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Lipid metabolism in Trypanosoma brucei: utilization of myristate and myristoyllysophosphatidylcholine for myristoylation of glycosyl phosphatidylinositols.

ABSTRACT: Myristate is the exclusive fatty acid species in the glycosyl phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG). [3H]Myristate can be incorporated into T. brucei GPIs by two distinct processes known as fatty acid remodelling and myristate exchange. Myristoyllysophosphatidylcholine (M-LPC) can also serve as a myristate donor for VSG in trypanosomes [Bowes, Samad, Jiang, Weaver and Mellors (1993) J. Biol. Chem. 268, 13885-13892]. We have studied in detail the myristoylation of GPIs using a [3H]M-LPC substrate. Labelling of VSG and free GPIs by [3H]M-LPC in cultured trypanosomes occurred at the same rate as with [3H]myristate. Concurrent with GPI labelling, there was rapid hydrolysis of [3H]M-LPC to generate extracellular [3H]myristate. Experiments in a trypanosomal cell-free system indicated that GPI labelling by fatty acid remodelling and myristate exchange was also equally efficient with [3H]M-LPC and [3H]myristate. Furthermore, both ATP and CoA are required for the myristoylation of GPIs by [3H]M-LPC. These experiments suggest that GPI myristoylation from M-LPC involves hydrolysis of M-LPC to free myristate. To address the physiological importance of myristate and M-LPC in VSG myristoylation, we radiolabelled trypanosomes in vivo with both substrates in medium containing serum, and found that [3H]myristate labelled VSG and GPIs more efficiently. Thus, VSG myristoylation by free myristate may be favoured in bloodstream trypanosome infections.

SUBMITTER: Werbovetz KA

PROVIDER: S-EPMC1217659 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Project description:Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

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Lipid metabolism in Trypanosoma brucei: utilization of myristate and myristoyllysophosphatidylcholine for myristoylation of glycosyl phosphatidylinositols.

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