Rompikuntal, P. K. | Search bioRxiv

bioRxiv2022

Identification of KNX-002, a novel inhibitor of Toxoplasma motility targeting the Class XIV myosin, TgMyoA

Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to test whether pharmacological inhibition of TgMyoA can alter disease progression in an animal model of infection. To this end, we sought to identify small molecule inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motors actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, KNX-002 was shown to slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. These data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target for toxoplasmosis. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.

microbiology↗

bioRxiv2020

Blocking palmitoylation of Toxoplasma gondii myosin light chain 1 disrupts glideosome composition but has little impact on parasite motility

Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin-based motor consisting of a Class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. The binding of TgMLC1 to TgGAP45 is therefore considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation disrupt the association of TgMLC1 with the membrane fraction of the parasite in phase partitioning experiments and completely block the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete. ImportanceGliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention and treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely-held "linear motor" model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of and between cells of the hosts they infect.

microbiology↗