Analyses for the cytopathic mechanism of cytocidal toxin from periodontpathic bacteria

牙周病细菌杀细胞毒素的细胞病变机制分析

• 批准号: 14370601
• 负责人: NAKAJIMA Takuma
• 金额: $ 8.96万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370601/
• 关键词: Tannerella forstyensis; Actinobacillus actinomyctemcomitans; cytocidal toxin(CCT); cytolethal istending loxin(CDT); Cell death; cytopathy; Bacterial pathogen; monoclonad antibody; Tannerella forsythensis; cellular toxicity; Cytolethal distending

[I] We had detected a novel activity of Tannerella forsyhtensis cytocidal toxin (Tf-CCT) from an extract of the periodontopathic bacteria that exhibited Cytolethal distending toxin (CDT)-like activity. To identify what is Tf-CCT, and to investigate the mechanism how Tf-CCT exhibits a cytopathy, we cloned its gene on the basis of amino acid sequence of purified protein, and subsequently, we confirmed the cytopathic activity of the cloned gene product. Moreover, we estimated a domain which contributes to the cytopathy of Tf-CCT. Details of the report are following;(a) Identification of Tf-CCT protein purified from T. forsytliensis extract. Tf-CCT protein purified by anion-exchange, size-exclusion and heparin-affinity was identified by the monoclonal antibody Be-24 that was established for neutralization against the cytopathy of T. forsydiensis extract. An 28.5 kDa fragment of Tf-CCT was chosen for the N-terminal amino acid sequence analysis, and a degenerative primer was prepared for the … More resulted sequence APQNMDVLL. The sequence analysis for both PCR product and cloned gene obtained from T.forsythensis genomic DNA library revealed that Tf-CCT was identical with prtH protease (prtH). However, the translation initiation site resides in upstream of the reported site hence the molecular size of natural Tf-CCT detected in the purified fraction was larger than estimated size of pt-Il-I.(b) To identify the translation initiation site, tirst (without ribosome binding motif; RBS), fifth (reported site with RBS) and eighth (with more typical RBS) were chosen as the first ATG to construct the recombinant gene, respectively. Albeit the extensive trial with different types of expression vectors for Tf-CCT was examined, no recombinant Tf-CCT was expressed in Esclmerichia coli whereas the significant expression was detected by in vitro expression system such as RTS and PURESYSTEM.(c) The recombinant Tf-CCT (rTf-CCT) expressed from fifth ATG (identical with reported prtH) exhibited the greatest cytopathy.(d) The protein BLAST revealed that Tf-CCT shares no similarity with any reported CDT besides a distinct homology with third domain of human vesicle transport-related protein slylp in the C-terminal side. Thus, the cytopathy of Tf-CCT is likely to exhibit through interaction with proteins that contribute regulation of intracellular membrane fusion.(e) To get the expression of rTf-CCT in vivo, we tried to fuse recombinant with several kinds of protein-tags, and eventually we found that C-terminal strep-tag fusion achieved sufficient expression of rTf-CCT in E. coli.(f) An ELISA system was examined for detection of anti-Tf-CCT antibody contained by humor in gingival sulcus from the patients of periodontitis.[II] We cloned and identified and cdtB gene of Actinobacillus actinomycetemcomilans serotype(a) (Aaa-cdtB gene). The amino acid sequence of Aaa-CdtB vas revealed to be identical with that of A. actinomycetemcotnitans serotype (b), while the DNA sequence contains two substitutions. We also examined an ELISA system for detection of anti-Aa-CdtB protein antibody contained by humor in gingival sulcus from the patients of periodontitis. Less

