Possibility of Involvement of Porphyromonas gingivalis in Coronary Heart Disease

Lee, Jin-Yong,Park, Byung-Lae,Yun, Hyun-Kyung,Park, Eun-Ah,Shin, Eun-Ah,Jue, Seong-Suk,Shin, Je-Won The Korea Society for Microbiology 2000 大韓微生物學會誌 Vol.35 No.3

Porphyromonas gingivalis has been implicated in periodontal diseases. Accumulating evidence suggests that cardiovascular disease is the most prevalent medical problem in patients with periodontal diseases. In order to check the possibility that P. gingivalis is involved in coronary heart disease, the present study was performed to observe P. gingivalis adherence and invasion of human coronary artery endothelial cells (HCAEC) and production of cytokines and growth factors by HCAEC upon P. gingivalis infection. $^3H$-labeled P. gingivalis 381 was incubated with HCAEC for 90 min. The radioactivity of the washed HCAEC was a measure of the absorbed (adhering and invading) P. gingivalis. The absorption radioactivity of the HCAEC infected by P. gingivalis was determined to be 59.58% of the input bacterial cells. In contrast, the absorption radioactivity of the cells infected by S. gordonii Challis which was employed as a control was negligible (0.59%). DPG3, a P. gingivalis mutant defective of fimbriae, appeared to be impaired to some extent in capability of adherence/invasion as compared to that of the parental strain 381, showing 43.04% of the absorption radioactivity. The absorption radioactivity of the HCAEC infected by P. gingivalis 381 in the presence of excessive fimbriae at the concentrations of $50\;{\mu}g$ and $100\;{\mu}g/ml$ was 57.27 and 45.44%, respectively. Invasion of HCAEC by P. gingivalis 381 was observed by an antibiotic (metronidazole) protection assay and transmission electron microscopy (TEM). In the antibiotic protection assay, invasion by the bacterium was measured to be 0.73, 1.09, and 1.51% of the input bacterial cells after incubation for 30, 60, and 90 min, respectively. Invasion by DPG3 was shown to be 0.16% after 90-min incubation. In comparison of invasion efficiency at 90 min of the incubation, the invasion efficiency of DPG3 was 0.37% while that of its parental strain 381 was 2.54%. The immunoblot analysis revealed fimbriae of P. gingivalis did not interact with the surface of HCAEC. These results suggest that fimbriae are not the major contribution to the adherence of P. gingivalis to HCAEC but may be important in the invasion of HCAEC by the bacterium. The presence of cytochalasin D ($1\;{\mu}g/ml$) and staurosporine ($1\;{\mu}M$) reduced the invasion of HCAEC by P. gingivalis 381 by 78.86 and 53.76%, respectively, indicating that cytoskeletal rearrangement and protein kinase of HCAEC are essential for the invasion. Infection of P. gingivalis induced HCAEC to increase the production of TNF-${\alpha}$. by 60.6%. At 90 min of the incubation, the HCAEC infected with P. gingivalis cells was apparently atypical in the shape, showing loss of the nuclear membrane and subcellular organelles. The overall results suggest that P. gingivalis may cause coronary heart disease by adhering to and invading endothelial cells, and subsequently damaging the cells.

Fusobacterium nucleatum modulates serum binding to Porphyromonas gingivalis biofilm

최점일,김성조,김수진,Choi, Jeom-Il,Kim, Sung-Jo,Kim, Soo-Jin The Korean Academy of Periodontoloy 2001 Journal of Periodontal & Implant Science Vol.31 No.4

