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Obligate intracellular apicomplexan parasites rely on gliding motion powered by their actomyosin system to disperse throughout tissues and to penetrate host cells. Toxoplasma gondii myosin A has been implicated in this process, but direct proof has been lacking. We designed a genetic screen to generate a tetracycline-inducible transactivator system in T. gondii. The MyoA gene was disrupted in the presence of a second regulatable copy of MyoA. Conditional removal of this myosin caused severe impairment in host cell invasion and parasite spreading in cultured cells, and unambiguously established the pathogenic function of this motor in an animal model.
Among the many vital functions of an obligate intracellular parasite, host cell invasion is a prerequisite for survival and replication, and this process is dependent on the ability of T. gondii to glide (1). Gliding motility requires an intact actin cytoskeleton (2) and is likely to be powered by the small unconventional myosin A (TgMyoA or simply MyoA) (3, 4). This motor is found right beneath the plasma membrane and exhibits the transient adenosine triphosphatase kinetics and biophysical properties necessary to generate fast movement (5). Additionally, this small myosin of the class XIV and its associated myosin light chain are extremely conserved throughout the Apicomplexa (57. To date, all attempts to disrupt the MyoA gene have failed. Thus, we tried to establish a system for conditional gene knockout to study this gene in vivo.
An inducible system based on the tetracycline repressor (TetR) has been reported for the control of gene expression in several protozoan parasites and is best optimized in Trypanosoma brucei (6). Indeed, existence of trans-splicing in kinetoplastida offers a unique opportunity to combine the tetracycline-dependent repression with the T7 polymerase transcription. However, the more potent and broadly used tet-transactivator system (tTA composed of TetR-VP16 fusion) (7) has not been used in parasites. The TetR can control gene expression in T. gondii (8) but the tTA system is totally inactive. The repression system is suitable for expression of toxic genes and of dominant-negative mutants but is inappropriate for the generation of conditional knockouts. Here, random integration was used to trap a transactivating domain functioning as tet-dependent transactivator when fused to TetR. The plasmid used for random insertion contained the dehydrofolate reductase-thymidylate synthase...