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

Sviluppo di un sensore basato su pHluorin2 per il pH glicosomiale 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.

Autore in primo piano: Progressi nel monitoraggio del pH glicosomiale in Trypanosoma brucei utilizzando il biosensore pHluorin2

Misurazione delle variazioni dinamiche del pH glisomiale nel Trypanosoma brucei vivente

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.

Preparazione del campione per la citometria a flusso per misurare la fluorescenza di pHluorin2 in Trypanosoma brucei vivente

Per iniziare, prendi le cellule di Trypanosoma brucei che esprimono pHluorin. Aggiungere tetraciclina o doxiciclina e incubare durante la notte per indurre l'espressione di pHluorina. Quindi trasferire tre millilitri di ciascuna coltura in provette separate da 15 millilitri.Quindi centrifugare le celle a temperatura ambiente per 10 minuti a 1000 xg. Rimuovere il surnatante e risospendere le cellule in un millilitro di PBS. Dopo aver centrifugato le cellule per la quarta volta, rimuovere il surnatante e risospendere il pellet cellulare in PBS con concentrazioni variabili di glucosio.Aggiungere ioduro di propidio o rosso tiazolo per distinguere le cellule morte da quelle vive. Quindi trasferire ogni campione in provette da cinque millilitri compatibili con il citometro a flusso.

sample-preparation-for-flow-cytometry-to-measure-phluorin2

Research

JoVE Journal

Immunology and Infection

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

46 Views.  Brigham Young University. Per iniziare, prendi le cellule di Trypanosoma brucei che esprimono pHluorin. Aggiungere tetraciclina o doxiciclina e incubare durante la notte per indurre l'espressione di pHluorina. Quindi trasferire tre millilitri di ciascuna coltura in provette separate da 15 millilitri.Quindi centrifugare le celle a temperatura ambiente per 10 minuti a 1000 xg. Rimuovere il surnatante e risospendere le cellule in un millilitro di PBS. Dopo aver centrifugato le cellule per la quarta volta, rimuover...

Video: Preparazione del campione per la citometria a flusso per misurare la fluorescenza di pHluorin2 in Trypanosoma brucei vivente

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. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics&#160;in the live cell setting.

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

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