P. gingivalis를 단독면역하거나 또는 Fusobacterium nucleatum 선면역 후 P. gingivalis 항혈청을 각각 얻어냈다. 두 종류의 항혈청이 P. gingivalis biofilm을 침투해 들어가는 능력을 confocal laser scanning microscope를 이용하여 비교 감증하였다. 항혈청의 P. gingivalis에 대한 avidity index도 측정하였다. 결과적으로 F. nucleatum의 선면역은 P. gingivalis 특이 항혈청에 대해 세균성 biofilm의 침투능력을 저하시키고, 동일한 세균에 대한 avidity도 감소시켰다. Anti-P. gingivalis immune sera were obtained from mice immunized with either P. gingivalis alone, or F. nucleaturm followed by P. gingivalis. Two groups of immune sera were examined for binding capacity to P. gingivalis biofilm by confocal laser scanning microscope, Antibody avidity index was also determined for each immune sera. The results indicated that prior immunization of mice with F. nucleaturm impaired P. gingivalis-specific immune sera in binding capacity to biofilm and antibody avidity to P. gingivalis. Elevated antibody responses in patients with destructive periodontal disease has often been related to suboptimal level of protective antibody $(opsonophagocytosis)^{1-3)}$ while post-immune sera obtained with experimental animals using a single periodontal pathogen demonstrated satisfactory levels of protective function against the homologous bacterial $challenge^{4,5)}$.The reason is unclear why elevated IgG responses in periodontal patients to periodontal pathogens do not necessarily reflect their protective function. Such an immune deviation might be derived from the fact that destructive periodontal disease is cumulative result of immunopathologic processes responding to an array of different colonizing microorganisms sequentially infecting in the subgingival environmental niche. Fusobacterium nucleaturm is one of the key pathogens in gingivitis, in the transitional phase of conversion of gingivitis into destructive periodontitk, and in adult $periodontitis^{6-8)}$. It also plays a central role in coaggregation with other important microbial species in subgingival $area^{6,9,10)}$ as well as in $biofilm^{11)}$, especially with Porphyromonas gingjvalis in synergism of virulence in human periodontal disease or in animal $models^{12-14)}$. This organism has also been reported to have immune modulating activity for secondary immune response to Actinobacillus $actinomycetemcomitans^{15)}$. It is presumed that sequential colonization and intermicrobial coaggregation between intermediate and late colonizers could potentially modulate the immune responses and development of specific T cell phenotypes in periodontal lesions. We have recently demonstrated the skewed polarization of P. gingivalis-specific helper T cell clones in mice immunized with F. nucleaturm followed by P. $gingivalis.^{16)}$. Consequently F. nucleaturm may initially prime the immune cells and modify their responses to the successive organism, P. gingivalis. This could explain why one frequently observes non-protective serum antibodies to P. gingivalis in periodontal patients in contrast with those obtained from animals that were immunized with $P.gingivalis\;alone^{17)}$. The present study was performed to investigate the immune modulating effect of F. nucleatum on serum binding to experimental biofilms and the avidity of anti-P. gingivalis antibody.

Porphyromonas gingivalis에 대한 노각나무 잎 추출물의 항균활성 및 생물막 형성 억제 효과

김혜수(Hye Soo Kim),박민정(Min Jeong Park),김수정(Soo Jeong Kim),김부경(Bu Kyung Kim),박준호(JunHo Park),김대현(DaeHyun Kim),조수정(Soo Jeong Cho) 한국생명과학회 2021 생명과학회지 Vol.31 No.3

