人および家畜に下痢症を引き起こす新規細菌毒素の構造解析と阻害剤の開発

• Project/Area Number: 16J09051
• Research Category: Grant-in-Aid for JSPS Fellows
• Allocation Type: Single-year Grants
• Section: 国内
• Research Field: Veterinary medical science
• Research Institution: Kyoto Sangyo University
• Principal Investigator: TONITI Waraphan (2017) 京都産業大学, 工学研究科, 特別研究員(DC2); TONITI WARAPHAN (2016) 京都産業大学, 工学研究科, 特別研究員(DC2)
• Project Period (FY): 2016-04-22 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 2017: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 2016: ¥700,000 (Direct Cost: ¥700,000)
• Keywords: enterotoxin / food-poisoning / Clostridium / binary toxin / CPILE / x-ray crystallography

Outline of Annual Research Achievements

The W5052 strain isolated from unusual Tokyo food-poisoning outbreaks did not harbor the cpe nor produce C. perfringens enterotoxin (CPE). On the other hand, they secreted a novel enterotoxin, CPILE. It is consisting of two components: CPILE-a, which acts as an enzymatic ADP-ribosyltransferase and CPILE-b, a membrane binding component.
The enterotoxicity of CPILE relies on endocytosis of the CPILE complex into the cytosol. CPILE-b binds to the specific cell receptor and assists translocation of CPILE-a into the target cell. How CPILE-b binds to the particular cell receptor is still unclear.
The purified CPILE-b was subjected to cleavage pro-sequence region by trypsin to trigger oligomerization. It should be noted that the SDS-PAGE of the purified CPILE-b showed both pro-sequence and CPILE-b bands. Then, the purified CPILE-b was concentrated and crystalized using standard crystallization kits via hanging drop vapor diffusion method. CPILE-b crystals found after one month screening and SDS-PAGE of selected crystals confirmed that they were CPILE-b crystals. However, poor data set was collected and failed to assign the correct space group even though the good shapes of CPILE-b crystals were used. The pro-sequence probably nicked to CPILE-b and disturbed good quality of crystal formation.
In conclusion, N-terminal GST-tag CPILE-b with regular purification procedure failed to separate pro-sequence region from CPILE-b body. The condition that triggers oligomerization of CPILE-b should be further investigation.

Research Progress Status

29年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

29年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin. (2017)
  • Author(s): Toniti, W., Yoshida, T., Tsurumura, T., Irikura, D., Monma, C., Kamata, Y., and Tsuge, H.
  • Journal Title: PLoS ONE Volume: 12 Issue: 2 Pages: e0171278-e0171278
  • DOI: 10.1371/journal.pone.0171278
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin (2017)
  • Author(s): Toniti Waraphan, Yoshida Toru, Tsurumura Toshiharu, Irikura Daisuke, Monma Chie, Kamata Yoichi, Tsuge Hideaki
  • Organizer: ETOX18 (European Workshop on Bacterial Protein Toxins)
  • Int'l Joint Research
• [Presentation] Characterization of actin ADP-ribosyltransferase from Clostridium perfringens iota-like enterotoxin. (2016)
  • Author(s): Toniti W., Yoshida T., Tsurumura T., Irikura D., Monma C., Kamata Y., Tsuge H.
  • Organizer: ICC05-AEM2016
  • Place of Presentation: 宇奈月（日本）
  • Year and Date: 2016-09-04
  • Int'l Joint Research
• [Remarks] 京都産業大学総合生命科学部ニュース
  • URL: http://www.kyoto-su.ac.jp/news/20170218_400n_news.html