A DHFR-based destabilizing domain enables inducible depletion of Aurora kinase 1 in Trypanosoma cruzi with target-dependent efficacy

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi , remains the most significant parasitic illness of the Americas, yet our ability to study essential genes in this organism has lagged decades behind that of related parasites. Here, we describe the implementation of a dihydrofolate reductase-based destabilizing domain (DDD) derived from Escherichia coli allowing for the control of protein stability by the small molecule trimethoprim (TMP). Through a systematic comparison of the original DDD and three temperature-sensitive (TS) variants, we identified the TS21 variant as the most effective in T. cruzi and showed that N-terminal degron fusions consistently outperform C-terminal fusions in inducible depletion. Using Aurora kinase 1 (AUK1) as a benchmark gene, we demonstrate that DDD-mediated AUK1 depletion is functional in both insect and mammalian stages of the parasite life cycle and halts parasite proliferation within 24 h of TMP washout with mitotic defects mirroring those observed in prior studies. Although AUK1 was strongly depleted, the efficiency of depletion was target dependent as three additional targets we tested (DRBD12, RBP10, and Kinesin-D) demonstrated little to no depletion following TMP removal. These findings establish the DDD as a proof-of-principle tool for inducible protein depletion in T. cruzi and define the target-to-target variability that users should anticipate.