Bidirectional silencing of RNA polymerase I transcription by a strand switch region in Trypanosoma brucei.

The procyclin genes in Trypanosoma brucei are transcribed by RNA polymerase I as part of 5-10 kb long polycistronic transcription units on chromosomes VI and X. Each procyclin locus begins with two procyclin genes followed by at least one procyclin-associated gene (PAG). In procyclic (insect midgut) form trypanosomes, PAG mRNA levels are about 100-fold lower than those of procyclins. We show that deletion of PAG1, PAG2 or PAG3 results in increased mRNA levels from downstream genes in the same transcription unit. Nascent RNA analysis revealed that most of the effects are due to increased transcription elongation in the knockouts. Furthermore, transient and stable transfections showed that sequence elements on both strands of PAG1 can inhibit Pol I transcription. Finally, by database mining we identified 30 additional PAG-related sequences that are located almost exclusively at strand switch regions and/or at sites where a change of RNA polymerase type is likely to occur.

Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse.

Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei.

A 'two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3' flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins.

Expression of a major surface protein of Trypanosoma brucei insect forms is controlled by the activity of mitochondrial enzymes.

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.