Discussion

A modified RNAi target sequencing approach, pooling 176 individual cell lines covering all predicted Trypanosoma brucei protein kinases (PKs), assessed essentially for bloodstream form proliferation in vivo. 48h after RNAi induction, 49 lines had a significant loss of fitness in vivo in two independent experiments, finding strong correlation between in vitro and in vivo results for the majority. Nine PKs were required for growth in vivo long before they were needed in vitro. Amongst these PKs were two MAP3Ks, involved osmotic shock resistance; several PKs with putative functions in stress response through PI3K/TOR, including BUD32-like, involved in translational regulation; VPS15, component of the PI3K complex with roles in autophagosome formation and vesicular trafficking; and CK2A2, a promiscuous PK capable of stress-induced mobilization. Three of the in vivo PKs have been implicated in repair of alkylation-induced cellular damage: SRPK1, a stress-response RNA splicing regulator; AUK2, a controller of entry in mitosis; and CAMKL, putatively involved in metabolic regulation. Finally, a parasite-specific pseudokinase which localizes to the flagellum attachment zone, FAZ20, was also found in that group. Identification of novel virulence-associated PKs provides new insights on parasite-host interaction with potential as drug targets. This strategy can be a valuable research tool to evaluate protein phosphorylation involvement in many biological processes.