DNA damage drives antigen diversification through mosaic VSG formation inTrypanosoma brucei

Abstract

SummaryAntigenic variation, using large genomic repertoires of antigen-encoding genes, allows pathogens to evade host antibody. Many pathogens, including the African trypanosomeTrypanosoma brucei,extend their antigenic repertoire through genomic diversification. While evidence suggests thatT. bruceirelies heavily on the generation of new variant surface glycoprotein (VSG) genes to maintain a chronic infection, a lack of experimentally tractable tools for studying this process has obscured its underlying mechanisms. Here, we present a highly sensitive targeted RNA sequencing approach for measuring VSG diversification. Using this method, we demonstrate that a Cas9-induced DNA double-strand break within the VSG coding sequence can induce VSG recombination with patterns identical to those observed during infection. These newly generated VSGs are antigenically distinct from parental clones and thus capable of facilitating immune evasion. Together, these results provide insight into the mechanisms of VSG diversification and an experimental framework for studying the evolution of antigen repertoires in pathogenic microbes.

Extravascular spaces are the primary reservoir of antigenic diversity inTrypanosoma bruceiinfection

“…To be sure that the increased VSG diversity we observed in tissues was not due to the derepression of silent VSGs within individual cells, we performed single-cell RNA sequencing using the SL-Smart-seq3xpress platform to quantify VSG expression in single cells from blood and tissue samples 33 . Because T. brucei can form new "mosaic" VSGs through recombination 4,8,16,34 , which are, by definition, absent from reference genomes, we initially used our VSG-Seq pipeline to de novo assemble VSGs in each cell. This approach accounts for the possibility that expressed VSGs might be absent from the reference genome, either as a result of mosaic VSG formation or as a result of an incomplete genome assembly, potentially affecting quantification and/or read mapping.…”