Variant surface glycoprotein RNA interference triggers a precytokinesis cell cycle arrest in African trypanosomes

Abstract

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...

Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest

“…In fact our results are reminiscent of the block of cytokinesis observed in a bloodstream T. brucei cell line ablated for the variable surface glycoprotein (VSG) (Sheader et al, 2005), because multinucleated cells did not accumulate in this case either. It has been suggested, that the observed precytokinesis arrest in this cell line is due to a cell-cycle checkpoint, the function of which would be to coordinate VSG synthesis with cell division (Sheader et al, 2005). Thus, analogous to this interpretation, we propose a model in which inhibition of mitochondrial fission by inactivation of TbDLP prevents completion of cytokinesis by triggering a cell-cycle checkpoint.…”

Bloodstream formTrypanosoma bruceidepend upon multiple metacaspases associated with RAB11-positive endosomes

“…This is because BSF parasites appear to lack a cell-cycle checkpoint that prevents additional rounds of S phase and mitosis prior to the completion of cytokinesis (Hammarton et al, 2003a). The precytokinesis block observed for triple MCA RNAi differs from that observed for RNAi of VSG, where a proposed VSG synthesis checkpoint prevents S phase reinitiation and cells accumulate with a 2N2K configuration (Sheader et al, 2005).…”

Developmental regulation and extracellular release of a VSG expression-site-associated gene product from Trypanosoma brucei bloodstream forms

“…3A). Moreover, ESAG9 expression was not upregulated in bloodstream forms arrested in G2 by VSGspecific RNAi ablation (Sheader et al, 2005; Gloria Rudenko, personal communication), eliminating non-specific elevation of ESAG9 mRNA upon cell cycle arrest, albeit in a distinct cell-cycle stage to stumpy forms. We conclude that ESAG9 expression is developmentally elevated in a stumpy-specific manner, that multiple members of the ESAG9 family are upregulated upon the transition to stumpy forms, and that different trypanosome lines express distinct repertoires of these genes.…”