N-linked glycosylation of proteins in the protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite of animal cells. Infection of humans is common and may result in devastating disease, especially in immunocompromised individuals. Despite previous reports that N-glycosylation of proteins may be a rare post-translational modification in this and related organisms, we demonstrate that it is actually quite prevalent in Toxoplasma. N-Glycosylation is completely inhibited by treatment of parasites with tunicamycin, but this does not appear to exert its major effect on the parasites until they have egressed from their host cells. Although the tunicamycin-treated parasites appear structurally normal at this time they are not motile and mostly incapable of invading new host cells. The few tunicamycin-treated parasites that do invade are severely affected in their ability to replicate and accumulate with a distended endoplasmic reticulum, deformed nuclei, and without recognizable late secretory organelles. We provide experimental evidence that indicate that Toxoplasma N-glycans differ structurally from those in other eukaryotes.

Immobilization of the type XIV myosin complex in Toxoplasma gondii.

The substrate-dependent movement of apicomplexan parasites such as Toxoplasma gondii and Plasmodium sp. is driven by the interaction of a type XIV myosin with F-actin. A complex containing the myosin-A heavy chain, a myosin light chain, and the accessory protein GAP45 is attached to the membranes of the inner membrane complex (IMC) through its tight interaction with the integral membrane glycoprotein GAP50. For the interaction of this complex with F-actin to result in net parasite movement, it is necessary that the myosin be immobilized with respect to the parasite and the actin with respect to the substrate the parasite is moving on. We report here that the myosin motor complex of Toxoplasma is firmly immobilized in the plane of the IMC. This does not seem to be accomplished by direct interactions with cytoskeletal elements. Immobilization of the motor complex, however, does seem to require cholesterol. Both the motor complex and the cholesterol are found in detergent-resistant membrane domains that encompass a large fraction of the inner membrane complex surface. The observation that the myosin XIV motor complex of Toxoplasma is immobilized within this cholesterol-rich membrane likely extends to closely related pathogens such as Plasmodium and possibly to other eukaryotes.