A trypanosome metacyclic VSG gene promoter with two functionally distinct, life cycle stage-specific activities.

In the mammalian bloodstream, African trypanosomes express the variant surface glycoprotein (VSG), continual switching of which allows evasion of the host immune response. Bloodstream VSG genes are transcribed from polycistronic bloodstream expression sites with promoters which are located 45-60 kb upstream. These promoters are not exclusively stage-regulated, being active in the insect midgut stage where VSG is not expressed. However, the metacyclic VSG (M-VSG) genes, a small subset activated when VSG synthesis begins in the metacyclic stage in the tsetse fly salivary glands, are transcriptionally activated specifically in that stage from promoters <3 kb upstream. Using deletion mapping and transient transfection, we show that the entire 1.22 M-VSG gene promoter region (171 bp) is required for full activity in metacyclic-derived trypanosomes. However, a subsidiary, bloodstream stage-specific activity is present in its 5' half which directs transcription initiation very close to the initiation site used in metacyclic-derived trypanosomes. Our results imply that the M-VSG gene promoter is longer and more complex than other VSG gene promoters.

VSG gene control and infectivity strategy of metacyclic stage Trypanosoma brucei.

As the metacyclic trypanosome stage develops in the tsetse fly salivary glands, it initiates expression of variant surface glycoproteins (VSGs) and does so by each cell activating, at random, one from a small subset of metacyclic VSG (M-VSG) genes. Whereas differential activation of individual VSG genes in the bloodstream occurs as a function of time, to evade waves of antibody, it is believed that the aim in the metacyclic stage is simultaneously to generate population diversity. M-VSG genes are activated in their telomeric loci and belong to monocistronic transcription units, unlike all other known trypanosome protein-coding genes, which appear to be transcribed polycistronically. The promoters of these metacyclic expression sites (M-ESs) have the unique property, in this organism, of being switched on and off in a life-cycle stage specific pattern. We have found that the 1.22 M-ES promoter is regulated according to life cycle stage, differential control being exerted through different elements of the promoter and under the influence of its genomic locus. We have characterized in detail the telomeres containing the 1.22 and 1.61 M-ESs. Upstream of the M-ES is a possibly haploid, non-transcribed region with some degenerate sequences homologous with expression site associated genes (ESAGs) that occur in bloodstream VSG expression sites. Further upstream (respectively, 22 and 13 kb upstream of the 1.22 and 1.61 VSG genes) are alpha-amanitin sensitive transcription units that may be polycistrons and are transcribed in all examined life cycle stages. They contain a number of genes. The differences between metacyclic and bloodstream ESs may have important consequences for life cycle regulation, genetic stability, phenotype complexity and adaptability of the metacyclic stage as it infects different host species.

Activity of a trypanosome metacyclic variant surface glycoprotein gene promoter is dependent upon life cycle stage and chromosomal context.

African trypanosomes evade the mammalian host immune response by antigenic variation, the continual switching of their variant surface glycoprotein (VSG) coat. VSG is first expressed at the metacyclic stage in the tsetse fly as a preadaptation to life in the mammalian bloodstream. In the metacyclic stage, a specific subset (<28; 1 to 2%) of VSG genes, located at the telomeres of the largest trypanosome chromosomes, are activated by a system very different from that used for bloodstream VSG genes. Previously we showed that a metacyclic VSG (M-VSG) gene promoter was subject to life cycle stage-specific control of transcription initiation, a situation unique in Kinetoplastida, where all other genes are regulated, at least partly, posttranscriptionally (S. V. Graham and J. D. Barry, Mol. Cell. Biol. 15:5945-5956, 1985). However, while nuclear run-on analysis had shown that the ILTat 1.22 M-VSG gene promoter was transcriptionally silent in bloodstream trypanosomes, it was highly active when tested in bloodstream-form transient transfection. Reasoning that chromosomal context may contribute to repression of M-VSG gene expression, here we have integrated the 1.22 promoter, linked to a chloramphenicol acetyltransferase (CAT) reporter gene, back into its endogenous telomere or into a chromosomal internal position, the nontranscribed spacer region of ribosomal DNA, in both bloodstream and procyclic trypanosomes. Northern blot analysis and CAT activity assays show that in the bloodstream, the promoter is transcriptionally inactive at the telomere but highly active at the chromosome-internal position. In contrast, it is inactive in both locations in procyclic trypanosomes. Both promoter sequence and chromosomal location are implicated in life cycle stage-specific transcriptional regulation of M-VSG gene expression.