Ji-Hoi Moon

Development and evaluation of new primers for PCR‐based identification of type II fimA of Porphyromonas gingivalis

For more accurate PCR-based identification of Porphyromonas gingivalis harboring genotype II fimA, the most prevalent type in periodontitis patients, a new primer set was developed and evaluated. The previous type II primers hybridized to the DNA of P gingivalis strains harboring type Ib as well as type II fimA, while the new primers specifically amplified only the DNA fragment of type II fimA. In the investigation using mixed bacterial culture and 155 clinical samples from peri-implantitis patients, the new primers increased the accuracy of PCR-based detection of type II fimA by excluding false-negatives as well as false-positives.

Antibacterial and antibiofilm effects of iron chelators against Prevotella intermedia

Prevotella intermedia, a major periodontopathogen, has been shown to be resistant to many antibiotics. In the present study, we examined the effect of the FDA-approved iron chelators deferoxamine (DFO) and deferasirox (DFRA) against planktonic and biofilm cells of P. intermedia in order to evaluate the possibility of using these iron chelators as alternative control agents against P. intermedia. DFRA showed strong antimicrobial activity (MIC and MBC values of 0.16 mg ml(-1)) against planktonic P. intermedia. At subMICs, DFRA partially inhibited the bacterial growth and considerably prolonged the bacterial doubling time. DFO was unable to completely inhibit the bacterial growth in the concentration range tested and was not bactericidal. Crystal violet binding assay for the assessment of biofilm formation by P. intermedia showed that DFRA significantly decreased the biofilm-forming activity as well as the biofilm formation, while DFO was less effective. DFRA was chosen for further study. In the ATP-bioluminescent assay, which reflects viable cell counts, subMICs of DFRA significantly decreased the bioactivity of biofilms in a concentration-dependent manner. Under the scanning electron microscope, P. intermedia cells in DFRA-treated biofilm were significantly elongated compared to those in untreated biofilm. Further experiments are necessary to show that iron chelators may be used as a therapeutic agent for periodontal disease.

Genotype analysis of Porphyromonas gingivalis fimA in Korean adults using new primers

Strains of Porphyromonas gingivalis, a periodontopathic bacterium, are classified into six genotypic variants based on nucleotide sequence differences in the fimA gene encoding FimA. A PCR assay using primer sets specific for each genotype has demonstrated that the most predominant fimA genotype in periodontitis patients is type II, which is now commonly referred to as the periodontitis-associated fimA genotype of P. gingivalis. However, the potential for false type II fimA positives caused by cross-hybridization of type II fimA-specific primers with type Ib fimA has complicated the genotyping. A previous study developed new primers that specifically amplified only the DNA fragment of type II fimA. The aim of the present study was to assess the prevalence of P. gingivalis fimA genotypes in Korean adults and to reconfirm the relationship between type II fimA and periodontitis using the new primers. Among 412 Korean adults, P. gingivalis was detected in 97.5 % of patients and 57.8 % of healthy subjects. Type II fimA was the most widely distributed type among healthy and periodontitis subjects. Organisms with types II, Ib and IV fimA had a significant frequency of occurrence in periodontitis subjects. Statistical analysis, however, revealed that a more significant correlation was found between periodontitis and the occurrence of type Ib fimA.

In vitro activity of deferoxamine against Porphyromonas gingivalis

Deferoxamine (DFO), an FDA-approved iron chelator used for treatment of iron poisoning, affects bacteria as iron availability is intimately connected with growth and several virulence determinants. However, little is known about the effect on oral pathogens. In this study, the effect of DFO on Porphyromonas gingivalis, a major periodontopathogen which has an essential growth requirement for hemin (Fe(3+)-protoporphyrin IX), was evaluated. The viability of P. gingivalis W83 was not affected by 0.06-0.24 mM DFO, whereas the doubling time of the bacterium was considerably prolonged by DFO. The inhibitory effect was evident at earlier stages of growth and reduced by supplemental iron. UV-visible spectra using the pigments from P. gingivalis cells grown on blood agar showed that DFO inhibited μ-oxo bisheme formation by the bacterium. DFO decreased accumulation and energy-driven uptake of hemin by P. gingivalis. Antibacterial effect of H(2)O(2) and metronidazole against P. gingivalis increased in the presence of DFO. Collectively, DFO is effective for hemin deprivation in P. gingivalis suppressing the growth and increasing the susceptibility of the bacterium to other antimicrobial agents such as H(2)O(2) and metronidazole. Further experiments are necessary to show that DFO may be used as a therapeutic agent for periodontal disease.

