Expression of a Major Surface Protein ofTrypanosoma bruceiInsect Forms Is Controlled by the Activity of Mitochondrial Enzymes

Abstract

In cycling between the mammalian host and the tsetse fly vector, trypanosomes undergo major changes in energy metabolism and surface coat composition. Early procyclic (insect) forms in the tsetse fly midgut are coated by glycoproteins known as EP and GPEET procyclins. EP expression continues in late procyclic forms, whereas GPEET is down-regulated. In culture, expression of GPEET is modulated by glycerol or glucose. Here, we demonstrate that a glycerol-responsive element of 25 nucleotides within the 3' untranslated region of GPEET mRNA also controls expression by glucose and during development in the fly. In trypanosomes, mitochondrial ATP is produced mainly by the acetate: succinate-CoA transferase/succinyl-CoA synthetase (ASCT) cycle, the citric acid cycle, and the cytochromes. Silencing of the pyruvate dehydrogenase or succinyl-CoA synthetase from the ASCT cycle by RNA interference induces reexpression of GPEET in late procyclic forms, whereas inhibition of the citric acid cycle or the cytochromes has no effect. In contrast, inhibition of the alternative oxidase, the second branch of the electron transport chain, with salicylhydroxamic acid overrides the effect of glucose or glycerol and causes a reduction in the level of GPEET mRNA. Our results reveal a new mechanism by which expression of a surface glycoprotein is controlled by the activity of mitochondrial enzymes.

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

“…Although neither EP2 nor GPEET procyclin is N-glycosylated, GPEET1 procyclin is phosphorylated on 6 out of 7 Thr residues (13)(14)(15). In culture, the procyclin expression profile depends on the carbon source (16) and metabolic state of the cells (17,18), and in the tsetse fly, there appears to be a program of procyclin expression such that GPEET procyclin is expressed early, giving way to EP1 and EP3 procyclin expression (16,19). GPEET and EP procyclins contain similar glycosylphosphatidylinositol (GPI) membrane anchors.…”

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei

“…Furthermore, the activity of mitochondrial enzymes was shown to control expression of GPEET [20]. A cis-regulatory element in the 3 untranslated region (UTR), known as the glycerol responsive element (GRE), destabilises GPEET mRNA in the fly gut and in culture in response to high glucose concentration or glycerol deprivation [20,21].…”

“…Furthermore, the activity of mitochondrial enzymes was shown to control expression of GPEET [20]. A cis-regulatory element in the 3 untranslated region (UTR), known as the glycerol responsive element (GRE), destabilises GPEET mRNA in the fly gut and in culture in response to high glucose concentration or glycerol deprivation [20,21]. Other environmental factors may also act via the GRE and hence lead to reactivation of GPEET in culture; for example, different serum components might explain the variation in GPEET expression observed between and within different strains of T. brucei [22].…”

“…Northern blot analysis of 10 g total RNA per lane was performed as described [11]. GPEET (Tb927.6.510) and EP mRNA were detected using Mega-Prime- 32 and MicroarrPROas (5 -GATTTCAGCGTTGCAGCACCAG) for both GPEET and EP1, flanking an internal region encoding the pentapeptide and dipeptide repeats, respectively [20]. Hybridisation was performed at 60 • C in solution containing 50% formamide and blots were washed at 55 • C in solution containing 0.2× SSC and 0.1% SDS.…”

“…The steady-state level and half-life of GPEET mRNA are reduced during differentiation and in late procyclic forms Distinct elements regulating mRNA stability and translation have been mapped within the procyclin 3 UTR [24,25,30,39]. The 3 UTR of GPEET harbours the GRE, a negative element conferring mRNA instability, thus leading to a transcript that is barely detected or even absent in late PF [16,20,23]. We decided to recapitulate these results and assess the relative steady-state levels and the stability of GPEET mRNA in both early and late PF.…”