Abstract
African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive.
Highlights
African trypanosomes are protozoan parasites that have dedicated more than 20% of their coding capacity [1,2] and 10% total cellular protein content [3] to a single biological function
The likely outcome of restricting this process of coat gene selection in natural infections would be a reduction in the chronic nature of African trypanosomiasis
Our findings suggest that the 70-bp repeat regions are required for the normal outcomes of Variant Surface Glycoproteins (VSG) switching, and the ability of T. brucei to survive in its host during a chronic infection
Summary
African trypanosomes are protozoan parasites that have dedicated more than 20% of their coding capacity [1,2] and 10% total cellular protein content [3] to a single biological function. To survive in the challenging environmental niche of the mammalian bloodstream, subspecies of Trypanosoma brucei must regularly change their antigenic glycoprotein coat In this manner, they are able to escape the antibody-mediated immune response of their host to cause a chronic infection of the bloodstream that results in death of both humans (African sleeping sickness) and livestock (nagana) if left untreated [4]. Addition of the Dimer_Rv sequence did not result in a significant increase in the selected VSG repertoire (pval = 0.205), some subtle differences between Δ70-ISceI and Dimer_Rv strains can be observed (Fig 5D) These data have identified a minimal 70-bp repeat region able to partially suppress the collection phenotypes (i.e. cell growth defect, cell cycle delay, increased VSG switching, and reduced VSG donor selection) associated with DSB formation proximal to a 70-bp repeat deletion mutant and result in phenotypes similar to lines harboring cognate 70-bp repeats
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