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

Ontwikkeling van een op pHluorin2 gebaseerde sensor voor glycosomale pH in Trypanosoma brucei

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.

Auteur in de schijnwerpers: Vooruitgang in glycosomale pH-monitoring in Trypanosoma brucei met behulp van pHluorin2 Biosensor

Meten van dynamische glycosomale pH-veranderingen bij levende Trypanosoma brucei

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.

Monstervoorbereiding voor flowcytometrie om pHluorin2-fluorescentie te meten bij levende trypanosoma brucei

Neem om te beginnen Trypanosoma brucei-cellen die pHluorine tot expressie brengen. Voeg tetracycline of doxycycline toe en incubeer een nacht om pHluorin-expressie te induceren. Breng vervolgens drie milliliter van elke cultuur over in afzonderlijke buizen van 15 milliliter.Centrifugeer de cellen vervolgens 10 minuten bij kamertemperatuur bij 1000 xg. Verwijder het supernatant en resuspendeer de cellen in één milliliter PBS. Nadat u de cellen voor de vierde keer hebt gecentrifugeerd, verwijdert u het supernatans en resuspendeert u de celpellet in PBS met verschillende glucoseconcentraties.Voeg propidiumjodide of thiazolrood toe om dode van levende cellen te onderscheiden. Breng vervolgens elk monster over in buisjes van vijf milliliter die compatibel zijn met de flowcytometer.

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. ...

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

Flowcytometrieprocedure om de relatieve pH van het glycosoom te meten bij levende trypanosoma brucei

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

Analyse van flowcytometriegegevens om de relatieve pH van het glycosoom in levende trypanosoma brucei te meten