Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

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. 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 in the live cell setting.

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 parasites are likewise d....

Using T. brucei brucei 90-13 BSF trypanosomes, a monomorphic parasite line, requires consideration of safety as they are considered Risk Group 2 organisms that should be handled in biosafety level 2 facilities.

1. Trypanosome culture and transfection

1. Culture T. brucei brucei 90-13 BSF trypanosomes in HMI-9 medium supplemented with 10% heat-inactivated FBS and 10% Nu-Serum at 37 °C in 5% CO₂¹⁰.
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pHLuorin2-PTS1 localization to glycosomes in BSF T. brucei
To assess the subcellular localization of the pHluorin2-PTS1, parasites were subjected to immunofluorescence assays. Signal from the transgene colocalized with anti-sera raised against a glycosome-resident protein, aldolase (TbAldolase) (Figure 2A). The average Pearson's correlation coefficient of colocalization between anti-TbAldolase and pHluorin2-PTS1 was 0.895, indicating that pHluorin2-PTS1 wa.......

Environmental perception and response mechanisms in the African trypanosome are poorly understood. Changes in nutrient availability are known to trigger diverse responses in the parasite, including acidification of glycosomes. Here, we have described a method to study glycosomal pH response to environmental perturbations in living cells using a heritable protein sensor, pHluorin2, and flow cytometry.

There are several critical steps in the use of the sensor. First, the characterization of tran.......

pHluorin2-PTS1 was cloned into pLEW100v5 by Twist Bioscience who provided the construct in a high-copy cloning vector; pLEW100v5 was a gift from Dr. George Cross. Antiserum raised against T. brucei aldolase is available from Dr. Meredith T. Morris, Clemson University, upon request. Work from the JCM and KAC laboratories was partially supported by an award from the National Institutes of Health (R01AI156382). SSP was supported by NIH 3R01AI156382.