A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii

Abstract

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. N early one-third of all deaths in the world today are caused by infectious disease. The development of new preventative and therapeutic strategies relies on an improved understanding of the interaction between pathogens and their hosts. In many pathogenic systems, this presents a formidable experimental challenge, because standard genetic tools are either rudimentary or unavailable. Biochemical, genomic, and other approaches exist, but these lack the assumption-free power of a genetic screen.An alternative nongenetic approach to studying mechanisms of host-pathogen interaction involves screening large structurally diverse collections of small molecules for those that disrupt the interaction. Once identified, the small molecules (or their derivatives) are used to determine the cellular components that function in the process (reviewed in refs. 1-3). The approach relies on the demonstrated ability of many small molecules to interact specifically with their targets (e.g., ref. 4). As with classical forward genetic screens, the approach is assumptionfree: by sampling large unbiased collections of structurally diverse small molecules, the screen ''selects'' structures that perturb the process under study. Such phenotype-based highthroughput screening has recently gained momentum in the academic setting due to technological advances and the av...

Molecular and functional aspects of parasite invasion

“…A recent high-throughput screen of small molecules that inhibit host cell invasion by T. gondii led to the identification of novel inhibitors that perturb different aspects of invasion, including gliding motility, micronemal secretion and conoid extension. Intriguingly, several of these inhibitors are also effective against Plasmodium invasion, illustrating the intimate functional similarities between the processes used by both parasites [28].…”

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

“…Gliding motility was visualized at 37°C on a Nikon TE300 inverted microscope with Nomarski optics (Melville, NY). Video data were collected with a VE1000SIT camera (Dage-MTI, Michigan City, IN).Conoid extension was assayed as previously described (Mondragon and Frixione, 1996;Carey et al, 2004b). At least 200 parasites from each parasite line (grown with or without Atc) were counted blindly, in each of two independent experiments.…”

“…Only the relevant (20 -25 kDa) portion of the blot is shown. Inhibitor 7 significantly blocks secretion, as previously described (Carey et al, 2004b) and was included as a control. The lanes shown were from the same blot and were exposed and adjusted for contrast and brightness identically.…”

The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa

“…Indeed, a recent in vivo screening based on a high-throughput invasion assay has identified a collection of small molecules that show noncytotoxic effects on T. gondii invasion [31]. These inhibitors of invasion have been dissected for their effect on motility, conoid extrusion, constitutive and induced microneme secretion, and the results have revealed surprising new findings.…”