Abstract

The mitotic machinery of protozoan parasites is often quite distinct from host. That of trypanosomes is particularly divergent, with most components showing no detectable similarity to proteins in models. They may also be very different functionally, but the extent of these differences/similarities is masked by our lack of understanding on how these divergent systems work. We have developed a novel genome-wide approach to functionally profile genes in trypanosomes. Direct RNAi-Fragment Sequencing (DRiF-Seq) robustly and sensitively measures competitive growth rates for >200,000 individual RNAi mutants in each experiment and can be used both in culture and in host. Quantitation of competitive growth rates shows that ~50% of non-VSG genes are associated with a significant loss-of-fitness in vitro – suggesting an essential genome in trypanosomes similar to that seen by Bar-Seq in Plasmodium. DRiF-Seq also reveals temporal hierarchy in gene knockdown fitness and allows comparison of fitness costs associated with specific components of biological processes. Comparison with genome-scale fitness datasets in Toxoplasma and Plasmodium shows conserved processes with differential fitness costs in Apicomplexa and Trypanosoma, and the method is sufficiently sensitive to highlight differential biology between different trypanosome species. Epistatic interactions between specific mitotic mutants and DRiF-Seq libraries suggests regulation of mitosis in trypanosomes has more similarity to model systems than sequence comparisons might suggest.

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