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 dramatically reduced rate (10-5-10-6) in laboratory-adapted strains1-3. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination (HR) initiated by a DNA lesion that is used by other pathogens (e.g. 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-bp repeats upstream of the transcribed VSG 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, suggesting 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 (BIR).

Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity

“…We performed a Ligation-Mediated PCR (LMPCR) assay to directly detect DSBs along BESs [10,43]. In this assay, a doublestranded adaptor with a blunt end and a staggered end was ligated to genomic DNA at the DSB followed by PCR using locus-specific primers and Southern analysis ( Figure 4A).…”

“…In contrast, deletion of TOPO3 and RMI1 leads to more frequent VSG switching [41,42]. Recent studies showed that inducing DSBs in the active BES increases the VSG switching rate, and different VSG switching mechanisms are used depending on the DSB position [10,43]. As high as 25% of DSBs downstream of the active VSG result in the loss of the entire active BES [10], and an accompanying in situ switch can give rise to ES loss coupled with in situ switchers (ES loss + in situ).…”

Telomere as an Important Player in Regulation of Microbial Pathogen Virulence

“…It is speculated that all active telomeres are prone to large telomere fragment deletions due to its active transcription state, but shorter telomeres is more likely to have a deletion landed in the subtelomeric region and to cause damage in the active VSG gene, which will force the parasite to go through VSG switching. Introducing a break at the I-SCE I site targeted immediately upstream of the active VSG gene led to a 250-fold increase in VSG switching frequency, confirming part of this theory that damage to the active VSG gene will force the parasite to switch (Boothroyd et al 2009). In consistent with this observation, deplete the active VSG using the RNAi approach also led to elevated VSG switching rate (Aitcheson et al 2005).…”

“…In this event, the donor can be any functional VSG gene in the genome. There is almost always a stretch of 70 bp repeats upstream of a VSG gene, in which homologous recombination can initiate as DNA double strand breaks (Boothroyd et al 2009). In rare occasions, several VSG donors have been identified in a single VSG switching event, where each donor contributes only a fragment of the gene, generating a new mosaic VSG gene product (Marcello & Barry 2007).…”

Meganucleases can restore the reading frame of a mutated dystrophin

“…25,26 MGNs were used to induce homologous recombination in a variety of cell types and organisms, including mammalian cells, mice, plants, Drosophila, Escherichia coli and trypanosome. 27,28 MGN-induced DSB can also be repaired by non-homologous end-joining (NHEJ), an error-prone process, which frequently results in micro-insertions or micro-deletions (indels) at the site of the break. 29 Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology.…”