RibonucleaseH1-targeted R-loops in surface antigen gene expression sites can direct trypanosome immune evasion

Abstract

Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp repeats in the actively transcribed ES, but spread throughout the active and inactive ES in the absence of RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching and replication-associated genome damage, some of which accumulates within the active ES. This work indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei immune evasion by transcription and recombination.\n\nAuthor summaryAll pathogens must survive eradication by the host immune response in order to continue infections and be passed on to a new host. Changes in the proteins expressed on the surface of the pathogen, or on the surface of the cells the pathogen infects, is a widely used strategy to escape immune elimination. Understanding how this survival strategy, termed antigenic variation, operates in any pathogen is critical, both to understand interaction between the pathogen and host and disease progression. A key event in antigenic variation is the initiation of the change in expression of the surface protein gene, though how this occurs has been detailed in very few pathogens. Here we examine how changes in expression of the surface coat of the African trypanosome, which causes sleeping sickness disease, are initiated. We reveal that specialised nucleic acid structures, termed R-loops, form around the expressed trypanosome surface protein gene and increase in abundance after mutation of an enzyme that removes them, leading to increased changes in the surface coat in trypanosome cells that are dividing. We therefore shed light on the earliest acting events in trypanosome antigenic variation.