα and Il6 in the placenta, and Hif1α in the umbilical cord, was significantly increased in the ED group. Microarray analysis of the umbilical cord revealed increased expression of several genes including Orm1, Mgl2, Rps6ka3 and Trim15 in the ED group.

Conclusions: Pg infection during the third trimester caused low birth weight and inflammation in the placenta and umbilical cord.
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http://dx.doi.org/10.1080/00016357.2018.1426876DOI Listing
August 2018

[Possible Involvement of Surface Antigen Protein 2 in the Morphological Transition and Biofilm Formation of Candida albicans].

Med Mycol J 2017 ;58(4):E139-E143

Department of Microbiology, Tokyo Dental College.

Surface antigen protein 2 (Csa2) is a member of the Candida albicans Common in Fungal Extracellular Membranes (CFEM) protein superfamily. We previously established its role in iron acquisition in C. albicans. However, the other roles of Csa2 remain unknown. Here, we compared growth, morphological transition, and biofilm formation among wild-type, Csa2-mutant, and complemented strains of C. albicans. Deletion of the Csa2 gene resulted in smaller and reduced colony growth, significant attenuation of the dimorphic transition under serum-inducing conditions, and reduced biofilm formation; complementation restored these levels to those of the wild-type. Our findings demonstrated that Csa2 participated in yeast-to-hyphae morphological switching under serum-inducing conditions and contributed to the biofilm formation of C. albicans. This work, therefore, provides novel insights into the potential roles of Csa2 in virulence of C. albicans.
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http://dx.doi.org/10.3314/mmj.17-00008DOI Listing
April 2018

Corrosion behavior of titanium in response to sulfides produced by Porphyromonas gingivalis.

Dent Mater 2018 02 20;34(2):183-191. Epub 2017 Oct 20.

Department of Dental Materials Science, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, 101-0061 Tokyo, Japan; Oral Health Science Center, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, 101-0061 Tokyo, Japan. Electronic address:

Objective: To investigate the effects of sulfides produced by Porphyromonas gingivalis (P. gingivalis) on the corrosion behavior of titanium.

Methods: Commercially pure titanium disks were mirror-polished and immersed in culture medium (BHI), spent medium after culturing P. gingivalis (BHI-S), and culture medium with P. gingivalis (BHI-P), and incubated aerobically at 37°C for 3-14 days. Titanium corrosion was evaluated through surface observation (using scanning electron microscope: SEM), color change (ΔE*ab), glossiness (Gs(20°)), chemical composition and state (using X-ray photoelectron spectroscopy: XPS), and titanium release.

Results: ΔE*ab and Gs(20°) did not significantly differ among specimens placed in test mediums for the study duration (p>0.05). SEM images of specimens showed no signs of localized or overall corrosion. XPS analysis indicated showed clear titanium metal state peaks on all specimens in addition to sulfide and sulfate on BHI-S and BHI-P specimens. Valency fraction of titanium decomposed from Ti2p spectrum of BHI-S and BHI-P specimens indicated no progression of oxidation. No significant levels of titanium release were found regardless of the mediums' sulfide content. Results suggested that sulfides produced by P. gingivalis attached on the surface of titanium specimens but did not cause titanium corrosion over the immersion period of 14 days.

Significance: It is imperative for dental practitioners to be aware of any elements which may influence the clinical success of titanium implants.
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http://dx.doi.org/10.1016/j.dental.2017.10.004DOI Listing
February 2018

Antimicrobial susceptibility profiles of oral Treponema species.

Anaerobe 2017 Dec 13;48:242-248. Epub 2017 Oct 13.

