Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. T. brucei multiplies extracellularly in the bloodstream, relying on antigenic variation of a dense variant surface glycoprotein (VSG) coat to escape antibody-mediated lysis. We investigated the role of VSG in proliferation and pathogenicity by using inducible RNA interference to ablate VSG transcript down to 1-2% normal levels. Inhibiting VSG synthesis in vitro triggers a rapid and specific cell cycle checkpoint blocking cell division. Parasites arrest at a discrete precytokinesis stage with two fulllength flagella and opposing flagellar pockets, without undergoing additional rounds of S phase and mitosis. A subset (<10%) of the stalled cells have internal flagella, indicating that the progenitors of these cells were already committed to cytokinesis when VSG restriction was sensed. Although there was no obvious VSG depletion in vitro after 24-h induction of VSG RNA interference, there was rapid clearance of these cells in vivo. We propose that a stringent block in VSG synthesis produces stalled trypanosomes with a minimally compromised VSG coat, which can be targeted by the immune system. Our data indicate that VSG protein or transcript is monitored during cell cycle progression in bloodstreamform T. brucei and describes precise precytokinesis cell cycle arrest. This checkpoint before cell division provides a link between the protective VSG coat and cell cycle progression and could function as a novel parasite safety mechanism, preventing extensive dilution of the protective VSG coat in the absence of VSG synthesis. antigenic variation ͉ Trypanosoma bruceiA frican trypanosomes, including Trypanosoma brucei, are unicellular parasites transmitted by tsetse flies in subSaharan Africa, which are responsible for debilitating disease in humans and livestock. African sleeping sickness is resurgent, with an estimated 300,000-500,000 cases a year, and it is invariably fatal if untreated (1). T. brucei multiplies extracellularly in the bloodstream, exposing it to immune attack. Each bloodstream-form cell is coated with Ϸ10 7 variant surface glycoprotein (VSG) molecules of a given antigenic type, making up Ϸ10% of total cellular protein (2, 3). Eventually the host mounts an antibody response against a given VSG variant. However, as parasites can switch between hundreds of antigenically diverse VSG coats during the course of a chronic infection, trypanosomes expressing a new VSG variant can escape the host antibody response against the old one (4, 5). This highly sophisticated strategy of antigenic variation makes African trypanosomes a paradigm for antigenic variation in general.Although a great deal is known about mechanisms mediating VSG switching, we know relatively little about the role of VSG itself in other aspects of immune evasion and pathogenicity. To investigate this, we inhibited VSG synthesis by performing inducible RNA interference (RNAi) both in vitro and in vivo. Inhibition of VSG synthesis in vitro triggers a rapid and specifi...

No abstract

The African trypanosome Trypanosoma brucei transcribes the active variant surface glycoprotein (VSG) gene from one of about 20 VSG expression sites (ESs). In order to study ES control, we made reporter lines with a green fluorescent protein gene inserted behind the promoter of different ESs. We attempted to disrupt the silencing machinery, and we used fluorescence-activated cell sorter analysis for the rapid and sensitive detection of ES up-regulation. We find that a range of treatments that either block nuclear DNA synthesis, like aphidicolin, or modify DNA-like cisplatin and 1-methyl-3-nitro-1-nitrosoguanidine results in up-regulation of silent ESs. Aphidicolin treatment was the most effective, with almost 80% of the cells expressing green fluorescent protein from a silent ES. All of these treatments blocked the cells in S phase. In contrast, a range of toxic chemicals had little or no effect on expression. These included berenil and pentamidine, which selectively cleave the mitochondrial kinetoplast DNA, the metabolic inhibitors suramin and difluoromethylornithine, and the mitotic inhibitor rhizoxin. Up-regulation also affected other RNA polymerase I (pol I) transcription units, as procyclin genes were also up-regulated after cells were treated with either aphidicolin or DNA-modifying agents. Strikingly, this up-regulation of silent pol I transcription units was bloodstream form-specific and was not observed in insect form T. brucei. We postulate that the redistribution of a limiting bloodstream form-specific factor involved in both silencing and DNA repair results in the derepression of normally silenced pol I transcription units after DNA damage.