본 연구에서는 천연물유래 구강건강 개선소재로써 노각나무의 이용 가능성을 알아보기 위해 노각나무 잎과 줄기를 에탄올에 추출한 다음 구강미생물에 대한 추출물의 항균활성을 조사하였다. 노각나무 잎과 줄기 추출물(1 mg/disc)은 구강미생물 중 P. gingivalis KCTC5352에 대해서만 항균활성을 나타내었으며 줄기보다는 잎 추출물의 항균활성이 우수하였다. 시판되고 있는 구강케어제품에 사용되고 있는 항균제와 노각나무 잎 추출물의 항균활성을 비교한 결과, P. gingivalis에 대한 노각나무 잎 추출물과 양성대조구로 사용한 triclosan의 항균활성은 유사하게 나타났으며. P. gingivalis에 대한 노각나무 잎 추출물의 MIC는 0.4 mg/ml이고 정균작용을 하였다. 노각나무 잎 추출물이 0.2-2.0 mg/ml 농도로 처리된 배양액에서 P. gingivalis KCTC5352의 생물막 형성과 세균 생육은 추출물의 농도가 증가할수록 농도의존적으로 억제되는 경향을 보였다. 또한 노각나무 잎 추출물(1 mg/ml) 처리가 P. gingivalis의 생물막 형성에 미치는 영향을 주사전자현미경으로 관찰한 결과에 의하면 추출물을 처리하지 않은 대조구는 추출물 처리구에 비해 P. gingivalis가 군집을 이루며 모여 있었고 세포 주변에서 생물막이 관찰되었지만 추출물을 처리한 처리구의 세포 주변에서는 생물막을 관찰할 수 없었다. qRT-PCR을 이용하여 생물막 형성 초기 과정에서 치면 부착에 필수적인 섬모(fimbriae)관련 mRNA 발현 양상을 0조사한 결과, 노각나무 잎 추출물이 0.2-2.0 mg/ml의 농도로 처리된 배양액에서 fimA와 mfa1 유전자 발현은 추출물의 농도가 높아질수록 농도의존적으로 억제되는 것을 확인할 수 있었다. 이상의 결과를 종합하면 노각나무 잎 추출물은 치주질환 원인균인 P. gingivalis에 대한 항균 활성과 생물막 형성 억제능이 우수하기 때문에 천연물유래 구강건강 개선소재로써 이용 가능성이 높을 것으로 판단된다. This study was conducted to investigate the potential of Stewartia koreana as oral healthcare materials. The antibacterial activity of ethanol extracts from leaves and branches of S. koreana against oral bacteria was confirmed. The leaf and branch extracts (1 mg/disc) showed antibacterial activity against P. gingivalis only among several tested oral bacteria. The leaf extracts showed higher antibacterial activity, with values similar to those of chlorhexidine, which was used as a positive control. The MIC of the leaf extract against P. gingivalis was 0.4 mg/ml and showed bacteriostatic action. The inhibitory effects of the extract on biofilm formation and on gene expression related to biofilm formation by P. gingivalis were determined by biofilm biomass staining, scanning electron microscopy (SEM), and qRT-PCR analysis. The biofilm production rate and cell growth of P. gingivalis in the cultures treated with 0.2-2.0 mg/ml of S. koreana leaf extracts were significantly decreased in a concentration-dependent manner. The inhibitory effect on the formation of P. gingivalis biofilms at concentrations of 1 mg/ml was confirmed by SEM. The qRT-PCR analysis showed concentration-dependent suppression of the fimA and fimB gene expression associated with fimbriae formation in the cultures treated with 0.2-2.0 mg/ml S. koreana leaf extract. These results support the conclusion that S. koreana leaf extracts can be used as oral healthcare materials derived from natural materials, as demonstrated by the antibacterial action and inhibition of biofilm formation of P. gingivalis.

<i>Porphyromonas gingivalis</i> traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells

Lee, Kyulim,Roberts, JoAnn S.,Choi, Chul Hee,Atanasova, Kalina R.,Yilmaz, Ö,zlem TaylorFrancis 2018 Virulence Vol.9 No.1

<P><B>ABSTRACT</B></P><P><I>Porphyromonas gingivalis</I>, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa. However, the precise characterization of the intracellular trafficking and fate of <I>P. gingivalis</I> in GECs remains incomplete. Therefore, we employed high-resolution three-dimensional-transmission-electron-microscopy to determine the subcellular location of <I>P. gingivalis</I> in human primary GECs upon invasion. Serial sections of infected-GECs and their tomographic reconstruction depicted ER-rich-double-membrane autophagosomal-vacuoles harboring <I>P. gingivalis</I>. Western-blotting and fluorescence confocal microscopy showed that <I>P. gingivalis</I> significantly induces LC3-lipidation in a time-dependent-manner and co-localizes with LC3, ER-lumen-protein Bip, or ER-tracker, which are major components of the phagophore membrane. Furthermore, GECs that were infected with FMN-green-fluorescent transformant-strain (PgFbFP) and selectively permeabilized by digitonin showed rapidly increasing large numbers of double-membrane-vacuolar-<I>P. gingivalis</I> over 24 hours of infection with a low-ratio of cytosolically free-bacteria. Moreover, inhibition of autophagy using 3-methyladenine or ATG5 siRNA significantly reduced the viability of intracellular <I>P. gingivalis</I> in GECs as determined by an antibiotic-protection-assay. Lysosomal marker, LAMP-1, showed a low-degree colocalization with <I>P. gingivalis</I> (∼20%). PgFbFP was used to investigate the fate of vacuolar- versus cytosolic-<I>P. gingivalis</I> by their association with ubiquitin-binding-adaptor-proteins, NDP52 and p62. Only cytosolic-<I>P. gingivalis</I> had a significant association with both markers, which suggests cytosolically-free bacteria are likely destined to the lysosomal-degradation pathway whereas the vacuolar-<I>P. gingivalis</I> survives. Therefore, the results reveal a novel mechanism for <I>P. gingivalis</I> survival in GECs by harnessing host autophagy machinery to establish a successful replicative niche and persistence in the oral mucosa.</P>

