sample preparation for flow cytometry to measure phluorin2 fluorescence in living trypanosoma brucei

트리파노소마 브루세이(Trypanosoma brucei)의 글리코솜 pH를 위한 pHluorin2 기반 센서 개발

Parasitic kinetoplastids, like most living organisms, rely on glucose as a fundamental component of central carbon metabolism. This group includes medically important organisms, such as the African trypanosome, Trypanosoma brucei; the American trypanosome, T. cruzi; and parasites of the genus Leishmania. Glucose metabolism is critical to parasite growth in the pathogenic lifecycle stages. For example, when deprived of glucose, the bloodstream form (BSF) of the African trypanosome dies rapidly. Notably, glycolysis serves as the sole source of ATP during this stage of infection1. Leishmania&#160;parasites are likewise dependent on glucose in the human host, with the amastigote lifecycle stage that resides in host macrophages reliant on this carbon source for growth2.
While these parasites have distinct lifestyles involving different insect vectors, they share many commonalities in how they respond to and consume glucose. For example, these parasites localize most glycolytic enzymes into modified peroxisomes called glycosomes. This kinetoplastid-specific organelle is related to mammalian peroxisomes based on conserved biosynthetic mechanisms and morphology3,4,5,6.
The compartmentalization of most of the glycolytic pathway enzymes into the glycosome offers parasite-specific means of regulation of the pathway. Using a chemical pH probe, we established that nutrient deprivation triggers a rapid acidification of procyclic form (PF) parasite glycosomes that results in altered glycolytic enzyme activity through exposure of an allosteric regulator binding site on the key glycolytic enzyme hexokinase7,8. In our previous work, the chemical probe required constant delivery for use, limiting its utility in other applications. Additionally, challenges maintaining the probe distribution in the glycosome in the BSF limited the utility of the approach for investigating glycosomal pH in that life stage.
In this study, we have used the fluorescent protein biosensor pHluorin2 to monitor glycosomal pH change in BSF T. brucei in response to environmental cues including glucose starvation9 (Figure 1). Results from this work suggest that BSF T. brucei acidifies glycosomes rapidly in response to starvation in a reversible fashion, similar to responses we have observed in PF parasites. We expect this biosensor will improve our understanding of glycolytic regulation in T. brucei and related parasites.

저자 스포트라이트: pHluorin2 Biosensor를 사용한 Trypanosoma brucei 의 당체 pH 모니터링의 발전

살아있는 트리파노소마 브루세이(Trypanosoma brucei)의 동적 당염색체 pH 변화 측정

Glucose metabolism is critical for Trypanosoma brucei, yet little is known about how the cells sense and respond to changing glucose levels in the environment. We have developed parasites expressing a pH biosensor that allows us to monitor glycosomal pH in live parasites, which will aid in the understanding of parasites'dynamic responses to nutrient fluctuation. A challenge for this experiment would be the availability of a flow cytometer equipped with a microtiter plate reader, as well as the appropriate filter sets to measure biosensor fluorescence.Additionally, T.brucei is a risk group two organism, so the cytometer must be housed within a biosafety level two facility. It has been discovered that the pH level of glycosomes can affect glycolysis as hexokinase and phosphofructokinase are pH sensitive. However, the pH probes that were previously used did not accurately target to the glycosome in the bloodstream form.A better tool was needed to study the relationship between glycosomal pH and glycolysis in this parasite life stage. The pHluorin2-PTS1 sensor is a tool that is better at localizing to the glycosome than chemical pH probes in this life stage. It's user friendly, as you only need to induce expression of the pH sensor overnight, and the cells will make and localize pHluorin2 to the glycosome.Using flow cytometry to measure pHluorin2 increases throughput and improves statistical measurements compared to microscopy.

살아있는 트리파노소마 브루세이(Trypanosoma brucei)에서 pHluorin2 형광을 측정하기 위한 유세포 분석을 위한 시료 전처리

먼저 pHluorin을 발현하는 Trypanosoma brucei 세포를 채취합니다. 테트라사이클린(tetracycline) 또는 독시사이클린(doxycycline)을 첨가하고 하룻밤 동안 배양하여 pHluorin 발현을 유도합니다. 그런 다음 각 배양액 3밀리리터를 별도의 15밀리리터 튜브에 옮깁니다.그런 다음 세포를 실온에서 1000xg로 10분 동안 원심분리합니다. 상층액을 제거하고 PBS 1밀리리터에 세포를 재현탁시킵니다. 세포를 네 번째로 원심분리한 후 상층액을 제거하고 다양한 농도의 포도당으로 PBS에 세포 펠릿을 재현탁시킵니다.프로피듐 요오드화물(propidium iodide) 또는 티아졸 레드(Thiazole Red)를 첨가하여 죽은 세포와 살아있는 세포를 구별합니다. 그런 다음 각 샘플을 유세포 분석기와 호환되는 5mL 튜브로 옮깁니다.

Research

JoVE Journal

Immunology and Infection

Sample Preparation for Flow Cytometry to Measure pHluorin2 Fluorescence in Living Trypanosoma brucei

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...

연구

면역학 및 감염병학

Flow Cytometry Procedure to Measure Relative pH of the Glycosome in Living Trypanosoma brucei

살아있는 Trypanosoma brucei에서 당체의 상대 pH를 측정하기 위한 유세포 분석 절차

Analysis of Flow Cytometry Data to Measure the Relative pH of the Glycosome in Living Trypanosoma brucei

살아있는 트리파노소마 브루세이(Trypanosoma brucei)에서 당체의 상대적 pH를 측정하기 위한 유세포 분석 데이터 분석