Genome-wide screens connect HD82 loss-of-function to purine analog resistance in African trypanosomes

Anna Trenaman, Michele Tinti , Abdelmadjid Atrih, David Horn

Antimicrobrial Chemostherapy mSphere 9:e00363-23.


Nucleoside analogs 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 metabolized by host-cell, viral, or parasite enzymes prior to incorporation into DNA, thereby inhibiting DNA replication. Here, we report genes that sensitize African trypanosomes to nucleoside analogs, including the guanosine analog, 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 sensitize Trypanosoma brucei to ganciclovir. Since ganciclovir activity depends upon phosphorylation by ectopically expressed viral thymidine kinase, we also tested the adenosine analog, ara-A, that may be fully phosphorylated by native T. brucei kinase(s). Both Tb927.6.2800 and HD82 knockdowns were resistant to this analog. Tb927.6.2800 knockdown increased sensitivity to hydroxyurea, while dNTP analysis indicated that HD82 is indeed a triphosphohydrolase with dATP as the preferred substrate. Our results provide insights into nucleoside/nucleotide metabolism and nucleoside analog metabolism and resistance in trypanosomatids. We suggest that the product of 6.2800 sensitizes cells to purine analogs through DNA repair, while HD82 does so by reducing the native purine pool.