Purification of periodontopatogenic bacterial toxin which induces apoptosis in B cells and identification of its signaling molecules

诱导B细胞凋亡的牙周病细菌毒素的纯化及其信号分子的鉴定

• 批准号: 13671906
• 负责人: NISHIHARA Tatsuji
• 金额: $ 1.92万
• 依托单位: Kyushu Dental College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671906/
• 关键词: exotoxin; periodontopathic bacteria; apoptosis; cell cycle arrest; CDT; juvenile periodontitis; intracellular signal transduction; p21; 難治性歯周炎; 歯周病原因子; B細胞

Cellular microbiology is a new science comprising microbiology, cell biology and molecular biology. Its primary focus is on the interactions between bacteria and host cells which give rise to pathology but which also may maintain health. Recent studies have shown that bacteria can control many aspects of eukaryotic cell behavior including cell movement and shape, cell proliferation and apoptosis. Actinobacillus actinomycetemcomitans, the etiologic agent of juvenile periodontitis, produces several exotoxins including leukotoxin and cytolethal distending toxin (CDT). We discovered a novel toxin (NTX) which affects the cell cycle of host cells (Infect. Immun. 1998 : 5980-7). In this study, we analyzed the mechanisms of cell cycle arrest induced by this NTX. NTX was purified from the culture supematant of A. actinomycetemcomitans, by four-step procedure : ammonium sulfate precipitation ; POROS HQ/M column chromatography ; polymyxin B matrix column chromatography ; and Mono-Q column chromatography. For the experimental control, recombinant CDT was obtained from the sonicate extracts of E.coli expressing A. actinomycetemcomitans CDT. Both NTX and CDT blocked the cell cycle progression at G2/M phase in mouse hybridoma HS-72 cells with induction of cell cycle regulatory protein p21 WAF1/CIP1. Intracellular responses of host cells against these exotoxins would be important for understanding how this periodontopathogen initiates periodontal pocket formation and plays a role in the development of periodotitis.

细胞微生物学是微生物学、细胞生物学和分子生物学相结合的一门新兴学科。它的主要重点是细菌和宿主细胞之间的相互作用，这些相互作用引起病理学，但也可能维持健康。最近的研究表明，细菌可以控制真核细胞的许多方面的行为，包括细胞的运动和形状，细胞增殖和凋亡。伴放线放线杆菌（Actinobacillusactinomycetemcomitans）是引起青少年牙周炎的病原菌，可产生白细胞毒素（leukotoxin）和细胞致死性膨胀毒素（cytolethalexpondingtoxin，CDT）等多种外毒素。我们发现了一种影响宿主细胞细胞周期的新型毒素（NTX）（Infect. Immun. 1998：5980-7）。在这项研究中，我们分析了这种NTX诱导的细胞周期阻滞的机制。从A.通过硫酸铵沉淀、波罗斯HQ/M柱层析、多粘菌素B基质柱层析和Mono-Q柱层析四步程序，从放线菌共生体中分离纯化放线菌。对于实验对照，从表达A.伴放线菌NTX和CDT均能通过诱导细胞周期调控蛋白p21 WAF 1/CIP 1的表达，将小鼠杂交瘤HS-72细胞周期阻滞于G2/M期。宿主细胞对这些外毒素的细胞内反应对于理解这种牙周病原体如何启动牙周袋形成并在牙周炎的发展中发挥作用是重要的。

项目成果

Nishihara, T., O.Kawai, K.Nakashima, M.Mori, S.Kato, and Y.Kowashi: "Effect of aging on interleukin-6 release from human gingival fibroblasts stimulated with interleukin-1β"Dentistry in Japan. 37. 20-25 (2001)

Nishihara, T.、O.Kawai、K.Nakashima、M.Mori、S.Kato 和 Y.Kowashi：“衰老对白细胞介素 1β 刺激的人牙龈成纤维细胞释放白细胞介素 6 的影响”日本牙科 37。 .20-25 (2001)

Nakashima, K., J.Tomioka, S.Kato, T.Nishihara and Y.Kowashi: "Nitric oxide-mediated protection of A. actinomycetemcomitans-infected murine macrophages against Apoptosis"Nitric Oxide : Biology and Chemistry. 6. 61-68 (2002)

Nakashima, K.、J.Tomioka、S.Kato、T.Nishihara 和 Y.Kowashi：“一氧化氮介导的 A. actinomycetemcomitans 感染的鼠巨噬细胞抗细胞凋亡的保护”一氧化氮：生物学和化学。

Saiki et al.: "Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans"Microbiol Immunol. 45. 497-506 (2001)

Saiki 等人：“Actinobacillus actinomycetemcomitans 的细胞致死膨胀毒素的重建和纯化”Microbiol Immunol。

Nakahara et al.: "Growth/differentiation factor-5 induces growth arrest and apoptosis in mouse B lineage cells with modulation by Smad"Cellular Signalling. 15. 181-187 (2003)

Nakahara 等人：“生长/分化因子 5 通过 Smad 的调节，诱导小鼠 B 谱系细胞生长停滞和细胞凋亡”细胞信号传导。

Nonaka et al.: "Involvement of caspase in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemconitans"J Periodontal Research. 36. 40-47 (2001)

Nonaka 等人：“半胱天冬酶参与感染放线杆菌放线菌的巨噬细胞凋亡细胞死亡”J 牙周研究。