De novo biosynthesis of sterols and fatty acids in the Trypanosoma brucei procyclic form: Carbon source preferences and metabolic flux redistributions

Abstract

De novo biosynthesis of lipids is essential for Trypanosoma brucei, a protist responsible for the sleeping sickness. Here, we demonstrate that the ketogenic carbon sources, threonine, acetate and glucose, are precursors for both fatty acid and sterol synthesis, while leucine only contributes to sterol production in the tsetse fly midgut stage of the parasite. Degradation of these carbon sources into lipids was investigated using a combination of reverse genetics and analysis of radio-labelled precursors incorporation into lipids. For instance, (i) deletion of the gene encoding isovaleryl-CoA dehydrogenase, involved in the leucine degradation pathway, abolished leucine incorporation into sterols, and (ii) RNAi-mediated down-regulation of the SCP2-thiolase gene expression abolished incorporation of the three ketogenic carbon sources into sterols. The SCP2-thiolase is part of a unidirectional two-step bridge between the fatty acid precursor, acetyl-CoA, and the precursor of the mevalonate pathway leading to sterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA. Metabolic flux through this bridge is increased either in the isovaleryl-CoA dehydrogenase null mutant or when the degradation of the ketogenic carbon sources is affected. We also observed a preference for fatty acids synthesis from ketogenic carbon sources, since blocking acetyl-CoA production from both glucose and threonine abolished acetate incorporation into sterols, while incorporation of acetate into fatty acids was increased. Interestingly, the growth of the isovaleryl-CoA dehydrogenase null mutant, but not that of the parental cells, is interrupted in the absence of ketogenic carbon sources, including lipids, which demonstrates the essential role of the mevalonate pathway. We concluded that procyclic trypanosomes have a strong preference for fatty acid versus sterol biosynthesis from ketogenic carbon sources, and as a consequence, that leucine is likely to be the main source, if not the only one, used by trypanosomes in the infected insect vector digestive tract to feed the mevalonate pathway.

Microbial Sterolomics as a Chemical Biology Tool

“…Dietary cholesterol 1 accounted for 20.0 %, and other components were 16 (0.1%), 30 (1.0%), 48 (1.0%), 57 (8.0%), and others (0.2%). 24,24-Dimethylcholesta-5,7,25(27)-trienol and 86 and protothecasterol 87 were not detected in this composition, but have been reported in subsequent studies [ 42 , 66 ], respectively.…”

“…The variability of labeling was potentially attributed to the equilibrium of acetyl-CoA pools in the mitochondria and cytosol [ 42 ]. Trypanosomal sterols protothecasterol 87 (ergosta-5,7,22 E ,25(27)-tetraenol), cholesta-5,7,24-trienol 82 , and ergosta-5,7,25(27)-trienol 83 have also been noted to incorporate isotope labeled from threonine [ 66 ].…”

Lipid hijacking: A unifying theme in vector-borne diseases

“…They may also acquire free lipids in the serum, including free fatty acids and lysophospholipids for their membranes ( Voorheis, 1980 ; Berman et al, 1987 ; Bowes et al, 1993 ). However, if there is an exogenous limitation on fatty acids, T. brucei may synthesize these lipids de novo from ketogenic carbon sources ( Millerioux et al, 2018 ; Figure 3B ). T. brucei may endogenously produce fatty acids from the type II fatty acid synthase pathway (FASII) or from the microsomal elongase pathway, which uses the enzyme acetyl coenzyme A carboxylase (ACC) ( Millerioux et al, 2018 ; Ray et al, 2018 ).…”

“…However, if there is an exogenous limitation on fatty acids, T. brucei may synthesize these lipids de novo from ketogenic carbon sources ( Millerioux et al, 2018 ; Figure 3B ). T. brucei may endogenously produce fatty acids from the type II fatty acid synthase pathway (FASII) or from the microsomal elongase pathway, which uses the enzyme acetyl coenzyme A carboxylase (ACC) ( Millerioux et al, 2018 ; Ray et al, 2018 ). The activity of this enzyme is regulated by lipid abundance in the tsetse fly, therefore maintaining a balance between lipid scavenging and synthesis ( Ray et al, 2018 ).…”

“Metabolic contest”, a new way to control carbon source preference

“…3 × 10 7 PCF of T. brucei were centrifuged at 1,400 g for 10 min, then the pellet was washed twice with PBS and the cells were incubated for 6 h at 27°C in 1.5 ml of incubation buffer (PBS supplemented with 5 g/l NaHCO 3 , pH 7.4) with 4 mM [U- 13 C]-glycerol and/or 4 mM glucose. This quantitative 1 H-NMR approach was previously developed to distinguish between [ 13 C]-enriched and non-enriched excreted molecules produced from [ 13 C]-enriched and non-enriched carbon sources, respectively (Bringaud et al, 2015; Mazet et al, 2013; Millerioux et al, 2018). The viability of the cells during the incubation was checked by microscopic observation.…”