A FRET flow cytometry method for monitoring cytosolic and glycosomal glucose in living kinetoplastid parasites

PLoS Neglected Tropical Diseases
Charles VoytonKenneth A Christensen

Abstract

The bloodstream lifecycle stage of the kinetoplastid parasite Trypanosoma brucei relies solely on glucose metabolism for ATP production, which occurs in peroxisome-like organelles (glycosomes). Many studies have been conducted on glucose uptake and metabolism, but none thus far have been able to monitor changes in cellular and organellar glucose concentration in live parasites. We have developed a non-destructive technique for monitoring changes in cytosolic and glycosomal glucose levels in T. brucei using a fluorescent protein biosensor (FLII12Pglu-700μδ6) in combination with flow cytometry. T. brucei parasites harboring the biosensor allowed for observation of cytosolic glucose levels. Appending a type 1 peroxisomal targeting sequence caused biosensors to localize to glycosomes, which enabled observation of glycosomal glucose levels. Using this approach, we investigated cytosolic and glycosomal glucose levels in response to changes in external glucose or 2-deoxyglucose concentration. These data show that procyclic form and bloodstream form parasites maintain different glucose concentrations in their cytosol and glycosomes. In procyclic form parasites, the cytosol and glycosomes maintain indistinguishable glucose levels (3.4 ±...Continue Reading

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Methods Mentioned

BETA
biosensors
FRET
transfection
biosensor
fluorescence microscopy
flow cytometry

Software Mentioned

Sigmaplot
FlowJo
BD attune cytometry

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African Trypanosomiasis

African trypanosomiasis, also known as sleeping sickness, is an insect-borne parasitic disease of humans and other animals. It is caused by protozoa of the species Trypanosoma brucei and almost invariably progresses to death unless treated. Discover the latest research on African trypanosomiasis here.