Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei

Abstract

Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4–5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4–5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.

TbLpn, a key enzyme in lipid droplet formation and phospholipid metabolism, is essential for mitochondrial integrity and growth of Trypanosoma brucei

“…A, top row of panels). To stimulate de novo formation of lipid droplets, resulting in larger and higher numbers of lipid droplets per cell, T. brucei procyclic forms can be incubated with oleate (Allmann et al ., ). When we applied this treatment to TbLpn RNAi cells, more lipid droplets were detected in control untreated and induced cells compared to non‐oleate‐treated parasites (Fig.…”

“…It has been shown before that the addition of oleate to T. brucei procyclic forms in culture results in increased levels of TAG and higher numbers of lipid droplets (Allmann et al ., ), demonstrating that fatty acid carbon can be used for TAG and lipid droplet formation in T. brucei . Oleate‐induced formation of lipid droplets has also been reported in mammalian cells and yeast (Singh et al ., ; Radulovic et al ., ).…”

“…Total parasite lipids were extracted (Bligh and Dyer, ) and separated by one‐dimensional thin layer chromatography (TLC) on Silica Gel 60 plates (Merck, Darmstadt, Germany) in solvent system 1 composed of chloroform:methanol:acetic acid:water (25:15:4:2; v/v/v/v) for phospholipid (Signorell et al ., ) or solvent system 2 composed of hexane:ethyl ether:acetic acid (90:15:2; v/v/v) for TAG (Allmann et al ., ) analysis. After separation, plates were dried and lipids were visualized by exposure to iodine vapor.…”

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha‐L‐iduronidase

“…For nanoLC/MS/MS analysis, samples were prepared as described in (Allmann et al ., ). The peptides were analysed on an Ultimate 3000 RSLC Nano‐UPHLC system (Thermo Scientific, Waltham, Massachusetts) coupled to a nanospray Q‐Exactive hybrid quadrupole‐Orbitrap mass spectrometer (Thermo Scientific).…”

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

“…The PCF of T. brucei EATRO1125.T7T (TetR-HYG T7RNAPOL-NEO) were cultivated in glucose conditions at 27°C in the presence of 5% CO 2 in SDM79 medium containing 10% (v/v) heat-inactivated fetal calf serum and 3.5 mg/ml hemin (Brun and Schonenberger, 1979). The glycerol-rich/glucose-free conditions were obtained by replacing glucose by glycerol in SDM79 and adding 50 mM N-acetyl-D-glucosamine that is a non-metabolized glucose analogue inhibiting glucose import (Azema et al, 2004), in order to prevent the consumption of the residual serum-derived glucose (final concentration in the medium: 0.5 mM) (Allmann et al, 2014). For the glycerol conditions, glycerol (10 mM) was added to the glucose/glycerol-depleted medium.…”