Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Area of Science:

• Parasitology
• Molecular Biology
• Biochemistry

Background:

• Glucose metabolism is vital for African trypanosome (Trypanosoma brucei) development and survival.
• Glycosomes, housing most glycolysis, are rapidly acidified upon glucose deprivation, impacting enzyme regulation.
• Previous pH measurement methods lacked precise localization, limiting their application.

Purpose of the Study:

• To develop and utilize a glycosomally localized pH biosensor in Trypanosoma brucei.
• To investigate cellular responses to changing environmental glucose levels.
• To establish a tool for real-time monitoring of glycosomal pH dynamics.

Main Methods:

• Development of transgenic Trypanosoma brucei expressing pHluorin2, a ratiometric pH biosensor, targeted to glycosomes.
• Confirmation of glycosomal localization using immunofluorescence and co-localization with aldolase.
• Calibration of pHluorin2 sensor responsiveness across a pH range of 4-8.
• Measurement of glycosomal pH using flow cytometry and validation in response to glucose deprivation.

Main Results:

• Successfully generated and localized the pHluorin2 biosensor within glycosomes of both bloodstream and procyclic form parasites.
• Demonstrated the sensor's ability to accurately report real-time changes in glycosomal pH, particularly acidification during glucose deprivation.
• Validated the biosensor's performance in live parasites, confirming known responses to metabolic stress.

Conclusions:

• The glycosomally localized pHluorin2 biosensor provides a robust tool for studying pH dynamics in Trypanosoma brucei.
• This technology enables deeper understanding of glucose metabolism regulation and parasite responses to environmental cues.
• The adaptable biosensor holds potential for high-throughput drug screening and broader applications in other organelles and trypanosomatids.