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

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Abstract
Sleeping sickness is caused by the trypanosome parasite. This parasite outwits the human immune system by periodically changing its coat protein in a process known as variant surface glycoprotein (VSG) switching. The favoured model to explain switching invokes a normal host process, called gene conversion, that is used to repair DNA. In this work, Nina Papavasiliou and colleagues establish the first in vitro system that recapitulates VSG switching. The data indicate that a spontaneous double-stranded DNA break upstream of the gene encoding the coat protein initiates the process. Sleeping sickness is caused by the parasite Trypanosoma brucei. This parasite outwits the human immune system by periodically changing its coat protein in a process known as VSG switching. Here, the first in vitro system that recapitulates VSG switching is established, indicating that a spontaneous double-stranded DNA break upstream of the gene encoding the code protein initiates the process. 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.