As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, onToxoplasma gondiiare now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Δku80strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Δku80strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Δku80strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novelT. gondiigenes.

Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca²⁺ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca²⁺ ionophores in both the presence and the absence of external Ca²⁺, while chelation of intracellular Ca²⁺ prevented release. Mobilization of intracellular calcium with thapsagargin or NH₄Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca²⁺ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore‐stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin–myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus‐coupled secretion system responsible for releasing adhesins involved in cell attachment.

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here...

Perforin-like proteins are expressed by many bacterial and protozoan pathogens, yet little is known about their function or mode of action. Here, we describe Toxoplasma perforin-like protein 1 (TgPLP1), a secreted perforin-like protein of the intracellular protozoan pathogen Toxoplasma gondii that displays structural features necessary for pore formation. After intracellular growth, TgPLP1-deficient parasites failed to exit normally, resulting in entrapment within host cells. We show that this defect is due to an inability to rapidly permeabilize the parasitophorous vacuole membrane and host plasma membrane during exit. TgPLP1 ablation had little effect on growth in culture but resulted in a reduction greater than five orders of magnitude of acute virulence in mice. Perforin-like proteins from other intracellular pathogens may play a similar role in microbial egress and virulence.

Vertebrate cells are highly susceptible to infection by obligate intracellular parasites such as Toxoplasma gondii , yet the mechanism by which these microbes breach the confines of their target cell is poorly understood. While it is thought that Toxoplasma actively invades by secreting adhesive proteins from internal organelles called micronemes, no genetic evidence is available to support this contention. Here, we report successful disruption of M2AP, a microneme protein tightly associated with an adhesive protein called MIC2. M2AP knockout parasites were >80% impaired in host cell entry. This invasion defect was likely due to defective expression of MIC2, which partially accumulated in the parasite endoplasmic reticulum and Golgi. M2AP knockout parasites were also unable to rapidly secrete MIC2, an event that normally accompanies parasite attachment to a target cell. These findings indicate a critical role for the MIC2–M2AP protein complex in parasite invasion.