치주염 유발 세균 Aggregatibacter actinomycetemcomitans와 Porphyromonas gingivalis에 의한 committed osteoclast precursor 분화 증가

박옥진 ( Ok-jin Park ),권영각 ( Yeongkag Kwon ),윤철희 ( Cheol-heui Yun ),한승현 ( Seung Hyun Han ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.4

치주질환은 만성염증성 질환으로 치조골소실을 일으켜 성인치아상실을 유발하는 요인 중 하나이다. 그람 음성세균인Aggregatibacter actinomycetemcomitans와 Porphyromonas gingivalis는 치주질환환자의 병소에서 쉽게 동정된다. 지질 다당체(Lipopolysaccharide; LPS)는 그람 음성세균의 핵심 독력인자로 알려져 있다. 이러한 세균과 LPS는 파골세포에 의한 골소실을 조절하는 요인 중 하나이다. 그러므로 본 연구에서는 동물모델을 활용하여 A. actinomycetemcomitans와 P. gingivalis의 의한 골소실 여부를 확인하고, 기전규명을 위하여 A. actinomycetemcomitans, P. gingivalis, A. actinomycetemcomitans와 P. gingivalis에서 분리한 LPS에 의한 파골세포분화 영향을 연구하였다. 열사멸한 A. actinomycetemcomitans (HKAa)와 열사멸한 P. gingivalis (HKPg)가 복강으로 투여된 쥐의 대퇴골은 대조군에 비해 감소된 골량을 보여주었다. 이러한 골소실의 증가가 파골세포분화 때문인지 확인하기 위해 파골세포분화를 연구한 결과, bone marrow-derived macrophage (BMM)의 RANKL-매개 파골세포분화를 감소시켰으나, committed osteoclast precursor의 파골세포분화를 유도함을 확인하였다. 세균에 의한 파골세포분화 결과와 동일하게 A. actinomycetemcomitans와 P. gingivalis에서 분리한 LPS 역시 RANKL-매개 파골세 포분화는 감소시키고, committed osteoclast precursor의 파골세포분화를 유도하였다. 결과적으로 치주원인균인 A. actinomycetemcomitans와 P. gingivalis는 committed osteoclast precursor의 파골세포분화를 증가시키는데, 이 세균들의 LPS가 핵심 역할을 수행하는 것으로 판단되며 이를 통해 골 흡수를 유발함을 알 수 있었다. Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis are gram-negative bacteria frequently found in lesions from patients with periodontitis manifesting alveolar bone loss. Lipopolysaccharides are a major virulence factor of gram-negative bacteria. Bone resorption is known to be regulated by bacteria and their virulence factors. In the present study, we investigated the effects of A. actinomycetemcomitans and P. gingivalis on bone resorption. Heat-killed A. actinomycetemcomitans (HKAa) and heatkilled P. gingivalis (HKPg) induced bone loss in the femurs of mice after intraperitoneal administration. HKAa and HKPg augmented the differentiation of committed osteoclast precursors into osteoclasts, while they inhibited the differentiation of bone marrow-derived macrophages into osteoclasts. Concordant with the effects of the heat-killed whole cells, LPS purified from A. actinomycetemcomitans and P. gingivalis also augmented osteoclast differentiation from committed osteoclast precursors but attenuated it from bone marrow-derived macrophages. Taken together, these results suggest that the whole cells and lipopolysaccharides of A. actinomycetemcomitans and P. gingivalis induce the differentiation of committed osteoclast precursors into osteoclasts, potentially contributing to bone resorption in vivo.

