Characterization of wild trypanosome coats using spliced-leader sequence enrichment and RNA-Seq

African Trypanosomes defend themselves against host antibody in the bloodstream and tissue through antigenic variation of a highly immunogenic coat of variant surface glycoproteins (VSG). These parasites persist in the host by utilizing a large genomic repertoire of VSG genes and pseudogenes to switch the expression of their surface coats throughout infection. Much of the current knowledge regarding antigenic variation in African trypanosomes is based on studies of VSG expression in lab-adapted strains of Trypanosoma brucei. The study of gene expression in wild trypanosome populations is particularly difficult due to the low parasitemia of natural infections; RNA isolated from whole blood or tissue contains a very small fraction of trypanosome RNA in a mixture of host RNA. To overcome this problem, we developed an RNA-seq library preparation protocol which depletes host ribosomal RNA and messenger RNAs. This protocol takes advantage of the 5’ spliced-leader (SL) sequence present on mature messenger RNA (mRNA)s in all trypanosomatid species. Using a biotin-streptavidin pulldown technique, we achieve specific enrichment of parasite transcripts from whole host blood RNA samples. Our parasite-enriched transcriptomes have the potential to allow us to characterize the VSG expression profile and identify other highly expressed putative surface proteins of African trypanosomes across geographically and temporally distinct natural infections. This SL enrichment technique can easily be adapted and applied to in vivo studies for any the African trypanosome species by using each of their unique SL sequences as biotinylated baits. Primary Reader: Dr. Monica Mugnier Secondary Reader: Dr. Janet Markl