DNA Rearrangements Associated with Multiple Consecutive Directed Antigenic Switches in Trypanosoma brucei

Abstract

Changes in variant surface glycoprotein (Vsg) expression allow Trypanosoma brucei to elude the immune response. The expressed vsg is always located at the telomeric end of a polycistronic transcription unit known as an expression site (ES). Although there are many ESs, only one is active at any particular time. The mechanisms regulating ES transcription and switching are unknown. Chromosome rearrangements within or upstream of the ES have been described to occur in occasional switch events, but no changes have been consistently associated with switching. We inserted the drug resistance genes neo and ble, conferring resistance to G418 and phleomycin, respectively, 1 kb downstream of "silent" ES promoters. This demonstrated that short-range transcription could be achieved from a silent ES promoter. From one initial transformant clone, panels of independent consecutive on-off-on switch clones were generated and analyzed. The first activation of the neo-targeted ES was always associated with deletion of the upstream tandem promoter in this ES, but no further rearrangements were detected in consecutive off-on switches of this ES. On the other hand, direct analysis of ES promoters showed that deletions and duplications occurred elsewhere. Activation of a ble-tagged 300-kb chromosome could not be achieved, but phleomycin-resistant clones could be obtained. One such clone arose from recombination between three ESs. Taken together, our experiments suggest that ES switching may occur after a period of chromosomal interactivity that may or may not leave tangible evidence in the form of detectable sequence changes.Trypanosoma brucei is an extracellular protozoan parasite that multiplies in the tissue fluids and vascular system of its vertebrate host, causing major veterinary and medical problems in Africa. The entire surface of the bloodstream form trypanosome is covered by a dense coat, which consists largely of a single molecular species known as the variant surface glycoprotein (Vsg) (8). Sequential expression of vsg genes allows the parasite to elude the immune response of the host (reviewed in references 9, 10, and 51). The expressed vsg is always located adjacent to a telomere, at the end of a polycistronic expression site (ES) (15). Hybridization analysis suggests the presence of approximately 20 ESs, whose sequences are highly conserved (13,54). Only one ES is active, yielding a single Vsg on the surface of the infective trypanosome. The ES contains a number of ES-associated genes (esag) (13, 51). ES transcription is driven by an ␣-amanitin-resistant polymerase (26, 27). The ES promoter is located 40 to 60 kb upstream of the telomeric vsg (25, 27). ES promoters sequenced so far show more than 90% sequence identity (18, 37, 53). The 5Ј end of the ES promoter is flanked by a region of imperfect direct repeats of 50 bp, which covers at least 10 kb in the vsg221 ES (54).Antigenic variation in T. brucei can occur by duplicative transposition of a new vsg into an already active ES, displacing the resident vsg. A switch in...

Bloodstream Form-specific Up-regulation of Silent VSG Expression Sites and Procyclin in Trypanosoma brucei after Inhibition of DNA Synthesis or DNA Damage

“…Partial activation of ESs in bloodstream form T. brucei has been achieved before in experiments targeting drug resistance genes into "silent" ESs, although the levels of transcription were much lower than we show here (19,64). These low levels of transcription were "erased" after activation and subsequent inactivation of the partially active ES, implying the resetting of an epigenetic state (19).…”

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

“…In support of this notion, promoters from active or inactive ESs share the same sensitivity to DNase (24), and antigenic variation still occurs properly if the promoter of the active ES is replaced by the ribosomal promoter (41,42). Other arguments include the detection of transcripts originating from inactive ESs such as RNA transcribed from the ES beginning in procyclic forms (29), RNA from antibiotic-resistance genes inserted in inactive ESs (25), or RNA from different ESAG6 from inactive ESs, accounting for up to 20% of the total ESAG6 transcripts (28). Taken together these observations support the notion of leaky ES transcription (27).…”

“…Previous experiments pointed to mixed conclusions. On the one hand, transcripts from inactive ESs could be detected in bloodstream clones and even in procyclic forms, which do not express VSG, suggesting that transcription initiation takes place in all ESs simultaneously (22)(23)(24)(25)(26)(27)(28)(29). On the other hand, experiments tagging ESs with antibiotic-resistance genes showed that RNA expression can be detected from two ES, although the expression was unstable (30).…”

Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity

“…VSG is the last gene in any BES, located less than 2 kb from the telomere and 40-60 kb downstream of the BES promoter [33]. T. brucei has multiple BESs (15-20 in the lister 427 strain used in this work), but only one is fully active, expressing a single type of VSG at any time [34,35].…”