Department of Microbiology, Tokyo Dental College, 2-1-14 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan; Oral Health Science Center, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan. Electronic address:

Treponemes occur in the microflora of the dental plaque. Certain Treponema species that are frequently isolated from chronic periodontitis lesions are involved in its initiation and progression. In addition to mechanical instrumentation, antimicrobial agents are used as an adjunctive treatment modality for periodontitis. Despite its importance for successful antimicrobial treatment, information about susceptibility is limited for Treponema species. The aim of this study was to assess the susceptibility of Treponema denticola strains, Treponema socranskii, and Treponema vincentii to eleven antimicrobial agents. The minimum inhibitory and minimum bactericidal concentrations of these antimicrobial agents revealed strain-specific variation. Doxycycline, minocycline, azithromycin, and erythromycin were effective against all Treponema species tested in this study, whereas fluoroquinolones only exhibited an equivalent effectiveness on T. socranskii. The susceptibility of one T. denticola strain, T. socranskii, and T. vincentii to kanamycin was influenced by prior exposure to aerobic conditions. The susceptibility to quinolone drugs varied among strains of T. denticola, although they share an amino acid sequence identity of greater than 99% for DNA gyrase (type II topoisomerase) subunit A. In addition, an ATP-binding cassette (ABC) transporter inhibitor assay for T. denticola indicated that the transport of quinolone drugs is partially related to this transporter, although there may be parallel transport mechanisms. Our results provide important insights into antimicrobial agent-Treponema dynamics and establish a basis for developing an appropriate adjunctive therapy for periodontal disease.
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http://dx.doi.org/10.1016/j.anaerobe.2017.10.005DOI Listing
December 2017

Effect of extracytoplasmic function sigma factors on autoaggregation, hemagglutination, and cell surface properties of Porphyromonas gingivalis.

PLoS One 2017 20;12(9):e0185027. Epub 2017 Sep 20.

Department of Microbiology, Tokyo Dental College, Chiyoda-ku, Tokyo, Japan.

Porphyromonas gingivalis is a bacterium frequently isolated from chronic periodontal lesions and is involved in the development of chronic periodontitis. To colonize the gingival crevice, P. gingivalis has to adapt to environmental stresses. Microbial gene expression is regulated by transcription factors such as those in two-component systems and extracytoplasmic function (ECF) sigma factors. ECF sigma factors are involved in the regulation of environmental stress response genes; however, the roles of individual ECF sigma factors are largely unknown. The purpose of this study was to investigate the functions, including autoaggregation, hemagglutination, gingipain activity, susceptibility to antimicrobial agents, and surface structure formation, of P. gingivalis ECF sigma factors encoded by SigP (PGN_0274), SigCH (PGN_0319), PGN_0450, PGN_0970, and SigH (PGN_1740). Various physiological aspects of the sigP mutant were affected; autoaggregation was significantly decreased at 60 min (p < 0.001), hemagglutination activity was markedly reduced, and enzymatic activities of Kgp and Rgps were significantly decreased (p < 0.001). The other mutants also showed approximately 50% reduction in Rgps activity. Kgp activity was significantly reduced in the sigH mutant (p < 0.001). No significant differences in susceptibilities to tetracycline and ofloxacin were observed in the mutants compared to those of the wild-type strain. However, the sigP mutant displayed an increased susceptibility to ampicillin, whereas the PGN_0450 and sigH mutants showed reduced susceptibility. Transmission electron microscopy images revealed increased levels of outer membrane vesicles formed at the cell surfaces of the sigP mutant. These results indicate that SigP is important for bacterial surface-associated activities, including gingipain activity, autoaggregation, hemagglutination, vesicle formation, and antimicrobial susceptibility.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185027PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607195PMC
October 2017

Effect of water containing organic acids on aspiration pneumonia-causative bacteria in the biofilm on the tooth surface.

J Dent Sci 2017 Sep 22;12(3):268-274. Epub 2017 Apr 22.

Department of Removable Prosthodontics and Gerodontology, Tokyo Dental College, Tokyo, Japan.

Background/purpose: The tooth surface is a source of oral microbes in dentulous individuals, it is difficult for elderly people requiring nursing care to perform mechanical tooth cleaning by themselves. The objective of this study was to investigate the antimicrobial effect of water containing organic acids (WOA) made by some organic acids as food additives on chemical cleaning for elderly people on aspiration pneumonia-causative bacteria in the biofilm on the tooth surface.

