Imaging the ATP metabolism in Trypanosoma cruzi intracellular amastigotes

• Project/Area Number: 19K07523
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 49040:Parasitology-related
• Research Institution: Nagasaki University
• Principal Investigator: Inaoka Ken Daniel 長崎大学, 熱帯医学研究所, 准教授 (10623803)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2021: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 寄生虫 / エネルギー代謝 / バイオセンサー / ATP動態 / ATEAM / ATPバイオセンサー / FRET / 代謝 / トリパノソーマ原虫 / ATeam / MaLion / Ateam / ミトコンドリア / グリコソーム / トリパノソーマ科原虫 / 細胞内寄生 / 創薬 / 細胞小器官

Outline of Research at the Start

Trypanosoma cruziはシャーガス病を引起す寄生原虫で、その生活環において、媒介昆虫内（EPI）、血流中（TRP）、細胞内（AMA）の3つのステージに大きく分けられる。臨床的に重要なのはAMAであるが、大量培養系が確立されておらず、解析が困難である事から、特に研究が進んでおらず、AMAで用いられる炭素源やエネルギー代謝における各細胞小器官の役割など、基礎的な情報すら未だ不明である。本研究の最終目的は、寄生現象を支えるエネルギー代謝の生理的役割を解明することである。

Outline of Final Research Achievements

The ATeam (a fluorescence resonance energy transfer (FRET)-based ATP biosensor) was developed to specifically visualize in real-time the ATP levels in a single living cell. We have attempted to generate transgenic Trypanosoma cruzi expressing ATeam in glycosome, cytosol and mitochondria. Despite of several attempt and effort, no transgenic parasite could survive during the selection process by G418. Moreover, similar experiment was conducted in T. brucei and after several attempt, T. brucei expressing glycosomal ATeam and cytosolic ATeam was obtained, but not the mitochondrial ATeam. Despite the effort, both transgenic parasites showed an aggregation pattern, indicating that ATeam when expressed in trypanosomatid parasites shows sign of toxicity. Moreover, purified ATeam made from trypanosomal ATP synthase epsilon-subunit did not show FRET signal in the presence of ATP.
Currently, we are developing transgenic parasites expressing MaLion, a novel ATP biosensor.

Academic Significance and Societal Importance of the Research Achievements

本研究では、様々な方法でATEAMの発現をT. cruziまたはT. bruceiで試みたが、原虫に対する毒性を示したため解析不可能であった。さらに、原虫由来のε-サブユニットを用いて作成したATcEAMおよびATbEAMではATPが結合せず、FRETを起こさないことが明らかとなった。そして、原虫ミトコンドリア内にはATP合成酵素以外に、ATP生産に寄与する新たな経路（ASCT/SCSサイクル）が明らかとなり、哺乳宿主の寄生環境に適した指摘温度であった。このことから、寄生虫は寄生環境に適応するためには、ウイルス、細菌と真菌といった病原体とは異なる、想定外な手段を用いる事が改めて判った。

Research Products

• [Int'l Joint Research] University of Sussex(英国)
• [Int'l Joint Research] Slovak Academy of Sciences(スロバキア)
• [Int'l Joint Research] BPPT(インドネシア)
• [Int'l Joint Research] University of Kinshasa(コンゴ共和国)
• [Journal Article] Mitochondria as a Potential Target for the Development of Prophylactic and Therapeutic Drugs against Schistosoma mansoni Infection (2021)
  • Author(s): Talaam Keith Kiplangat、Inaoka Daniel Ken、Hatta Takeshi、Tsubokawa Daigo、Tsuji Naotoshi、Wada Minoru、Saimoto Hiroyuki、Kita Kiyoshi、Hamano Shinjiro
  • Journal Title: Antimicrobial Agents and Chemotherapy Volume: 65 Issue: 10
  • DOI: 10.1128/aac.00418-21
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma gondii (2021)
  • Author(s): Acharjee Rajib、Talaam Keith、Hartuti Endah、Matsuo Yuichi、Sakura Takaya、Gloria Bundutidi、Hidano Shinya、Kido Yasutoshi、Mori Mihoko、Shiomi Kazuro、Sekijima Masakazu、Nozaki Tomoyoshi、Umeda Kousuke、Nishikawa Yoshifumi、Hamano Shinjiro、Kita Kiyoshi、Inaoka Daniel
  • Journal Title: International Journal of Molecular Sciences Volume: 22 Issue: 15 Pages: 7830-7830
  • DOI: 10.3390/ijms22157830
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase (2021)
  • Author(s): Hartuti Endah Dwi、Sakura Takaya、Tagod Mohammed S. O.、Yoshida Eri、Wang Xinying、Mochizuki Kota、Acharjee Rajib、Matsuo Yuichi、Tokumasu Fuyuki、Mori Mihoko、Waluyo Danang、Shiomi Kazuro、Nozaki Tomoyoshi、Hamano Shinjiro、Shiba Tomoo、Kita Kiyoshi、Inaoka Daniel Ken
  • Journal Title: International Journal of Molecular Sciences Volume: 22 Issue: 13 Pages: 7236-7236
  • DOI: 10.3390/ijms22137236
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Weak O2 binding and strong H2O2 binding at the non-heme diiron center of Trypanosome Alternative Oxidase (2021)
  • Author(s): S.Yamasaki, M.Shoji, M.Kayanuma, V.Sladek, D.K.Inaoka, Y.Matsuo, T.Shiba, L.Young, A.L.Moore, K.Kita, Y.Shigeta
  • Journal Title: Biochimica et Biophysica Acta - Bioenergetics Volume: 1862 Issue: 4 Pages: 148356-9
  • DOI: 10.1016/j.bbabio.2020.148356
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei. (2020)
  • Author(s): Mochizuki K, Inaoka DK, Mazet M, Shiba T, Fukuda K, Kurasawa H, Millerioux Y, Boshart M, Balogun EO, Harada S, Hirayama K, Bringaud F, Kita K.
  • Journal Title: Biochim Biophys Acta Bioenerg Volume: 1861(11) Issue: 11 Pages: 148283-148283
  • DOI: 10.1016/j.bbabio.2020.148283
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target (2020)
  • Author(s): Sato D, Hartuti ED, Inaoka DK, Sakura T, Amalia E, Nagahama M, Yoshioka Y, Tsuji N, Nozaki T, Kita K, Harada S, Matsubayashi M, Shiba T.
  • Journal Title: Genes (Basel) Volume: 11(12) Issue: 12 Pages: 1468-1468
  • DOI: 10.3390/genes11121468
  • Peer Reviewed / Open Access
• [Remarks] 分子感染ダイナミックス解析分野の研究内容
  • URL: http://www.tm.nagasaki-u.ac.jp/molecdyna/research.html