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 ± 0.4mM and 3.4 ± 0.5mM glucose respectively) at a 6.25mM external glucose concentration. In contrast, bloodstream form parasites maintain glycosomal glucose levels that are ~1.8-fold higher than the surrounding cytosol, equating to 1.9 ± 0.6mM in cytosol and 3.5 ± 0.5mM in glycosomes. While the mechanisms of glucose transport operating in the glycosomes of bloodstream form T. brucei remain unresolved, the methods described here will provide a means to begin to dissect the cellular machinery required for subcellular distribution of this critical hexose.
Highlights
Glucose is an important metabolite for members of the class Kinetoplastea that includes Trypanosoma brucei, T. cruzi, and parasites of the genus Leishmania
African sleeping sickness is caused by Trypanosoma brucei
To normalize experiments between PCF and bloodstream form (BSF) cells and to mitigate media effects on cells, studies were carried out in phosphate buffered saline (PBS; 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, and 1.8 mM KH2PO4) adjusted to pH 7.4, and containing glucose at the concentration required for the experiment. pH calibration buffer used for intracellular FLII12Pglu-700μδ6 calibration was carried out in HEPES and MOPS buffered saline (HM-PBS; 137mM NaCl, 2.7mM KCl, 25mM MOPS, 25mM HEPES, 10mM Na2HPO4, and 1.8 mM KH2PO4) adjusted to the desired pH, and titrated with glucose to the desired concentration for calibration
Summary
Glucose is an important metabolite for members of the class Kinetoplastea that includes Trypanosoma brucei, T. cruzi, and parasites of the genus Leishmania. Human African trypanosomiasis (HAT) is caused by a bloodstream infection with T. brucei. The disease is endemic to subSaharan Africa where it is estimated that 60 million people live at risk for contracting the disease [1]. Despite the millions of people at risk, adequate treatment options, especially for late stage disease, are lacking, and can be accompanied by severe drug toxicity [2]. Resistance to existing drugs has been reported [3], [4]. For these reasons, we are expanding the repertoire of analytical methods used to study T. brucei metabolism to advance our understanding of HAT and enable development of new and more effective anti-trypanosomal treatment(s)
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