PROJECT SUMMARYThe Apicomplexa are a large phylum of intracellular pathogens that cause substantial disease in humans andanimals worldwide. Their ability to infect their hosts, survive in their intracellular niche, and cause disease isstrictly dependent on a series of unique organelles that are common to this group of pathogens. As most of theprotein constituents of these organelles are not present in their human hosts, they represent intense areas ofinvestigation in the search for new drug targets. Toxoplasma gondii has served as a model system for thestudy of apicomplexan parasites due of its ease of genetic manipulation and extensive set of molecular toolsthat have been developed for its study. While these molecular tools have focused on dissecting function at theindividual gene level, the next big advances will require new approaches that are better able to evaluateprecisely how proteins interact in complexes and how they participate in networks within the parasite to enableinfections. To develop new tools that function at the protein level, we have been developing Toxoplasmastrains with an expanded genetic code that can incorporate unnatural amino acids (UAAs) to study protein-protein interactions in vivo. This strategy uses an amber stop codon suppression system that enables strains toincorporate a photoreactive UAA at specific sites into a bait protein, which crosslink to binding partners whenactivated by UV light. We have demonstrated that parasites engineered to express an orthogonal ambersuppressor tRNA and aminoacyl-tRNA synthetase pair efficiently incorporate the photoactivatable UAA p-azidophenylalanine (Azi) into control proteins and have also shown that we can obtain robust photoreactivecrosslinking using this approach. In this application, we will engineer our system to enable incorporation of asecond photoreactive UAA, p-benzoylphenylalanine. We will then utilize these systems to determine theprecise interactions of the TgARO complex, which is critical for the function of the secretory rhoptries, as wellas the interactions of a newly discovered protein in the conoid, which is important for invasion. Thus, thisapplication develops new tools for protein-protein interaction studies in Toxoplasma and applies these tools tothe study of critical protein complexes in the parasite.