Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii

We have developed a control system for regulating gene activation in Toxoplasma gondii. The elements of this system are derived from the Escherichia coli tetracycline resistance operon, which has been widely used to tightly control gene expression in eukaryotes. The tetracycline repressor (tetR) Interferes with transcription initiation while the chimeric transactivator, composed of the tetR fused ito the activating domain of VP16 transcriptional factor, allows tet-dependent transcription. Accordingly, tetracycline derivatives such as anhydrotetracycline, which we found to be well tolerated by T.gondii, can serve as effector molecules, allowing control of gene expression in a reversible manner. As a prerequisite to functionally express the tetR in T.gondii, we used a synthetic gene with change of codon frequency. Whereas no activation of transcription was achieved using the synthetic tetracycline-controlled transactivator, tTA2(s), the TetR(s) modulates parasite transcription over a range of similar to 15-fold as measured for several reporter genes. We show here that the tetR-dependent induction of the T.gondii myosin A transgene expression drastically down-regulates the level of endogenous MyoA. This myosin is under the control of a tight feedback mechanism, which occurs at the protein level.