Imaging the ATP metabolism in Trypanosoma cruzi intracellular amastigotes

2021 Fiscal Year Final Research Report

• 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
• Keywords: 寄生虫 / エネルギー代謝 / バイオセンサー / ATP動態 / ATEAM

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.

Free Research Field

生化学

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。