digitonin and Trypanosomiasis--African

The isoprenoid biosynthetic pathway is a very complex route that entails multiple steps and generates a high number of end-products that are essential for cell viability such as sterols, dolichols, coenzyme Q, heme and prenylated proteins. In parasites from the Trypanosomatidae family this pathway provides new potential drug targets for exploitation in the search for improved therapies, and indeed compounds such as ketoconazole, aminobisphosphonates or terbinafine have been shown to have antiprotozoal activity both in vitro and in vivo. However, despite the high therapeutic importance of the pathway, the subcellular compartmentalization of the different steps of isoprenoid biosynthesis is not known in detail. Here we have analysed the intracellular location of the enzymes 3-hydroxy-3-methyl-glutaryl Coenzyme A (HMG-CoA) synthase (HMGS) and mevalonate kinase (MVAK) in Leishmania major promastigotes as well as in Trypanosoma brucei procyclic and bloodstream forms. For this purpose we generated specific polyclonal antibodies against both highly purified recombinant proteins and used those in indirect immunofluorescence and digitonin titration experiments. Results show that sterol biosynthesis is distributed in multiple intracellular compartments and provide evidence indicating that in trypanosomatids the production of HMG-CoA from acetyl Coenzyme A and generation of mevalonate occur mainly in the mitochondrion while further mevalonate phosphorylation is almost exclusively located in glycosomes. Furthermore, we have determined that peroxin 2 (PEX2) is involved in efficient targeting of MVAK and that the enzyme is relocated to the cytosol upon depletion of this peroxin involved in glycosomal matrix protein import.

Topics: Animals; Antibodies, Protozoan; Biosynthetic Pathways; Digitonin; Fluorescent Antibody Technique; Humans; Hydroxymethylglutaryl-CoA Synthase; Leishmania major; Leishmaniasis; Microbodies; Mitochondria; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Protozoan Proteins; Rabbits; Terpenes; Trypanosoma brucei brucei; Trypanosomiasis, African

1. The production of pyruvate, glycerol and glycerol-3-phosphate by intact and digitonin-permeabilized Trypanosoma brucei brucei has been studied with glucose or the glycolytic intermediates as substrates. 2. Under aerobic conditions hexosephosphates gave maximal glycolysis in the presence of 40-60 micrograms digitonin/10(8) trypanosomes while the triosephosphates gave it at 20-30 micrograms digitonin/10(8) trypanosomes. 3. In the presence of salicylhydroxamic acid, and the glycolytic intermediates, permeabilized trypanosomes produced equimolar amounts of pyruvate and glycerol-3-phosphate and no glycerol. Under the same conditions, glucose catabolism produced glycerol in addition to pyruvated and glycerol-3-phosphate. 4. In the presence of salicylhydroxamic acid and ATP or ADP intact trypanosomes produced equimolar amounts of pyruvate and (glycerol plus glycerol-3-phosphate) with glucose as substrate. 5. A carrier for ATP and ADP at the glycosomal membrane is implicated. 6. It is apparent that glycerol formation is regulated by the ATP/ADP ratio and that it needs intact glycosomal membrane and the presence of glucose.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Anaerobiosis; Animals; Cell Membrane Permeability; Digitonin; Glucose; Glycerol; Glycerophosphates; Glycolysis; Pyruvates; Rats; Salicylamides; Trypanosoma brucei brucei; Trypanosomiasis, African