ena-STUDY-School of Life Sciences University of Dundee-18-09-2020-23:03:53:361-70
Trypanosoma brucei VSG Switching - CRISPR/Cas9
Several important and persistent pathogens employ antigenic variation to continually evade host adaptive immune responses. African trypanosomes, for example, express one super-abundant variant surface glycoprotein (VSG) at a time, and switch expression at low frequency, exploiting a reservoir of silent VSG genes. It has been known for more than thirty years that populations expressing different VSGs emerge in a hierarchical order, but our understanding of how order emerges remains incomplete. A recent mathematical model and analysis of in vivo data from a mouse model suggested that cells expressing different length VSGs grow at different rates. Here, we demonstrate that cells expressing longer VSG do indeed make the major contribution to dynamics, in contemporary switched populations, and in a host-independent manner. High-frequency VSG gene recombination and switching were triggered in in vitro culture using inducible CRISPR-Cas9 to target the active VSG, followed by RNA-seq analysis to quantitatively monitor post-switching VSG abundance. VSG diversity was dynamic, yet highly reproducible, revealing a significant correspondence between early changes in VSG abundance and VSG length. Remarkably, cells expressing longer VSGs copied from silenced transcription units typically diminished in abundance over time, while the opposite was true for cells expressing longer VSGs copied from minichromosomes, which lack promoters. Our findings explain features of antigenic variation dynamics observed in vivo and indicate that the VSGs themselves impact variant abundance over time. VSG length-dependent growth provides a mechanism by which parasites can prolong immune evasion with a limited set of variant antigens.
Several important and persistent pathogens employ antigenic variation to continually evade host adaptive immune responses. African trypanosomes, for example, express one super-abundant variant surface glycoprotein (VSG) at a time, and switch expression at low frequency, exploiting a reservoir of silent VSG genes. It has been known for more than thirty years that populations expressing different VSGs emerge in a hierarchical order, but our understanding of how order emerges remains incomplete. A recent mathematical model and analysis of in vivo data from a mouse model suggested that cells expressing different length VSGs grow at different rates. Here, we demonstrate that cells expressing longer VSG do indeed make the major contribution to dynamics, in contemporary switched populations, and in a host-independent manner. High-frequency VSG gene recombination and switching were triggered in in vitro culture using inducible CRISPR-Cas9 to target the active VSG, followed by RNA-seq analysis to quantitatively monitor post-switching VSG abundance. VSG diversity was dynamic, yet highly reproducible, revealing a significant correspondence between early changes in VSG abundance and VSG length. Remarkably, cells expressing longer VSGs copied from silenced transcription units typically diminished in abundance over time, while the opposite was true for cells expressing longer VSGs copied from minichromosomes, which lack promoters. Our findings explain features of antigenic variation dynamics observed in vivo and indicate that the VSGs themselves impact variant abundance over time. VSG length-dependent growth provides a mechanism by which parasites can prolong immune evasion with a limited set of variant antigens.