Streptococcus mutans와 Porphyromonas gingivalis에 대한 상백피(Morus alba root bark) 에탄올 추출물의 항균 효과

박충무(Chung Mu Park),윤현서(Hyun-Seo Yoon) 한국차학회 2023 한국차학회지 Vol.29 No.4

본 연구는 구강질환을 유발하는 대표적인 두 균주인 Streptococcus mutans와 Porphyromonas gingivalis에대한 상백피 에탄올 추출물(MAEE)의 항균 활성을 분석하였다. Griess reaction과 WST-1 assay를 통해lipopolysaccharide (LPS)로 자극한 생쥐대식세포인 RAW 264.7 세포에서 MAEE의 nitric oxide (NO) 억제 및 세포독성 효과를 분석하였다. 그리고 디스크 확산법, 최소억제농도(minimum inhibition concentration, MIC), 최소살균농도(minimum bactericidal concentration, MBC), 성장억제 효과 분석을 통해 MAEE의 항균활성을 평가하였다. 그 결과 MAEE는 LPS로 자극한 RAW 264.7 세포에서 세포독성없이 농도 의존적으로 NO 생성을 억제하였다. 그리고 MAEE는 강력한 항균 활성을 나타냈는데, 100 mg/mL의 MAEE는 S. mutans와 P. gingivalis에 대해 각각 13.94 mm와 15.65 mm의 clear zone을 형성하는 것으로 보다 MAEE는 P. gingivalis보다 S. mutans에 더강한 항균활성을 보이는 것으로 생각된다. 그리고, S. mutans와 P. gingivalis의 MIC는 각각 0.4 mg/mL와 0.4~0.8 mg/mL였고, 두 병원균의 MBC는 각각 0.4 mg/mL와 0.8 mg/mL로 나타났다. 또한 MAEE 처리는 두 병원균의성장을 유의하게 억제하였으나 농도 의존적이지는 않았다. 특히, 24시간동안 MAEE를 처리한 S. mutans의OD600이 3.2 mg/mL에서 0.146인 반면 대조군의 흡광도는 0.509를 보였다. 그리고 같은 용량의 MAEE 처리된 P.gingivalis를 24시간 동안 배양했을 때 대조군의 흡광도 0.486 대비 0.154로 나타나 강한 성장억제효과를 보였다.결론적으로, MAEE는 두 구강 내 병원균에 대해 강력한 항균 활성을 보였으며, MAEE는 P. gingivalis보다 S.mutans에 대해 더 강력한 항균 활성을 나타내는 것을 확인할 수 있었다. 이러한 결과를 종합해 볼 때 상백피는다양한 형태로 가공하여 음용 시 항당뇨, 고혈압, 콜레스테롤 개선 외에도 본 연구를 통해 치아우식증과 치주질환의 예방 및 치료제로 활용 가능성을 검증하였기에 상백피의 차로 활용성을 높인 것으로 생각된다. This study aimed to analyze the antibacterial activity of the ethanol extract of Morus alba root bark (MAEE) against two critical oral pathogens, Streptococcus mutans and Porphyromonas gingivalis. The Griess reaction and WST-1 assays were used to analyze the nitric oxide (NO) inhibitory and cytotoxic effects of MAEE on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The antibacterial activity of MAEE was evaluated using a disk diffusion assay and a growth inhibition assay by determining minimum inhibition concentrations (MICs) and minimum bactericidal concentrations (MBCs). MAEE treatment dose-dependently inhibited NO production and had no cytotoxic effect on LPS-stimulated RAW 264.7 cells. Furthermore, MAEE exhibited potent antibacterial activity and more potently generated a clear zone in the presence of S. mutans than P. gingivalis. The MICs of MAEE for S.mutans and P. gingivalis were 0.4 and 0.4~0.8 mg/mL, and its MBCs were 0.4 and 0.8 mg/mL, respectively. In addition, MAEE significantly inhibited the growths of both pathogens, but no dose-dependency was observed. The OD600 of S. mutans treated with MAEE at 3.2 mg/mL for 24 h was 0.146 compared to a control OD600 of 0.509. Absorbance at 600 nm for P. gingivalis treated with MAEE at 3.2 mg/mL for 24 h was 0.154 compared to a control OD600 of 0.486. In conclusion, MAEE showed potent antibacterial activity against both oral pathogens but affected S.mutans stronger than P. gingivalis. The study indicates that MAEE might help prevent dental caries and periodontal diseases.

