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

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 10–10 per population doubling in recent isolates from nature, but at a markedly reduced rate (10–10) in laboratory-adapted strains. 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. The T. brucei genome contains .1,000 VSG genes and pseudogenes, yet the single transcribed VSG gene is invariably found in 1 of ,15 large (40–60 kb) telomeric expression sites. VSG switching can be achieved by shifting transcription from one expression site to another (in situ switch) or by reciprocal translocations between two expression sites (telomere exchange), but most switching occurs by copying a new VSG gene into the actively transcribed expression site by duplicative gene conversion. Antigenic switching by gene conversion has been proposed to be initiated by a DSB within or upstream of the actively transcribed VSG gene, but physical evidence for a DSB has been lacking. To determine whether a DSB within the transcribed expression site is sufficient to precipitate an antigenic switch, we introduced the heterologous recognition sequence for the yeast mitochondrial endonuclease I-SceI adjacent to the 70-bp repeat region upstream of the VSG 221 locus (70.II cell line; Fig. 1a). I-SceI has previously been used to introduce targeted DSBs in several organisms, including T. brucei. Regulation of the I-SceI enzyme was achieved through stable transfection under the control of an inducible promoter. The activity of I-SceI was monitored by induction of the enzyme for 1.5 and 2.5 days and subsequent quantitative Southern blotting (Fig. 1b). As expected, an ,9 kb XhoI/XhoI fragment (Fig. 1a) was reduced to ,1.3 kb in response to I-SceI induction (Fig. 1b, lanes 2 and 3), which corresponds to the size shift seen when genomic DNA was digested with XhoI and recombinant I-SceI (Fig. 1b, lane 1). This smaller fragment was not seen when DNA from uninduced cells was digested with XhoI alone (Fig. 1b, lane 4). By measuring the intensity of the bands, we estimated that the action of I-SceI leads to a DSB in ,1% of the cells. To measure changes in switching frequency upon I-SceI induction accurately, we developed a magnetic-activated cell sorting (MACS) assay in conjunction with conventional flow cytometry. MACS was optimized to enrich for trypanosomes that had switched their VSG (see Methods). The induction of a DSB increased the switching frequency ,250-fold compared to cells without an I-SceI recognition sequence or in the absence of I-SceI induction (1.53 10, 5.93 10 and 1.53 10 per population, respectively; Fig. 1c, d). This far exceeds any switching frequency reported for laboratoryadapted strains, and is more representative of switching frequencies seen in the early stages of a natural infection. The results indicate that roughly half the cells in which a DSB was generated switched their VSG. The increased switching frequency was not observed when a DSB was induced in the VSG pseudogene upstream of the 70-bp repeat region (PS cell line; Fig. 1a, c), indicating that the location of the DSB adjacent to the 70-bp repeats is critical to the high frequency of switching seen here. Repetitive sequences can provide homology for homologous recombination. In T. brucei, all expression-site-associated VSG genes (and probably most silent VSG genes) are found downstream of imperfect 70-bp repeats, which have been mapped to the upstream border of VSG gene switching events. Removal of the 70-bp repeats, however, did not decrease an already low rate of VSG switching. To determine whether the 70-bp repeats are necessary for the high frequency of DSB-induced switching, we replaced them with an I-SceI recognition sequence (–70 cell line; Fig. 1a). A DSB in the absence of the 70-bp repeats did not increase VSG switching (Fig. 1c), indicating that the 70-bp repeats do facilitate VSG switching. The order in which VSG genes are expressed during the course of an infection has been described as ‘semi-predictable’ and is thought to be key to protracted illness. Telomere-proximal VSG genes, such as those in silent expression sites or mini-chromosomes, are activated first, followed by those in sub-telomeric arrays. To determine the chromosomal location of the donor VSG gene and to elucidate whether switching occurred by duplication, reciprocal telomere exchange or in situ switching, we cloned the progeny and identified the expressed VSG gene in 42 switched clones from several independent experiments, further characterizing 18 clones by rotating agarose gel electrophoresis (RAGE) and Southern blotting. As shown in Fig. 2 and Supplementary Table 1, all of the switchers showed loss of VSG 221 and