Materials And Methods: Ninety-six specimens made from bovine incisors were divided into four groups and incubated with one of four aspiration pneumonia-causative bacteria. Each group was further divided into six subgroups according to treatment as follows: control group (DW), chlorhexidine gluconate solution group (CHX), WOA group (WOA), ultrasonic treatment in distilled water group (DW-U), ultrasonic treatment in chlorhexidine gluconate solution group (CHX-U) or ultrasonic treatment in WOA group (WOA-U). After treatment, the levels of viable microbes in the biofilm were evaluated by quantitative adenosine triphosphate analysis and compared among the six groups.

Results: For every evaluated microbe, there were significant differences between DW and WOA, and DW and WOA-U. However, there was no significant difference among the WOA, DW-U, CHX-U and WOA-U groups. These results suggested that the antimicrobial effect of WOA on microbes attached to the tooth surface was similar to that of ultrasonic cleaning.

Conclusion: WOA has an antimicrobial effect on microbes in the biofilm on the tooth surface.
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http://dx.doi.org/10.1016/j.jds.2017.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400008PMC
September 2017

Elevation of pro-inflammatory cytokine levels following anti-resorptive drug treatment is required for osteonecrosis development in infectious osteomyelitis.

Sci Rep 2017 04 7;7:46322. Epub 2017 Apr 7.

Department of Orthopedic Surgery Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan.

Various conditions, including bacterial infection, can promote osteonecrosis. For example, following invasive dental therapy with anti-bone resorptive agents, some patients develop osteonecrosis in the jaw; however, pathological mechanisms underlying these outcomes remain unknown. Here, we show that administration of anti-resorptive agents such as the bisphosphonate alendronate accelerates osteonecrosis promoted by infectious osteomyelitis. Potent suppression of bone turnover by these types of agents is considered critical for osteonecrosis development; however, using mouse models we found that acceleration of bone turnover by teriparatide injection did not prevent osteonecrosis but rather converted osteoclast progenitors to macrophages expressing inflammatory cytokines, which were required for osteonecrosis development. In fact, we demonstrate that TNFα-, IL-1α/β- or IL-6-deficient mice as well as wild-type mice administered a TNFα-inhibitor were significantly resistant to development of osteonecrosis accompanying infectious myelitis, even under bisphosphonate treatment. Our data provide new insight into mechanisms underlying osteonecrosis and suggest new ways to prevent it.
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http://dx.doi.org/10.1038/srep46322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384218PMC
April 2017

SigCH, an extracytoplasmic function sigma factor of Porphyromonas gingivalis regulates the expression of cdhR and hmuYR.

Anaerobe 2017 Feb 8;43:82-90. Epub 2016 Dec 8.

Oral Health Science Center, Tokyo Dental College, Tokyo, Japan; Department of Microbiology, Tokyo Dental College, Tokyo, Japan. Electronic address:

Extracytoplasmic function (ECF) sigma factors play an important role in the bacterial response to various environmental stresses. Porphyromonas gingivalis, a prominent etiological agent in human periodontitis, possesses six putative ECF sigma factors. So far, information is limited on the ECF sigma factor, PGN_0319. The aim of this study was to investigate the role of PGN_0319 (SigCH) of P. gingivalis, focusing on the regulation of hmuY and hmuR, which encode outer-membrane proteins involved in hemin utilization, and cdhR, a transcriptional regulator of hmuYR. First, we evaluated the gene expression profile of the sigCH mutant by DNA microarray. Among the genes with altered expression levels, those involved in hemin utilization were downregulated in the sigCH mutant. To verify the microarray data, quantitative reverse transcription PCR analysis was performed. The RNA samples used were obtained from bacterial cells grown to early-log phase, in which sigCH expression in the wild type was significantly higher than that in mid-log and late-log phases. The expression levels of hmuY, hmuR, and cdhR were significantly decreased in the sigCH mutant compared to wild type. Transcription of these genes was restored in a sigCH complemented strain. Compared to the wild type, the sigCH mutant showed reduced growth in log phase under hemin-limiting conditions. Electrophoretic mobility shift assays showed that recombinant SigCH protein bound to the promoter region of hmuY and cdhR. These results suggest that SigCH plays an important role in the early growth of P. gingivalis, and directly regulates cdhR and hmuYR, thereby playing a potential role in the mechanisms of hemin utilization by P. gingivalis.
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http://dx.doi.org/10.1016/j.anaerobe.2016.12.006DOI Listing
February 2017

Antimicrobial Susceptibility of Microorganisms Isolated from Periapical Periodontitis Lesions.

