• The Journal of the Korean Society for Microbiology
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• v.21 no.4
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• pp.487-501
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• 1986

Previous studies from our laboratory suggested that Korean LJP patients might habor A. actinomycetemcomitans of different serotype from Caucasian LJP patients in whom serotype b was predominant. In order to observe the prevalence and serotype distribution of A. actinomycetemcomitans in localized juvenile periodontitis patients and to evaluate leukotoxic activity of oral isolates, this study was performed. A. actinomycetemcomitans was isolated by using a selective medium(tryptic soy agar supplemented with 10% serum, $75{\mu}g$ of bacitracin and $5{\mu}g$ of vancomycin per ml). Using immunoabsorbed, ammonium sulfate-fractionated serotype-specific antisera, a total of 69 strains were serologically categorized by ELISA. Leukotoxicity was monitored biochemically by measuring lactate dehydrogenase indicator of cell viability in culture supernatant of PMNL plus viable A. actinomycetemcomitans mixture. The results were as follows: 1. A. actinomycetemcomitans was detected in 75% of 16 LJP patients, and 71% in the LJP lesions and 6% in the control sites. 2. Presence or absence of A. actinomycetemcomitans in the sampled disease sites has no in fluence on clinical measurements. 3. Three serotypes were approximately equally distributed in overall 9 patients. Three patients harbored 2 different serotypes of A. actinomycetemcomitans in the same disease site or different disease sites. 4. The proportion of leukotoxic oral isolates was 22% of a total of 46 strains and the prevalence was 69% in 13 sampled sites. The same disease site could harbor both leukotoxic and nonleukotoxic strains. 5. Distribution of leukotoxic strains in 3 serotypes were not different.

• Journal of Periodontal and Implant Science
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• v.39 no.sup2
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• pp.231-237
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• 2009

Purpose: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite $AB_2$ toxin (CdtB: the enzymatic A subunit; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for $GM_1$ ganglioside. Methods: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to $GM_1$ ganglioside was checked through ELISA. Results: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to $GM_1$ ganglioside, while CdtA alone did not. Conclusions: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.

• Bulletin of the Korean Chemical Society
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• v.31 no.2
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• pp.275-280
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• 2010

Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus bacterium that is associated with several human diseases, including endocarditis, meningitis, osteomyelitis, subcutaneous abscesses and periodontal diseases. A full-length Omp1-like protein gene from A. actinomycetemcomitans was cloned into a pQE30 vector and overexpressed in Escherichia coli BL21(DE3) cells. The protein revealed sequence homologies to Seventeen kilodalton proteins (Skp) from Pasteurella multocida and E. coli that have been characterized as periplasmic chaperones. This soluble Omp1-like protein was successfully purified to homogeneity for further folding and functional studies. The purity, identity, and conformation of the protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, fluorescence spectroscopic, and differential scanning calorimetric studies. We showed that the protein formed an oligomer larger than a tetramer. We found, further, that it is comprised of mostly $\alpha$-helices and boasts high thermal stability.

• Journal of Periodontal and Implant Science
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• v.37 no.sup2
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• pp.339-351
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• 2007

Cytolethal distending toxin(CDT)은 세포 주기 중 G2에서 M 기로의 전환을 막아 세포의 증식을 억제할 수 있는 세균 단백 독소의 일종이다. 구강 미생물 중 유일하게 Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans)만이 이 CDT를 생성 할 수 있는 것으로 알려져 있다. A. actinomycetemcomitans는 localized aggressive periodontitis (LAP)의 원인균으로 여겨지며 비 운동성의 그람 음성 구간균이고 $37^{\circ}C$, 5% $CO_2$ 하에 성장이 왕성하다. A. actinomycetemcomitans의 CDT는 3개의 인접한 유전자인 cdtA, cdtB, cdtC에 의해 형성 되며 각각의 유전자에 대한 단백질의 기능은 아직 완전히 밝혀지지 않았다. 현재까지 연구에 의하면 cdtA는 CDT의 세포부착과 관련이 있는 것으로 여겨지며 이 유전자의 기능 이상 시 CDT의 독성 효과가 현저히 감소한다고 알려져 있다. 따라서 본 연구는 A. actinomycetemcomitans의 cdtA 유전자를 클로닝, 단백질 발현하여 향후 치주질환의 발병 과정에서 CdtA의 역할을 규명하고 질환의 예방 및 치료법에 도움을 주고자 하였다. A. actinomycetemcomitans Y4균주를 cdtA 유전자 클로닝을 위해 사용하였다. A. actinomycetemcomitans의 genomic DNA는 genomic DNA 추출 kit를 사용하여 분리하고 cdtA에 특이적인 primer를 이용하여 PCR을 통해 cdtA 유전자를 증폭하였다. 증폭된 cdtA 유전자를 T-vector에 클로닝 하였으며, 클로닝 된 cdtA 유전자는 단백질 발현을 위해 pRSET Avector에 서브클로닝 한 후 발현 균주인 BL21(DE3)를 이용하여 발현시켰다. 발현 후 Ni-NTA AP conjugate를 이용한 Western blot을 통해 pRSET-CDTA를 확인하였다.

