title: VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection.
abstract: Trypanosoma brucei relies on antigenic variation of its variant surface glycoprotein (VSG) coat for survival. We show that VSG switching can be efficiently studied in vitro using VSG RNAi in place of an immune system to select for switch variants. Contrary to models predicting an instant switch after inhibition of VSG synthesis, switching was not induced by VSG RNAi and occurred at a rate of 10(-4) per division. We find a highly reproducible hierarchy of VSG activation, which appears to be capable of resetting, whereby more than half of the switch events over 12 experiments were to one of two VSGs. We characterized switched clones according to switch mechanism using marker genes in the active VSG expression site (ES). Transcriptional switches between ESs were the preferred switching mechanism, whereby at least 10 of the 17 ESs identified in T. brucei 427 can be functionally active in vitro. We could specifically select for switches mediated by DNA rearrangements by inducing VSG RNAi in the presence of drug selection for the active ES. Most of the preferentially activated VSGs could be activated by multiple mechanisms. This VSG RNAi-based procedure provides a rapid and powerful means for analysing VSG switching in African trypanosomes entirely in vitro.
authors.affiliation: Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford OX1 3SY, UK.
authors.forename: Niall
authors.initials: N
authors.lastname: Aitcheson
authors.forename: Suzanne
authors.initials: S
authors.lastname: Talbot
authors.forename: Jesse
authors.initials: J
authors.lastname: Shapiro
authors.forename: Katie
authors.initials: K
authors.lastname: Hughes
authors.forename: Carl
authors.initials: C
authors.lastname: Adkin
authors.forename: Thomas
authors.initials: T
authors.lastname: Butt
authors.forename: Karen
authors.initials: K
authors.lastname: Sheader
authors.forename: Gloria
authors.initials: G
authors.lastname: Rudenko
mesh_terms: D000818:Animals; D000940:Antigenic Variation; D003470:Culture Media; D008969:Molecular Sequence Data; D010641:Phenotype; D034622:RNA Interference; D017422:Sequence Analysis, DNA; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000254:growth & development / Q000276:immunology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics* / Q000378:metabolism
chemical_list: D003470:Culture Media; D014643:Variant Surface Glycoproteins, Trypanosoma