Genome-wide knockdown screens connect a nuclear HD-domain protein to purine analogue resistance in African trypanosomes

Nucleoside analogues have been used extensively as anti-infective agents, particularly against viral infections, and have long been considered promising anti-parasitic agents. These pro-drugs are metabolised by host-cell, viral or parasite enzymes prior to incorporation into DNA, thereby inhibiting DNA replication. Here, we report genes that sensitise African trypanosomes to nucleoside analogues, including the guanosine analogue, ganciclovir. We applied ganciclovir selective pressure to a trypanosome genome-wide knockdown library, which yielded nucleoside mono and diphosphate kinases as hits, validating the approach. The two most dominant hits to emerge, however, were Tb927.6.2800 and Tb927.6.2900, which both encode nuclear proteins; the latter of which is HD82, a SAMHD1 related protein and a putative dNTP triphosphohydrolase. We independently confirmed that HD82, which is conserved among the trypanosomatids, can sensitise Trypanosoma brucei to ganciclovir. Since ganciclovir activity depends upon phosphorylation by ectopic expression of a viral thymidine kinase, we also tested the adenosine analogue, ara-A, that may be fully phosphorylated by native T. brucei kinase(s). Both Tb927.6.2800 and HD82 were found to sensitise T. brucei to this analogue. Only Tb927.6.2800 increased tolerance to hydroxyurea, which is thought to reduce deoxynucleoside triphosphate (dNTP) pools. Our results provide insights into nucleoside/nucleotide metabolism and nucleoside analogue resistance in trypanosomatids.Sample descriptionTrypanosoma brucei strain Lister 427 bloodstream form parasites were used in two high throughput RNAi target sequencing (RIT-seq) screens using the thymidine analogue ganciclovir. The two screens used an inducible negative selectable marker (HSV-TK) with either the aldolase 3' UTR or the VSG 3' UTR sequence. Ganciclovir selection was applied at either 2 x EC50 or 4 x EC50.