Development of new tools to study how T. gondii rhoptry effector proteins are delivered into host cells during invasion

Toxoplasma gondii is a protozoan parasite that infects 30% of the human population and can cause life- threatening disease in immunocompromised individuals and the developing fetus. Like the related apicomplexan parasites that cause malaria and cryptosporidiosis, T. gondii must invade cells of its hosts to propagate and cause disease. During the early stages of invasion, proteins are exocytosed from club-shaped apical organelles known as rhoptries. The necks of the rhoptries are not docked directly to the parasite plasma membrane; rather, they dock to a small apical vesicle (AV) at the extreme apical end of the parasite. Exocytosis of the rhoptry contents therefore involves two membrane fusion events: rhoptry-to-AV, and AV-to- parasite plasma membrane. After being exocytosed, these rhoptry “effector” proteins are translocated across the host cell plasma membrane and into the host cell cytosol where they function in invasion, subversion of the host cell’s innate immune response, and manipulation of host cell gene expression to the parasite’s advantage. Virtually nothing is known about how rhoptry effector proteins are translocated into the host cell during invasion. We have shown that the parasite generates a transient, rhoptry exocytosis-dependent perforation of the host cell plasma membrane early in the invasion process. Electrophysiological characterization of the perforation suggests that it corresponds to the insertion or activation of multiple pore- like structures in the host cell membrane. We hypothesize that this host cell perforation is the pathway through which rhoptry effector proteins are translocated into the host cell during invasion. The preliminary data raise a number of important questions about the mechanism and function of host cell perforation that require new assays to address. The development and first application of these assays is the goal of this developmental project. The Aims are to: (1) Develop an assay to test whether rhoptry effector proteins need to be unfolded in order to be translocated into the host cell cytosol. (2) Develop an assay to monitor host cell perforation, rhoptry effector delivery, and invasion in real time; use this assay to test whether AV exocytosis is sufficient for host cell perforation. Successful completion of these Aims will significantly advance our understanding of how rhoptry effector proteins are delivered into the host cell during invasion and provide new approaches to identification of the perforating agent. A better understanding of the translocation process is not only of fundamental cell biological interest, but may generate a new therapeutic paradigm: rather than targeting a single translocated rhoptry effector protein, targeting the rhoptry protein delivery mechanism will simultaneously disrupt transfer into the host cell of many of the effector proteins that play such a central role in the pathogenesis of the devastating diseases caused by T. gondii and other apicomplexan parasites.