구강 질환 유발균인 Streptococcus mutans와 Porphyromonas gingivalis에 대한 모링가(Moringa oleifera) 잎의 항균 효과

박충무,현숙경,윤현서 한국차학회 2023 한국차학회지 Vol.29 No.3

구강질환을 유발하는 두 균주인 Streptococcus mutans와 Porphyromonas gingivalis에 대한 모링가(Moringa oleifera) 잎 에탄올 추출물 (M. oleifera ethanol extract, MOEE)의 항균 효과를 확인하고자 하였다. MOEE의 항균활성은 디스크 확산법, 최소억제농도(minimum inhibition concentration, MIC), 최소살균농도(minimum bactericidal concentration, MBC), 성장억제 효과 분석을 통해서, RAW 264.7 cell에서의 nitric oxide (NO) 생성 억제효과와 세포독성은 Griess reaction과 WST-1 assay를 통해 분석하였다. 실험 결과, lipopolysaccharide (LPS)에 의해유도된 NO 생성은 MOEE의 농도가 높아짐에 따라 유의미하게 억제되었고, 세포독성도 강하게 나타나지 않았다. 그리고 MOEE는 S. mutans와 P. gingivalis에 대해 300 mg/mL에서 19.72 mm, 17.22 mm로 나타나 MOEE는 P. gingivalis를 더 강하게 억제하였다. 또한 S. mutans와 P. gingivalis의 MIC는 0.8 mg/mL과 0.4~0.8 mg/mL였고, MBC는 두 균주 모두에서 0.8 mg/mL로 나타났다. 성장억제 효과는 S. mutans와 P. gingivalis를 0.8 mg/mL의MOEE 농도에서 24시간 배양하였을 때 OD600이 각각 0.219, 0.125로 나타나 MOEE가 P. gingivalis의 성장을 더 강하게 억제하는 것으로 나타났다. 결론적으로, MOEE는 구강질환을 유발하는 두 균주에 대해 유의미한 항균 효과를 나타내었고, 특히 S. mutans와 P. gingivalis의 성장을 더 강하게 억제하였다. 추후 생물막 생성 억제와 다양한 용매 추출물에 대한 활성 검증을 통해 모링가 잎이 치아우식증과 치주질환 예방을 위한 기능성 소재로서 활용될 수있을 기초를 제공할 수 있을 것으로 생각한다. This study examined the antibacterial activity of Moringa oleifera ethanol extract (MOEE) against oral pathogens, Streptococcus mutans and Porphyromonas gingivalis. The antibacterial activity of MOEE was analyzed using a disk diffusion assay, minimum inhibition concentration (MIC), minimum bactericidal concentration (MBC), and growth inhibition assay, and the inhibitory effect of nitric oxide synthesis and cytotoxicity was assessed using the Griess reaction and WST-1 assay in RAW 264.7 cells. NO production by LPS stimulation was inhibited dose-dependently without cytotoxicity. MOEE potently inhibited the disk diffusion of P. gingivalis than S. mutans, which was 19.72 mm and 17.22 mm at 300 mg/mL, respectively. In addition, the MIC of S. mutans and P. gingivalis was 0.8 mg/mL and 0.4~0.8 mg/mL, and the MBC of both pathogens was 0.8 mg/mL for both. MOEE also potently inhibited the growth of P. gingivalis and S. mutans, which was OD600 of 0.8 mg/mL MOEE-treated BHI broth was 0.219 and 0.125, respectively. Consequently, MOEE exhibited potent antibacterial activity, which was stronger against P. gingivalis than S. mutans. MOEE might be used as a functional material for preventing dental caries and periodontal diseases when the inhibitory activity of the biofilm and extracts from various solvents will be conducted in future studies.

