A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei

Abstract

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10-2–10-3 per population doubling in recent isolates from nature, but at a markedly reduced rate (10-5–10-6) in laboratory-adapted strains1,2,3. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication.

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Figure 1: Antigenic switching is induced by a single I-SceI-generated DSB.
Figure 2: I-SceI-induced antigenic switching occurs by duplicative gene conversion.
Figure 3: PCR and sequencing analyses of recipient ( VSG 221 expression site) and donor ( VSG 224 expression site).
Figure 4: Wild-type trypanosomes incur staggered DSBs specifically at the 70-bp repeat regions of the active expression site.

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Acknowledgements

This work was supported by awards from the C. H. Revson Foundation (to C.E.B.), the German National Academic Foundation (to K.I.L.), the Otto Ritter Foundation (to K.I.L.), the W. M. Keck Foundation (to F.N.P.), the Irma T. Hirschl Foundation (to F.N.P.) and by grant number R01AI021729 from the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health (to G.A.M.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID or the NIH.

Author Contributions All authors conceived of and designed the experiments. C.E.B. and T.L. are primarily responsible for the experiments shown in Fig. 1. C.E.B. and O.D. are primarily responsible for the experiments shown in Figs 2 and 3. F.N.P. is primarily responsible for the experiments in Fig. 4. C.E.B., O.D., G.A.M.C. and F.N.P. wrote the manuscript.

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Correspondence to F. Nina Papavasiliou.

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Boothroyd, C., Dreesen, O., Leonova, T. et al. A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei. Nature 459, 278–281 (2009). https://doi.org/10.1038/nature07982

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