Antibacterial effects of N-acetylcysteine against endodontic pathogens

The success of endodontic treatment depends on the eradication of microorganisms from the root canal system and the prevention of reinfection. The purpose of this investigation was to evaluate the antibacterial and antibiofilm efficacy of N-acetylcysteine (NAC), an antioxidant mucolytic agent, as an intracanal medicament against selected endodontic pathogens. Minimum inhibitory concentrations (MICs) of NAC for Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were determined using the broth microdilution method. NAC showed antibacterial activity, with MIC values of 0.78–1.56 mg/ml. The effect of NAC on biofilm formation of each bacterium and a multispecies culture consisting of the four bacterial species was assessed by crystal violet staining. NAC significantly inhibited biofilm formation by all the monospecies and multispecies bacteria at minimum concentrations of 0.78–3.13 mg/ml. The efficacy of NAC for biofilm disruption was evaluated by scanning electron microscopy and ATP-bioluminescence quantification using mature multispecies biofilms. Preformed mature multispecies biofilms on saliva-coated hydroxyapatite disks were disrupted within 10 min by treatment with NAC at concentrations of 25 mg/ml or higher. After 24 h of treatment, the viability of mature biofilms was reduced by > 99% compared with the control. Moreover, the biofilm disrupting activity of NAC was significantly higher than that of saturated calcium hydroxide or 2% chlorhexidine solution. Within the limitations of this in vitro study, we conclude that NAC has excellent antibacterial and antibiofilm efficacy against endodontic pathogens and may be used as an alternative intracanal medicament in root canal therapies.

Characterization of FimA in Porphyromonas gingivalis genotype IV

It has been reported that a large majority of periodontitis patients carry organisms with either type II or IV-fimA, while type I is the most prevalent fimA genotype among Porphyromonas gingivalis-positive healthy adults. Here we report characterization of recombinant fimbrial protein (rFimA) produced in Escherichia coli from genotype IV-fimA. In SDS-PAGE and immunoblot analysis after partial dissociation, type IV-rFimA showed a ladder-like pattern representing oligomeric/polymeric forms of native fimbrial structure. Unlike anti-type I-native fimbriae which can only recognize conformational epitopes of the respective proteins, both anti-type IV-native fimbriae and anti-type IV-rFimA antibodies recognized conformational as well as linear epitopes in type IV-fimbriae. These results suggest that the type IV-rFimA proteins retain the native fimbrial antigenicity and the antigenicity of type IV-fimbriae is different from that of type I-fimbriae.

Removal and killing of multispecies endodontic biofilms by N-acetylcysteine

Removal of bacterial biofilm from the root canal system is essential for the management of endodontic disease. Here we evaluated the antibacterial effect of N-acetylcysteine (NAC), a potent antioxidant and mucolytic agent, against mature multispecies endodontic biofilms consisting of Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans and Enterococcus faecalis on sterile human dentin blocks. The biofilms were exposed to NAC (25, 50 and 100 mg/mL), saturated calcium hydroxide or 2% chlorhexidine solution for 7 days, then examined by scanning electron microscopy. The biofilm viability was measured by viable cell counts and ATP-bioluminescence assay. NAC showed greater efficacy in biofilm cell removal and killing than the other root canal medicaments. Furthermore, 100 mg/mL NAC disrupted the mature multispecies endodontic biofilms completely. These results demonstrate the potential use of NAC in root canal treatment.

TMEM126A, a CD137 ligand binding protein, couples with the TLR4 signal transduction pathway in macrophages.

