Requirement for acetyl‐CoA carboxylase in Trypanosoma brucei is dependent upon the growth environment

Abstract

Summary Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses two fatty acid synthesis pathways: a major de novo synthesis pathway in the ER and a mitochondrial pathway. The 2-carbon donor for both pathways is malonyl-CoA, which is synthesized from acetyl-CoA by Acetyl-CoA Carboxylase (ACC). Here, we show that T. brucei ACC shares the same enzyme architecture and moderate ~30% identity with yeast and human ACCs. ACC is cytoplasmic and appears to be distributed throughout the cell in numerous puncta distinct from glycosomes and other organelles. ACC is active in both bloodstream and procyclic forms. Reduction of ACC activity by RNA interference (RNAi) resulted in a stage-specific phenotype. In procyclic forms, ACC RNAi resulted in 50-75% reduction in fatty acid elongation and a 64% reduction in growth in low lipid media. In bloodstream forms, ACC RNAi resulted in a minor 15% decrease in fatty acid elongation and no growth defect in culture, even in low lipid media. However, ACC RNAi did attenuate virulence in a mouse model of infection. Thus the requirement for ACC in T. brucei is dependent upon the growth environment in two different life cycle stages.

Trypanosoma brucei: Inhibition of acetyl-CoA carboxylase by haloxyfop

“…We assayed ACC activity as described (Vigueira and Paul, 2011). Inhibitors were prepared in filter-sterilized dimethyl sulfoxide (DMSO) and used as 100X stocks.…”

“…Metabolic labeling was performed essentially as described (Paul et al , 2004, Vigueira and Paul, 2011). Briefly, after 4 days of haloxyfop treatment, ~1 × 10 8 PF cells were labeled with 25 μCi of [11,12- 3 H]laurate (C12:0; 60 mCi/mmol) for 2 h in a 28°C CO 2 incubator.…”

“…Streptavidin blotting can detect the biotin prosthetic group on ACC and was performed essentially as described (Vigueira and Paul, 2011). Briefly, PF parasites were treated for 4 days with haloxyfop.…”

“…Previously, we reported that T. brucei acetyl-CoA carboxylase (TbACC 1 ) is required to efficiently establish and maintain an infection in a mouse model (Vigueira and Paul, 2011). Knockdown of TbACC by RNA interference (RNAi) nearly doubled the mean time until death, suggesting TbACC is a suitable candidate for investigation as a drug target.…”

Mitochondrial calcium transport in trypanosomes

“…This pathway is known to result in the generation of lipoic acid and myristic acid, and is essential in these trypanosomes [106]. Alternatively, acetyl-CoA could be used to generate acetate [107] that could be transferred to the cytosol where is converted back to acetyl-CoA by acetyl-CoA synthetase [100] and can be used for fatty acid synthesis, which appears to the essential for the bloodstream forms when they are in their mammalian hosts [108]. Fig.…”