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Abstract
In eukaryotes, membrane contact sites (MCS) are defined as specialized regions between two organelles in close apposition to each other. These regions enable inter-organellar communication by creating microdomains maintained via protein complexes. Toxoplasma gondii (T. gondii), an obligate intracellular protozoan parasite capable of infecting virtually any nucleated cell, is the causative agent of one of the most prevalent parasitic infections in the world, disseminated toxoplasmosis. Disseminated toxoplasmosis is clinically relevant for the mothers of unborn fetuses and immunocompromised individuals. T. gondii goes through a complex life cycle that involves intracellular and extracellular forms and numerous hosts. The parasite harbors two essential organelles: the apicoplast, a non-photosynthetic plastid conserved in most apicomplexans, and the mitochondrion. The two organelles have been observed in close proximity for many years and share essential metabolic pathways. Despite their close apposition and their metabolic exchanges, little is known about the nature of their interaction. We hypothesize that apicomplexan-specific MCS proteins could mediate the interaction between the apicoplast and the mitochondrion of T. gondii. To identify those interactors, we are creating a reporter system using Split-GFP, which consists of two portions of a green fluorescent protein (GFP) that do not fluoresce on their own but can self-complement and fluoresce when they interact. This Split-GFP system has been used as a fluorescent tag to study protein-protein interactions in other organisms. First, as a proof of concept, we are generating T. gondii strains with each component of the Split-GFP system expressed in different subcellular compartments. Once established, we will then use the Split-GFP components as a reporter system in a genetic screen to identify MCS components between the apicoplast and mitochondrion of T. gondii. Understanding apicomplexan-specific MCS will shed light on inter-organelle communication in these organisms and on essential proteins that may be targeted in the on-going search to find new therapeutic drug targets against T. gondii infections.