RNA-seq analysis of contemporary switched T. brucei clones expressing different VSGs.

Several persistent pathogens employ antigenic variation to continually evade mammalian host adaptive immune responses. African trypanosomes use variant surface glycoproteins (VSGs) for this purpose, transcribing one telomeric VSG expression site (ES) at a time, with strong repression of other telomeric ESs. In Trypanosoma brucei, VSG switching occurs at low frequency, primarily exploiting a reservoir of (sub)telomeric VSG templates to replace the active VSG. It has been known for over fifty years that VSGs emerge in a semi-predictable order. The hierarchy has primarily been explained by differential activation rates and subsequent host immune responses; VSGs from repressed ESs (ES-VSGs) typically precede VSGs from minichromosomes (MC-VSGs), for example. Here, we show that trypanosomes grow at different and predictable rates depending upon the VSG that is activated. We triggered high-frequency VSG gene recombination and switching in in vitro culture using inducible CRISPR-Cas9 to target the active VSG. VSG dynamics, that were independent of host immune selection, were then assessed using RNA-seq. Although ES-VSGs were typically activated at higher rates, cells that activated MC-VSGs displayed a competitive advantage and came to dominate the population; an advantage that was particularly pronounced for longer VSGs. Differential growth was also associated with wider transcriptome differences, affecting genes encoding glycolytic enzymes, tRNA synthetases, and other expression site-associated genes. We conclude that VSG template location and length contribute to trypanosome antigenic variation dynamics. VSG dependent differential growth likely facilitates the establishment and maintenance of an infection, prolonging parasite immune evasion with a limited set of variant antigens.