• Restorative Dentistry and Endodontics
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• v.25 no.4
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• pp.606-618
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• 2000

Dental pulp infection is most commonly caused by extensive dental caries, and some bacterial species invade root canals; bacterial components and products are thought to be associated with the pathogenesis of periapical periodontitis. A principle driving force behind pulpal disease response appears to lie in the host immune system's to bacteria and their products. We examined the production of interleukin $1{\beta}$ (IL-$1{\beta}$) and tumor necrosis factor ${\alpha}$(TNF-${\alpha}$) from human peripheral mononuclear cells, lymphocytes and monocytes stimulated by heat-killed Acitnobacillus actinomycetemcomitans (ATCC 29523), Porphyromonas gingivalis (ATCC 33277) and Prevotella intermedia (ATCC 25611), and also by their sonicated bacterial extracts (SBE), respectively. The effects of three strains of heat-killed bacteria and their SBEs on the morphology of cultured blood cell lines HL-60 (KCLB 10240) and J774A.1 (KCLB 40067) were observed under the inverted microscope. Ultrastructural changes of J774A.1 exposed to heat-killed P. intermedia and its SBE were investigated using transmission electron microscopy. Production of IL-$1{\beta}$ was reduced in human peripheral mononuclear cells after stimulation by sonic bacterial extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. Heat-killed and sonic extract of P. gingivalis inhibited the production of TNF-${\alpha}$ in peripheral mononuclear cells. Production of TNF-${\alpha}$ was inhibited in peripheral monocytes after stimulation by sonic extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. HL-60 and J 774A.1 cells showed granular degeneration after treatment with heat-killed and sonic extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia Chromatin margination and shrinkage were observed in 774A.1 treated with heat-killed P. intermedia. Cell wall structure and organelles were destroyed and vacuoles were formed in cytoplasm in J774A.1 treated with P. intermedia sonic extract. These results suggest that A actinomycetemcomitans, P gingivalis and P intermedia may have an important role in the formation and progression of pulpal diseases via both modulation of production of IL-$1{\beta}$ and TNF-${\alpha}$ from blood mononuclear cells and cytopathic effects.

• Journal of Periodontal and Implant Science
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• v.38 no.sup2
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• pp.335-342
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• 2008

Purpose: Cytolethal distending toxin (CDT) considered as a key factor of localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis is composed of five open reading frames (ORFs). Among of them, the individual role of CdtA and CdtC is not clear; several reports presents that CDT is an AB2 toxin and they enters the host cell via clathrin-coated pits or through the interaction with GM3 ganglioside. So, CdtA, CdtC, or both seem to be required for the delivery of the CdtB protein into the host cell. Moreover, recombinant CDT was suggested as good vaccine material and antibody against CDT can be used for neutralization or for a detection kit. Materials and Methods: We constructed the pET28a-cdtC plasmid from Aggregatibacter actinomycetemcomitans Y4 by genomic DNA PCR and expressed in BL21 (DE3) Escherichia coli system. We obtained the antibody against the recombinant CdtC in mice system. Using the anti-CdtC antibody, we test the native CdtC detection by ELISA and Western Blotting and confirm the expression time of native CdtC protein during the growth phase of A. actinomycetemcomitans. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and generated the anti CdtC antibody against recombinant CdtC subunit expressed in E. coli system. Our anti CdtC antibody can be interacting with recombinant CdtC and native CDT in ELISA and Western system. Also, CDT holotoxin existed at 24h but not at 48h meaning that CDT holotoxin was assembled at specific time during the bacterial growth. Conclusion: In conclusion, we thought that our anti CdtC antibody could be used mucosal adjuvant or detection kit development, because it could interact with native CDT holotoxin.

