Identifying genes associated with acute lethality in Toxoplasma

The development of effective antiparasitic interventions relies on the identification of protein targets whose inhibition is lethal to the parasite to enhance the potential for rapid and sustained clinical impact. The use of genetic disruption to categorize genes as essential does not allow us to distinguish between two sources of fitness defects: genes whose disruption causes cell death (cidal), versus genes whose disruption simply halts or slows growth (static), diluting the mutant in the population without causing cell death. Recent recombination-based, screening modalities afford new opportunities to investigate gene function without causing complete gene disruption. By utilizing a high-throughput tagging (HiT) approach we can label each fitness-conferring protein with a synthetic sequence encoding the minimal auxin-inducible degron (mAID) and induce transient protein depletion to distinguish between perturbations that kill the parasite, versus ones that allow recovery and subsequent replication once the inhibition period concludes. Towards the goal of highlighting proteins with cidal effects and contributing to the development of selective therapies, we have curated a list of 700 fitness-conferring genes that are shared across apicomplexans but absent in vertebrates. To efficiently degrade mAID-tagged proteins and reduce non-induced protein degradation, we have improved the degron system by introducing only one copy of the auxin receptor TIR1 into the parasite genome. Lastly, to gain functional insights for each fitness-conferring protein, we are developing an in-situ sequencing approach to enable linkage between each cell's morphology and the mutation it carries, in a pooled library of genetic perturbations. The next steps include performing the transient inhibition screen and expanding the in-situ sequencing approach to perform optical pooled screens in T. gondii. As most of these fitness-conferring genes have never been functionally annotated, this scheme will not only reveal proteins with parasite-killing potential but also provide expansive functional insight into uncharted territories of apicomplexan biology.