Development of a new therapeutic agent that inhibits the production of exosome-associated Shiga toxin 2

• Project/Area Number: 18K07128
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 49050:Bacteriology-related
• Research Institution: Doshisha University
• Principal Investigator: Takahashi Miho 同志社大学, 生命医科学部, 助教 (00446569)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: 志賀毒素 / エキソソーム（エクソソーム） / 小胞輸送 / プロテインキナーゼ / エキソソーム / ペプチドアレイ / エクソソーム / キナーゼ / 基質ペプチドアレイ / Shia toxin (Stx)

Outline of Final Research Achievements

After being incorporated into target cells, Shiga toxin 2 (Stx2) was actively released from the cells, and part of released Stx2a was associated with exosomes (refer to as exo-Stx2). To determine the intracellular molecules that regulate the production of exo-Stx2, we examined the effect of knockdown of Rabs (Rab11a, Rab11b, Rab27a, Rab27b, and Rab35) on the release of Stx2 in the culture medium. The results showed that the release of Stx2 was inhibited by Rab11b knockdown but not by the other Rabs knockdown, indicating that exo-Stx2 is released by Rab11b dependent machinery. By using a kinase substrate peptide array, we analyzed the hundreds of protein kinases activations in Stx treated cells, and found that the types of activated protein kinases were different between the case with Stx1 and Stx2 in the intracellular transport of Stx.

Academic Significance and Societal Importance of the Research Achievements

Stx2が細胞内に取り込まれた後、その一部がエキソソームに結合した状態（exo-Stx2）で細胞外へ放出されるが、このexo-Stx2をマウスに静脈投与した場合、遊離型Stx2に比べマウスに対する個体毒性が増強することがわかっている。従って、exo-Stx2がどのような分子機構で産生されているのかを明らかにし、さらにその産生を制御する分子を同定できれば、Stx2の強毒性に関わる分子を標的とした新しい腸管出血性大腸菌感染症治療薬が開発可能となることが期待される。

Research Products

• [Journal Article] A nontoxigenic form of Shiga toxin 2 suppresses the production of amyloid b by altering the intracellular transport of amyloid precursor protein through its receptor-binding B-subunit (2021)
  • Author(s): Waka Sato, Miho Watanabe-Takahashi, Takashi Hamabata, Koichi Furukawa, Satoru Funamoto, Kiyotaka Nishikawa
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 557 Pages: 247-253
  • DOI: 10.1016/j.bbrc.2021.04.015
  • Peer Reviewed
• [Journal Article] Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin (2021)
  • Author(s): Miho Watanabe-Takahashi, Masakazu Tamada, Miki Senda, Masahiro Hibino, Eiko Shimizu, Akiko Okuta, Atsuo Miyazawa, Toshiya Senda, Kiyotaka Nishikawa
  • Journal Title: Communications biology Volume: 538 Pages: 1-7
  • DOI: 10.1038/s42003-021-02068-3
  • Peer Reviewed / Open Access
• [Journal Article] Exosome-associated Shiga toxin 2 is released from cells and causes severe toxicity in mice (2018)
  • Author(s): Watanabe-Takahashi, M., Yamasaki, S., Murata, M., Kano, F., Motoyama, J., Yamate, J., Omi, J., Sato, W., Ukai, H., Shimasaki, K., Ikegawa, M., Tamura-Nakano, M., Yanoshita, R., Nishino, Y., Miyazawa, A., Natori, Y., Toyama-Sorimachi, N., Nishikawa, N.
  • Journal Title: Sci Rep. Volume: 8 Pages: 10776-10776
  • DOI: 10.1038/s41598-018-29128-9
  • Peer Reviewed / Open Access
• [Presentation] Exosome-associated Shiga toxin 2 is released from cells and causes severe toxicity in mice. (2018)
  • Author(s): Miho Watanabe-Takahashi, Jun Motoyama, Jyoji Yamate, Shinji Yamasaki and Kiyotaka Nishikawa
  • Organizer: VTEC2018
  • Int'l Joint Research