Characterisation of the OTU domain deubiquitinase complement of<i>Toxoplasma gondii</i>

Abstract

The phylum Apicomplexa contains several parasitic species of medical and agricultural importance. The ubiquitination machinery remains, for the most part, uncharacterised in apicomplexan parasites, despite the important roles that it plays in eukaryotic biology. Bioinformatic analysis of the ubiquitination machinery in apicomplexan parasites revealed an expanded ovarian tumour domain-containing (OTU) deubiquitinase (DUB) family in <i>Toxoplasma</i>, potentially reflecting functional importance in apicomplexan parasites. This study presents comprehensive characterisation of <i>Toxoplasma</i> OTU DUBs. AlphaFold-guided structural analysis not only confirmed functional orthologues found across eukaryotes, but also identified apicomplexan-specific enzymes, subsequently enabling discovery of a cryptic OTU DUB in <i>Plasmodium</i> species. Comprehensive biochemical characterisation of 11 <i>Toxoplasma</i> OTU DUBs revealed activity against ubiquitin- and NEDD8-based substrates and revealed ubiquitin linkage preferences for Lys6-, Lys11-, Lys48-, and Lys63-linked chain types. We show that accessory domains in <i>Toxoplasma</i> OTU DUBs impose linkage preferences, and in case of apicomplexan-specific TgOTU9, we discover a cryptic ubiquitin-binding domain that is essential for TgOTU9 activity. Using the auxin-inducible degron (AID) to generate knockdown parasite lines, TgOTUD6B was found to be important for <i>Toxoplasma</i> growth.