Abstract
Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite’s life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by “click” chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite’s myosin motor.
Highlights
Parasites of the phylum Apicomplexa are responsible for an enormous amount of morbidity and mortality worldwide; members include Plasmodium spp., which cause malaria, and Toxoplasma gondii, which infects approximately one-third of the world’s population and can cause life-threatening disease in the developing fetus and immunocompromised individuals
To generate sufficient quantities of the different electrophoretic forms of TgMLC1 for biochemical/proteomic analysis, we tested whether recombinant FLAG-tagged TgMLC1 expressed in insect cells was sensitive to compound treatment
TachypleginA and its analogues were recently identified as inhibitors of T. gondii invasion and motility [16,17], and we show here that TgMLC1 is one of the biologically relevant targets of these compounds using a combination of ‘‘click’’ chemistry, mass spectrometry and mutational analysis
Summary
Parasites of the phylum Apicomplexa are responsible for an enormous amount of morbidity and mortality worldwide; members include Plasmodium spp., which cause malaria, and Toxoplasma gondii, which infects approximately one-third of the world’s population and can cause life-threatening disease in the developing fetus and immunocompromised individuals. A functional myosin motor complex is important for efficient invasion and egress from infected host cells, as well as for parasitedriven dissemination throughout the body [1] Components of this motor complex include the unconventional class XIV myosin TgMyoA, its associated regulatory light chain, TgMLC1 [2], an essential light chain (TgELC1) [3], and gliding-associated proteins TgGAP40, TgGAP45, TgGAP50 and TgGAP70 [4,5]. These proteins are localized to the space between the parasite plasma membrane and the flattened vesicles of the inner membrane complex (IMC) [6,7,8]. While many proteins that interact directly and indirectly with TgMyoA have been identified and their physical interactions characterized, how the motor complexes are spatially organized within the parasite and how the components coordinate to produce translational motion are not well understood
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