Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Abstract

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

Fig. 1. : subcellular distribution of percent activities of seven enzymes in Trypanosoma evansi determined by selective membrane permeabilisation with digitonin. Top (activity graph): release of enzymes after treatment with several concentrations of digitonin; bottom (scan of polyvinylidene difluoride membrane): western blot analysis (40 µg of protein in each well) of pellet (P) and supernatant (S), using antisera against trypanosome hexokinase (HK), for several digitonin concentrations. The last well (TX-100) was loaded with Triton X-100 (0.1%) solubilised parasite cells. ENO: enolase; G3PDH: glycerol-3-phosphate dehydrogenase; GPD: glucose-6-phosphate dehydrogenase; MD: malate dehydrogenase; PGI: glucose-6-phosphate isomerase; PGK: phosphoglycerate kinase.

Fig. 2. : distribution of specific activities of 14 enzymes and percent protein (total = 298.5 mg) in five Trypanosoma evansi subcellular fractions resulting from differential centrifugation. The polyvinylidene difluoride membrane scan (top left) corresponds to the western blot (40 µg of protein in each well) of fractions and purified glycosomes (PG) using antisera against trypanosome hexokinase (HK). ALD fructose-bisphosphate aldolase; CF: cytosolic fraction; ENO: enolase; G3PDH: glycerol-3-phosphate dehydrogenase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOT: glutamate-oxaloacetate transaminase; GPD: glucose-6-phosphate dehydrogenase; LGF: large granular fraction; MD: malate dehydrogenase; MF: microsomal fraction; NF: nuclear fraction; PFK: phosphofructokinase; PGI: glucose-6-phosphate isomerase; PGK: phosphoglycerate kinase; PGM: phosphoglycerate mutase; PK: pyruvate kinase; sa: specific activity; SGF: small granular fraction; TIM: triosephosphate isomerase.

Fig. 3. : detrended correspondence analysis of differential centrifugation data (Fig. 2): relative position of 14 enzymes (open circles) and five subcellular fractions (solid triangles) in the plane defined by axes 1 and 2. ALD fructose-bisphosphate aldolase; CF: cytosolic fraction; ENO: enolase; G3PDH: glycerol-3-phosphate dehydrogenase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOT: glutamate-oxaloacetate transaminase; GPD: glucose-6-phosphate dehydrogenase; HK hexokinase; LGF: large granular fraction; MD: malate dehydrogenase; MF microsomal fraction; NF nuclear fraction; PFK: phosphofructokinase; PGI: glucose-6-phosphate isomerase; PGK: phosphoglycerate kinase; PGM: phosphoglycerate mutase; PK: pyruvate kinase; SGF: small granular fraction; TIM: triosephosphate isomerase.