We showed previously that a novel protein, transmembrane protein 126A (TMEM126A), binds to CD137 ligand (CD137L, 4-1BBL) and couples with its reverse signals in macrophages. Here, we present data showing that TMEM126A relays TLR4 signaling. Thus, up-regulation of CD54 (ICAM-1), MHC II, CD86 and CD40 expression in response to TLR4 activation was diminished in TMEM126A-deficient macrophages. Moreover in TMEM126A-deficient RAW264.7 cells, LPS/TLR4-induced late-phase JNK/SAPK and IRF-3 phosphorylation was abolished. These findings indicate that TMEM126A contributes to the TLR4 signal up-regulating the expression of genes whose products are involved in antigen presentation.

Effects of sodium tri- and hexameta-phosphate in vitro osteoblastic differentiation in Periodontal Ligament and Osteoblasts, and in vivo bone regeneration

The present study was designed to assess the effects and underlying mechanism of two poly(P) compounds, sodium triphosphate (STP, Na5P3O10) and sodium hexametaphosphate (SHMP, Na15P13O40~Na20P18O40) on osteoblastic differentiation of human periodontal ligament cells (PDLCs) and osteoblasts in vitro, and bone formation in vivo. Differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and mRNA expression for marker genes. To examine the osteogenic potential to regenerate bone, the critical-sized mouse calvarial defect model was utilized. Incubation of PDLCs and osteoblasts with STP and SHMP resulted in a dose- and time-dependent increase in growth, alkaline phosphatase (ALP) activity, mineralization and mRNA expression for marker genes. STP and SHMP increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), Akt, and mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPK). Treatment with the mTOR inhibitor, rapamycin, attenuatted STP- and SHMP-induced osteoblastic differentiation. Micro-CT and histologic analysis showed that STP significantly increased new bone formation in calvarial defects, compared with SHMP and control group. Collectively, this is the first study to demonstrate that STP and SHMP promotes the osteoblastic differentiation in vitro, whereas STP only stimulated bone repair in vivo. Therefore, STP may be useful therapeutic approach for the regeneration of bone or periodontal tissue.

Potent in vitro and in vivo activity of plantibody specific for Porphyromonas gingivalis FimA

ABSTRACT Fimbrial protein fimbrillin (FimA), a major structural subunit of Porphyromonas gingivalis, has been suggested as a vaccine candidate to control P. gingivalis-induced periodontal disease. Previously, cDNAs encoding IgG monoclonal antibodies (MAbs) against purified FimA from P. gingivalis 2561 have been cloned, and the MAbs have been produced in rice cell suspension. Here we examined the biological activities of the plant-produced MAb specific for FimA (anti-FimA plantibody) of P. gingivalis in vitro and in vivo. The anti-FimA plantibody recognized oligomeric/polymeric forms of native FimA in immunoblot analysis and showed high affinity for native FimA (KD = 0.11 nM). Binding of P. gingivalis (108 cells) to 2 mg of saliva-coated hydroxyapatite beads was reduced by 53.8% in the presence of 1 μg/ml plantibody. Anti-FimA plantibody (10 μg/ml) reduced invasion of periodontal ligament cells by P. gingivalis (multiplicity of infection, 100) by 68.3%. Intracellular killing of P. gingivalis opsonized with the anti-FimA plantibody by mouse macrophages was significantly increased (77.1%) compared to killing of bacterial cells with irrelevant IgG (36.7%). In a mouse subcutaneous chamber model, the number of recoverable P. gingivalis cells from the chamber fluid was significantly reduced when the numbers of bacterial cells opsonized with anti-FimA plantibody were compared with the numbers of bacterial cells with irrelevant IgG, 66.7% and 37.1%, respectively. These in vitro and in vivo effects of anti-FimA plantibody were comparable to those of the parental MAb. Further studies with P. gingivalis strains with different types of fimbriae are needed to investigate the usefulness of anti-FimA plantibody for passive immunization to control P. gingivalis-induced periodontal disease.