[I]我们已经检测到一种新的活性坦纳氏菌cytocidal toxin（Tf-CCT）从牙周病细菌的提取物，表现出细胞致死膨胀毒素（CDT）的活性。为了鉴定Tf-CCT是什么，并研究Tf-CCT表现出细胞病变的机制，我们根据纯化蛋白的氨基酸序列克隆了其基因，随后，我们证实了克隆的基因产物的细胞病变活性。此外，我们估计了一个结构域，有助于Tf-CCT的细胞病变。（a）从T.黑木耳提取物经阴离子交换、分子排阻和肝素亲和纯化的Tf-CCT蛋白，用抗T.山茱萸提取物选择Tf-CCT的28.5kDa片段用于N-末端氨基酸序列分析，并制备用于Tf-CCT的变性引物。 ...更多信息 结果序列APQNMDVLL。对PCR产物和克隆的基因进行序列分析，结果表明Tf-CCT与prtH蛋白酶（prtH）完全一致。然而，翻译起始位点位于报告位点的上游，因此在纯化级分中检测到的天然Tf-CCT的分子大小大于pt-Il-I的估计大小。(b)为了确定翻译起始位点，分别选择第三（无核糖体结合基序; RBS）、第五（有RBS的报告位点）和第八（有更典型的RBS）作为第一个ATG构建重组基因。尽管对不同类型的Tf-CCT表达载体进行了广泛的试验，但在大肠杆菌中没有表达重组Tf-CCT，而通过体外表达系统如RTS和PURESYSTEM检测到显著表达。(c)从第五ATG（与报道的prtH相同）表达的重组Tf-CCT（rTf-CCT）表现出最大的细胞病变。(d)BLAST分析表明，Tf-CCT除了与人囊泡转运相关蛋白slylp的第三结构域在C端有明显的同源性外，与其他已报道的CDT没有相似性。因此，Tf-CCT的细胞病变可能通过与有助于调节细胞内膜融合的蛋白质相互作用而表现出来。(e)为了获得rTf-CCT的体内表达，我们尝试将重组体与多种蛋白标签融合，最终发现C末端strep-tag融合可以实现rTf-CCT在大肠杆菌中的充分表达。杆菌(f)应用ELISA法检测牙周炎患者龈沟液中抗转铁蛋白-CCT抗体。[II]克隆并鉴定了伴放线放线杆菌血清型（a）的cdtB基因（Aaa-cdtB基因）。Aaa-CdtB的氨基酸序列与A.放线菌血清型（B），而DNA序列含有两个取代。我们还建立了一种检测牙周炎患者龈沟液中抗Aa-CdtB蛋白抗体的ELISA系统。少

项目成果

Hasebe A, Yoshimura A, Kataoka H, Tanaka S, Arakawa S, Ishikura H, Golenbock D.T, Sugaya T, Tsuchida N, Kawanami M, Hara Y, Shibata K: "Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease"Infection and Immunity.

Hasebe A、Yoshimura A、Kataoka H、Tanaka S、Arakawa S、Ishikura H、Golenbock D.T、Sugaya T、Tsuchida N、Kawanami M、Hara Y、Shibata K：“福赛斯拟杆菌脂蛋白及其在牙周病中可能的病理作用”感染

Gao CF, Ren S, Wang J, Zhang SL, Jin F, Nakajima T, Ikeda M, Tsuchida N.: "P130 and its truncated form mediate p53-induced cell cycle arrest in Rb(-/-) Saos2 cells"Oncogene.. 21・49. 7569-7579 (2002)

Gau CF、Ren S、Wang J、Zhang SL、Jin F、Nakajima T、Ikeda M、Tsuchida N.：“P130 及其截短形式介导 Rb(-/-) Saos2 细胞中 p53 诱导的细胞周期停滞”癌基因。 21・49。7569-7579（2002）

Fukuyo Y, Mogi K, Tsunematsu Y, Nakajima T: "E2FBP1/hDrill modulates cell growth through downregulation of promyelocytic leukemia bodies"Cell Death and Differentiation. 11(March 12)(Advanced on-line press). 1-3 (2004)

Fukuyo Y、Mogi K、Tsunematsu Y、Nakajima T：“E2FBP1/hDrill 通过下调早幼粒细胞白血病小体调节细胞生长”细胞死亡和分化。

Ishikura H, Arakawa S, Nakajima T, Tsuchida N, Ishikawa I: "Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme."Journal of Medical Microbiology. 52. 1-7 (2003)

Ishikura H、Arakawa S、Nakajima T、Tsuchida N、Ishikawa I：“福赛坦纳菌（福赛拟杆菌）siaHI 基因的克隆和唾液酸酶的纯化。”医学微生物学杂志。

Fukuyo Y, Mogi K, Tsunernatsu Y, Nakaj.ia T.: "E2FBP1/hDrill modulates cell growth through downregulation of proninyelocytic leukemia bodies"Cell Death and Differentiation. 11-(Advanced-on-line press, March 12).. 1-13 (2004)

Fukuyo Y、Mogi K、Tsunernatsu Y、Nakaj.ia T.：“E2FBP1/hDrill 通过下调原粒细胞白血病体来调节细胞生长”细胞死亡和分化。