Bull Tokyo Dent Coll 2016 ;57(3):133-42

Department of Oral and Maxillofacial Surgery, Tokyo Dental College.

Periapical periodontitis usually results from microbial infection, with these microorganisms occasionally migrating to the root canal, which can lead to further, potentially life-threatening, complications. Here, the susceptibility of 27 bacterial strains to various antimicrobial agents was evaluated. These strains comprised 13 species; 16 of the strains were clinical isolates from periapical lesions. Each strain was inoculated onto blood agar plates containing one of the antimicrobial agents. The plates were incubated anaerobically at 37°C for 96 hr and the minimal inhibitory concentrations (MICs) determined. Ten strains required an MIC of 32 μg/ml or greater for amoxicillin, 6 for cefmetazole, and 5 for cefcapene among β-lactam antibiotics; 8 strains required an MIC of 32 μg/ml or greater for clindamycin, 4 for azithromycin, and 11 for clarithromycin among macrolide antibiotics; 3 strains required an MIC of 32 μg/ml or greater for ciprofloxacin and 2 for ofloxacin among fluoroquinolones. The effect of cefcapene on 5 strains was evaluated after biofilm formation to investigate the relationship between biofilm formation and susceptibility. All strains showed a decrease in susceptibility after biofilm formation. The results revealed that several antimicrobial agents commonly used in a clinical setting, including amoxicillin, cefmetazole, and clindamycin, are potentially effective in the treatment of orofacial odontogenic infections. The development of resistant strains, however, means that this can no longer be guaranteed. In addition, azithromycin, ciprofloxacin, and ofloxacin were more effective than the 3 β-lactam antibiotics tested. These results suggest that sensitivity testing is needed if odontogenic infections are to be treated safely and effectively.
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http://dx.doi.org/10.2209/tdcpublication.2015-0018DOI Listing
January 2018

Characterization of a potential ABC-type bacteriocin exporter protein from Treponema denticola.

BMC Oral Health 2016 Jul 16;17(1):18. Epub 2016 Jul 16.

Department of Microbiology, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo, 101-0061, Japan.

Background: Treponema denticola is strongly associated with the development of periodontal disease. Both synergistic and antagonistic effects are observed among bacterial species in the process of biofilm formation. Bacteriocin-related genes have not yet been fully characterized in periodontopathic bacteria. The aim of this study was to detect and characterize bacteriocin-associated proteins in T. denticola.

Methods: The whole genome sequence of T. denticola ATCC 35405 was screened with a Streptococcus mutans bacteriocin immunity protein (ImmA/Bip) sequence. The prevalence of homologous genes in T. denticola strains was then investigated by Southern blotting. Expression of the genes was evaluated by qRT-PCR.

Results: In the genome sequence of T. denticola, an amino acid sequence coded by the open reading frame TDE_0719 showed 26 % identity with the S. mutans ImmA. Furthermore, two protein sequences encoded by TDE_0425 and TDE_2431 in T. denticola ATCC 35405 showed ~40 % identity with that coded by TDE_0719. Therefore, TDE_0425, TDE_0719, and TDE_2431 were designated as tepA1, A2, and A3, respectively. Open reading frames showing similarity to the HlyD family of secretion proteins were detected downstream of tepA1, A2, and A3. They were designated as tepB1, B2, and B3, respectively. A gene harboring a bacteriocin-like signal sequence was detected upstream of tepA1. The prevalence of tepA1 and A2 differed among Treponema species. Susceptibility to chloramphenicol and ofloxacin was slightly decreased in a tepA2 mutant while that to kanamycin was increased. Expression of tepA3-B3 was increased in the tepA2 mutant.

Conclusion: These results indicate that T. denticola ATCC 35405 has three potential bacteriocin export proteins and that the presence of these genes differs among the Treponema strains. TepA3-B3 of the corresponding proteins may be involved in resistance to chloramphenicol.
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http://dx.doi.org/10.1186/s12903-016-0243-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947327PMC
July 2016

Treponema denticola invasion into human gingival epithelial cells.

