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 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 approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 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.

Figure 1. Antigenic switching is induced by a single I-SceI-generated DSB

a, Schematic of the telomeric region of the VSG 221 ES (WT). An I-SceI RS was introduced adjacent to the 70-bp repeat region (70.II), within the pseudogene (PS), and in place of the 70-bp repeats (-70). b, I-SceI cuts in vivo. DNA was cut with recombinant I-SceI and XhoI in lane 1 and XhoI in lanes 2, 3, and 4. The Southern blot was probed with a Puro probe (underlined in red in a). c, Switching frequency in 70.II is increased ∼250× above levels in the absence of an RS (-RS) or without I-SceI induction (70.II -dox). This increase was not observed for PS or −70. Error bars represent s.e.m. for ≥3 experiments. d, Representative flow cytometry plots for uninduced (-dox) and induced (+dox) 70.II cells. Events in the lower left (221^-) and right (221⁺) quadrants represent switchers and cells not bound by the column, respectively.

Figure 2. I-SceI-induced antigenic switching occurs by duplicative gene conversion

Chromosomes were separated by RAGE and analyzed by Southern blotting. Representative clones are shown. VSG 221 is present in the parental (PA) strain and lost upon I-SceI induction (221 panels). In all switchers (clone numbers are marked on top of each lane), the lost VSG 221 gene is replaced by a VSG gene duplicated from a, a silent ES (224, bR-2, c11, 121, c5, VO-2) or b, a mini-chromosome (MC) (31, 42, 28) that is copied into the ES previously occupied by VSG 221 (arrowheads). Multiple bands represent >1 copy of the VSG gene in the genome.

Figure 3. PCR and sequencing analyses of recipient (VSG 221 ES) and donor (VSG 224 ES)

a, PCR and sequencing analyses indicate loss of the I-SceI RS, exonucleolytic degradation and DSB processing, and invasion of the first homologous region in the VSG 224 ES proximal to the VSG. Primers used for PCR are indicated by red arrows. The transcribed ES is indicated by a dotted arrow. For sequence data see Supplementary Fig. S1. b, PCR showing loss of the VSG 221 subtelomeric region (black arrows in panel a) in the switched clones. PA, parental; tubulin is shown as a control.

Figure 4. Wild-type trypanosomes incur staggered DSBs specifically at the 70-bp repeat regions of the active ES

a, LM-PCR over the active ES reveals DSBs within the 70-bp repeat region. A schematic appears over the autoradiogram and the location of LM-PCR primers and DNA probe are indicated as follows: red arrow, DSB-specific (linker-specific) primer; black arrow, locus-specific primer; gray bar, probe. Triangles denote 5-fold dilutions of input DNA from 2 VSG 221-expressing (221a, 221c) and 1 VSG 1.13-expressing (113) cell lines. Bars indicate location of 100-bp ladder. b, Top: probing the active ES for DSBs upstream of the pseudogene reveals infrequent breaks. The sizes of the amplicons indicate that the breakpoints are within the upstream 70-bp repeat region (70-bp I). Bottom: amplification of the pseudogene locus with the forward and reverse primers used for LM-PCR in (a) and (b) serves as loading control. c and d, Top: LM-PCR for the presence of DSBs at a silent ES (VSG 224) and chromosome internal locus (histone-3 variant). Bottom: loading control.