flow cytometry procedure to measure relative ph of the glycosome in living trypanosoma brucei

Trypanosoma bruceiにおけるグリコソームpHのためのpHluorin2を用いたセンサの開発

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.

著者スポットライト:pHluorin2バイオセンサーを使用した Trypanosoma brucei のグリコソームpHモニタリングの進歩

生きているTrypanosoma bruceiの動的グリコソームpH変化の測定

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.

生きているトリパノソーマ・ブルーセイのグリコソームの相対pHを測定するためのフローサイトメトリー手順

まず、フローサイトメーターソフトウェアを起動します。新しいテストを開きます。必要な数のサンプルを追加し、名前を付けます。次に、YL2-HまたはRL1-Hチャンネルヒストグラム、前方散乱面積対側方散乱面積のドットプロット、前方散乱面積対前方散乱高さのドットプロット、BL1-HとVL2-Hのチャンネルドットプロットを設定します。未染色の野生型Trypanosoma bruceiコントロール細胞をサンプル注入ポートにセットし、ステージ位置を調整します。ファイルサイズを小さくするには、録音を意図していないチャンネルのチェックを外します。調整を可能にするために、低流量から開始します。次に、[実行]をクリックしてデータ取得を開始します。YL2またはRL1の電圧を微調整して、10〜3〜10〜4番目の相対蛍光強度単位内のメインピークを揃えます。前方散乱光と側方散乱の電圧を調整して、イベントの90%以上がドットプロット範囲内にあるようにします。前方散乱閾値を設定して、生細胞を省略せずにデブリを除外します。VL 2 および BL 1 チャンネル設定を変更して、未染色野生型コントロールの一次ピークを 10 から 2 から 10 から 4 番目の相対蛍光強度単位以内に配置します。[記録] をクリックし、少なくとも 10, 000 のイベントを測定します。次に、ヨウ化プロピジウムまたはチアゾールレッドで染色した最初の誘導pHluorin発現トリパノソーマサンプルをサンプル注入ポートに配置します。ここでも、すすぎを行った後、低流量でデータ取得を開始してください。各プロットを監視して、90%を超えるイベントが正しくキャプチャされ、VL2-HチャネルとBL1-Hチャネルが飽和していないことを確認します。各サンプルのデータをFCSファイル形式で保存し、分析の準備をします。

Research

JoVE Journal

Immunology and Infection

Flow Cytometry Procedure to Measure Relative pH of the Glycosome 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. ...

免疫・感染学

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

生きているトリパノソーマ・ブルーセイのpHluorin2蛍光を測定するためのフローサイトメトリー用サンプル調製

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

生きたトリパノソーマ・ブルーセイのグリコソームの相対pHを測定するためのフローサイトメトリーデータの解析