Microb Pathog 2016 May 22;94:104-11. Epub 2016 Jan 22.

Oral Health Science Center, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo 101-0064, Japan; Department of Microbiology, Tokyo Dental College, 2-1-14 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan. Electronic address:

Host cell invasion is important for periodontal pathogens in evading host defenses and spreading into deeper areas of the periodontal tissue. Treponema denticola has been implicated in a number of potentially pathogenic processes, including periodontal tissue penetration. Here we tested the ability of T. denticola strains to invade human gingival epithelial cells (HGEC). After 2 h infection, intracellular location of T. denticola cells was confirmed by confocal laser scanning microscopy (CLSM). Results from an antibiotic protection assay following [(3)H]uridine labeling indicated that invasion efficiency reached a maximum at 2 h after infection. Internalized T. denticola cells were still observed in HGEC at 24 h by CLSM. A dentilisin deficient mutant exhibited significantly decreased invasion (p < 0.05) compared with the wild-type strain. In inhibition assays, phenylmethylsulfonyl fluoride and metabolic inhibitors such as methyl-β-cyclodextrin and staurosporine significantly reduced T. denticola invasion. Under CLSM, T. denticola colocalized with GM-1 ganglioside-containing membrane microdomains in a cholesterol-dependent manner. These results indicated that T. denticola has the ability to invade into and survive within HGECs. Dentilisin activity of T. denticola and lipid rafts on HGEC appear to play important roles in this process.
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http://dx.doi.org/10.1016/j.micpath.2016.01.010DOI Listing
May 2016

Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea.

PLoS One 2016 22;11(1):e0147114. Epub 2016 Jan 22.

Department of Microbiology, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo, Japan.

Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147114PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4723239PMC
July 2016

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation.

Appl Environ Microbiol 2016 Jan 4;82(6):1756-1766. Epub 2016 Jan 4.

Department of Microbiology, Tokyo Dental College, Tokyo, Japan

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.
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http://dx.doi.org/10.1128/AEM.03452-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784043PMC
January 2016

Inhibitory effects of a novel cationic dodecapeptide [CL(14-25)] derived from cyanate lyase of rice on endotoxic activities of LPSs from Escherichia coli and periodontopathic Aggregatibacter actinomycetemcomitans.

Microb Pathog 2016 May 28;94:2-11. Epub 2015 Aug 28.

Laboratory of Chemistry, Tokyo Dental College, Tokyo 101-0062, Japan. Electronic address:

Objective: CL(14-25), a dodecapeptide of cyanate lyase from rice, is a novel cationic α-helical antimicrobial peptide. In this study, we examined inhibitory ability of CL(14-25) against endotoxic activities of lipopolysaccharides (LPSs) from Escherichia coli and periodontal pathogenic Aggregatibacter actinomycetemcomitans.

Methods: Endotoxin-neutralizing activity of CL(14-25) was evaluated by inhibition to induction of cytokine and nitric oxide in human aortic endothelial cells (HAECs) and RAW264 mouse macrophage cells, respectively. Protective effect of CL(14-25) was determined in mice against lethal toxicity of LPS.

Results: IL-6 in HAECs was induced by stimulation with LPS preparations of A. actinomycetemcomitans and E. coli tested in this study, and addition of CL(14-25) to the medium caused inhibition of their induction in a dose-dependent manner. CL(14-25) inhibited NO induction in RAW264 cells by a smooth type LPS of E. coli O55:B5 and an Rc type LPS of E. coli J5 as well as lipid A of E. coli R515 in a dose-dependent manner. Simultaneous injection of E. coli O55:B5 LPS and CL(14-25) in BALB/c mice resulted in prevention of lethal toxicity of the former. The results of a Limulus amebocyte lysate assay and surface plasmon resonance analysis of interaction between CL(14-25) and E. coli LPS or lipid A showed that CL(14-25) specifically binds to a lipid A moiety of LPS.

Conclusion: The results of present study suggest that CL(14-25) has a potential to be used as a nutraceutical agent for periodontal therapy.
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http://dx.doi.org/10.1016/j.micpath.2015.08.011DOI Listing
May 2016
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