Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is metabolized to pyruvate. Aerobic metabolism depends on the activities of glycosomal triosephosphate isomerase and a mitochondrial glycerophosphate oxidase, and on glycerophosphate ↔ dihydroxyacetone phosphate exchange across the glycosomal membrane. Using a combination of genetics and computer modelling, we show that triosephosphate isomerase is probably essential for bloodstream trypanosome survival, but not for the insect-dwelling procyclics, which preferentially use amino acids as an energy source. When the enzyme level decreased to about 15% of that of the wild-type, the growth rate was halved. Below this level, a lethal rise in dihydroxyacetone phosphate was predicted. Expression of cytosolic triosephosphate isomerase inhibited cell growth. Attempts to knockout the trypanosome alternative oxidase genes (which are needed for glycerophosphate oxidase activity) were unsuccessful, but when we lowered the level of the corresponding mRNA by expressing a homologous double-stranded RNA, oxygen consumption was reduced fourfold and the rate of trypanosome growth was halved.
- metabolic control
- RNA interference
- trypanosome alternative oxidase
Abbreviations used: Abbreviations used in the text, and/or in equations and Scheme 1: general; SHAM, salicylhydroxamic acid; ds, double-stranded; gene names: NEO, neomycin phosphotransferase marker (fused to a short leader from trypanosome tubulin); HYG, hygromycin-resistance marker; BLE, bleomycin-resistance marker; BST, blasticidin-resistance marker; TPI, triosephosphate isomerase gene; TPITi, tetracycline-inducible triosephosphate isomerase gene; TAO, trypanosome alternative oxidase gene; TETR, tetracycline repressor gene; metabolic intermediates: Glc-6-P, glucose 6-phosphate; Fru-6-P, fructose 6-phosphate; Fru-1,6-P2, fructose 1,6-bisphosphate; GA-3-P, glyceraldehyde 3-phosphate; PYR, pyruvate; DHAP, dihydroxyacetone phosphate; BPGA, bisphosphoglycerate; 3-PGA, 3-phosphoglycerate; 2-PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Gly-3-P, glycerol 3-phosphate; enzymes: ALD, aldolase; HXK, hexokinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase [GAPDH1 is glycosomal and GAPDH2 is cytosolic]; GDH, glycerol-3-phosphate dehydrogenase; GLYK, glycerol kinase; GPO, glycerol-3-phosphate oxidase; PGI (‘phosphoglucose isomerase’), glucose-6-phosphate isomerase; PGK, phosphoglycerate kinase; PYK, pyruvate kinase; PFK, phosphofructokinase; TAO, trypanosome alternative oxidase; TPI, triosephosphate isomerase; ZMBH, Zentrum für Molekularbiologie der Universität Heidelberg.
- The Biochemical Society, London ©2001