A Small-Molecule Approach to Studying Invasive Mechanisms of Toxoplasma gondii

Toxoplasma gondii is the most common protozoan parasite of humans. Infection with T. gondii can lead to life-threatening disease as a result of repeated cycles of host cell invasion, parasite replication, and host cell lysis. Relatively little is known about the invasive mechanisms of T. gondii and related parasites within the Phylum Apicomplexa (including Plasmodium spp., the causative agents of malaria), due to difficulties associated with studying genes essential to invasion in haploid obligate intracellular organisms. To circumvent this problem, we have developed a highthroughput microscope-based assay, which we have used to screen a collection of 12,160 structurally diverse small molecules for inhibitors of T. gondii invasion. A total of 24 noncytotoxic invasion inhibitors were identified. Secondary assays demonstrated that different inhibitors perturb different aspects of invasion, including gliding motility, secretion of host cell adhesins from apical organelles (the micronemes), and extension of a unique tubulinbased structure at the anterior of the parasite (the conoid). Unexpectedly, the screen also identified six small molecules that dramatically enhance invasion, gliding motility, and microneme secretion. The small molecules identified here reveal a previously unrecognized complexity in the control of parasite motility and microneme secretion, and they constitute a set of useful probes for dissecting the invasive mechanisms of T. gondii and related parasites. Small-molecule-based approaches provide a powerful means to address experimentally challenging problems in host-pathogen interaction, while simultaneously identifying new potential targets for drug development.