• Journal of Periodontal and Implant Science
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• v.20 no.1
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• pp.147.1-147.1
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• 1990

• International Journal of Oral Biology
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• v.41 no.4
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• pp.199-208
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• 2016

The aim of this study was to identify the non-Aggregatibacter actinomycetemcomitans bacteria grown on the tryptic soy-serum-bacitracin-vancomycin (TSBV) medium, an A. actinomycetemcomitans selective medium. A total of 82 unidentified bacterial isolates from the oral cavities of a Korean population were kindly provide by the Korean Collection for Oral Microbiology. All the clinical isolates were grown on TSBV medium and bacterial DNA purified from each isolate was subjected to PCR with universal primers specific for bacterial 16S rRNA genes (16S rDNAs) sequence. The each bacterial 16S rDNA was amplified by PCR and the nucleotide sequences of it was determined by the dideoxynucleotide chain termination method. They were identified by 16S rDNA sequence comparison method at the specie-level. The data showed that Neisseria spp. (42 strains), Fusobacterium spp. (10 strains), Capnocytophaga spp. (8 strains), Propionibacterium acnes (5 strains), Aggregatibacter aprophilus (4 strains), Campylobacter spp. (5 strains), Veillonella dispar (3 strains), Streptococcus sp. (1 strain), Haemophilus parainfluenzae (1 strain), Leptotrichia wadei (1 strain), Morococcus sp./Neisseria sp. (1 strain), and Staphylococcus sp. (1 strain) were identified. These results could be used to develop a new A. actinomycetemcomitans-selective medium which is more effective than the TSBV medium in future studies.

• Journal of dental hygiene science
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• v.9 no.2
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• pp.249-255
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• 2009

This study was carried out for the purpose of comparing bacterial culture method, single PCR, and multiplex PCR for identification of F. nucleatum and A. actinomycetemcomitans in subgingival plaque of adult periodontitis. Targeting 20 patients with adult periodontitis, the subgingival plaque was collected in teeth, respectively, for #16, #36, #44. A bacillus was cultivated by painting it over the solid selective media of F. nucleatum and A. actinomycetemcomitans. Bacterial species were detected in 0 tooth with 12 pieces, respectively. Through single PCR and multiplex PCR, the positive reaction was indicated in 43 teeth with 45 pieces, respectively, as for F. nucleatum, and in 1 tooth with 4 pieces, respectively, as for A. actinomycetemcomitans. In the comparative analysis between bacterial identification methods. F. nucleatum showed the more statistically significant difference(p=0.0(0) in comparison between single PCR and multiplex PCR. Even A. actinomycetemcomitans was indicated significantly(p=0.067) in a case that is based on 0.1 in significant level in the comparison between single PCR and multiplex PCR. In conclusion, as a result of comparing the bacterial identification methods, the detection frequency was indicated to be higher in PCR than in bacterial culture method. Single PCR and multiplex PCR showed the mutually similar detection frequency. Accordingly, given thinking of economic efficiency, quickness, and reduction in labor force, it is thought to be more efficient method to use single PCR as the bacterial identification method.

• Journal of The Korean Society of Integrative Medicine
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• v.7 no.2
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• pp.141-151
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• 2019

Purpose : Hypochlorous acid (HOCl), a major inorganic bactericidal compound of innate immunity, is effective against a broad range of microorganisms. In particular, HOCl is well-known as a non-antibiotic antimicrobial substance. However, effects of HOCl as an antimicrobial agent are still needed to study these functions against various specific type of microorganisms. In this study, we investigated the antimicrobial effect of hypochlorous acid (HOCl) in S. mutans and A. actinomycetemcomitans to cause dental caries and periodontal disease. Experiments were conducted to observe whether HOCl become effective replacement of disinfectant. Methods : To observe antimicrobial effect of HOCl, stabilized HOCl is prepared in the form of a physiologically balanced solution in pre-conditioned and post-conditioned HOCl solution. As a control, commercially available disinfectant MAXCLEAN was used as positive control. Moreover, S. mutans and A. actinomycetemcomitans distribution in gagrin, filtered tap water, and culture media. Cell viability were measured by viable cell count methods and disk diffusion test. Results : Our results showed that treatment of HOCl have no effect against antimicrobial effect compare to control group especially gagrin in disk diffusion test. HOCl tended to reduced viability against S. mutans in group of post-conditioned than pre-conditioned of HOCl solution however, there was no significant difference as well as no effect in A. actinomycetemcomitans. Conclusion : HOCl showed tendency to reduce viability against S. mutans in group of post-conditioned of HOCl solution and no effect of antimicrobial effect. Although HOCl is well known as effective against a broad range of microorganisms, HOCl seems to have diversity following type of species to be used as antimicrobial drug following our results. Therefore, it is necessary to be rigidly controlled and regulated in using HOCl solution clinically.