P. gingivalis에 특이적으로 작용하는 앱타머에 관한 연구

신애리(Ae-Ri Shin) 한국콘텐츠학회 2021 한국콘텐츠학회논문지 Vol.21 No.4

본 연구는 치주질환의 주 원인균인 P. gingivalis에 선택적으로 작용하는 특이적 앱타머를 선별하고 선별된 앱타머와 결합하는 단백질 분자를 정제 및 동정함으로써 P. gingivalis에 관한 작용기전을 규명하고자 하였다. 이를 위하여 39개의 random sequence를 갖는 DNA library를 제조하여 SELEX 방법을 이용하여 P. gingivalis에 특이성을 가진 앱타머를 선별하였으며 PCR2.1 cloning vector를 활용한 cloning을 시행하여 염기서열을 분석했다. 8종의 각기 다른 염기서열을 가진 앱타머를 선별하였고 직접적으로 작용하는 단백질을 밝혀내고자 선별된 앱타머 중 APG-3를 이용하여 modified weston blot을 시행하고 단백질을 분석한 결과 P. gingivalis에 선택적으로 결합하는 11종의 단백질을 분리, 동정하였다. 이와 같은 결과로 앱타머가 치주질환의 원인균인 P. gingivalis의 당 대사 및 세포활성억제와 관련된 단백질에 선택적으로 결합하여 부착함으로써 치주질환의 진단을 위한 센서로 가능성을 제시했다. In this study, by selecting specific aptamers that selectively detection on P. gingivalis , the main cause of periodontal disease, and purifying and identifying protein molecules that bind to the selected aptamers, the mechanism of action of P. gingivalis was investigated. A DNA library having 39 random sequences was prepared, and aptamers with specificity for P. gingivalis were selected using the SELEX method, and the nucleotide sequence was analyzed by cloning using PCR2.1 cloning vector. 8 of aptamers with different nucleotide sequences were selected, and modified weston blot was performed using APG-3 among the selected aptamers to identify 11 proteins that act directly, and proteins were analyzed. As a result, a protein that selectively binds to P. gingivalis was isolated and identified. Therefore, aptamer selectively binds and attaches to proteins related to inhibition of sugar metabolism and cell activity of P. gingivalis , suggesting the possibility of a sensor for diagnosis of periodontal disease.

Xylitol Mitigate Neutrophil Inflammatory Response Against Porphyromonas gingivalis Infection

Hee Sam Na,YuRi Song,Yoon Hee Choi,Jin Chung 대한구강생물학회 2018 International Journal of Oral Biology Vol.43 No.3

Periodontitis is generally a chronic disorder characterized by breakdown of tooth-supporting tissues, producing dentition loss. Porphyromonas gingivalis (P. gingivalis), a Gram- negative anaerobic rod, is one of the major pathogens associated with periodontitis. Neutrophils are first line defense cells in the oral cavity that play a significant role in inflammatory response. Xylitol is a known anti-caries agent and has anti-inflammatory effects. In this study, we conducted experiments to evaluate anti-inflammatory effects of xylitol on P. gingivalis infected neutrophils for possible usage in prevention and treatment of periodontal infections. P. gingivalis was intraperitoneally injected and peritoneal lavage was collected for cytokine determination. For in vitro study, neutrophils were collected from mouse peritoneal cells after zymosan injection or bone marrow cells. Neutrophils were stimulated with live P. gingivalis and ELISA was used to determine the effect of xylitol on P. gingivalis induced cytokine production. IL-1β, IL-6, TNF-α concentration and neutrophil population in the peritoneal lavage was increased in P. gingivalis-infected mouse. Peritoneal cells infected with live P. gingivalis revealed significantly increased production of IL-1β, IL-6 and TNF-α at multiplicity of infection of 10. Neutrophils from bone marrow and peritoneal lavage revealed increased production of IL-1β, IL-6 and TNF-α. Xylitol significantly mitigated P. gingivalis induced cytokine production in neutrophils. Findings indicate that xylitol is an anti-inflammatory agent in neutrophils infected with live P.gingivalis, that suggests its use in periodontitis management.

Development and Optimization of a Rapid Colorimetric Membrane Immunoassay for Porphyromonas gingivalis

( Jiyon Lee ),( Myoung-kwon Choi ),( Jinju Kim ),( Sechul Chun ),( Hong-gyum Kim ),( Hosung Lee ),( Jinsoo Kim ),( Dongwook Lee ),( Seung-hyun Han ),( Do-young Yoon ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 Journal of microbiology and biotechnology Vol.31 No.5

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer’s disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3′,5,5′-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 10³-10⁵ bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without cross-reactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.