Laying the groundwork for protein-protein interaction experiments for Toxoplasma gondii Dihydroorotate Dehydrogenase: preparation and preliminary design

Abstract

Toxoplasma gondii is an intracellular parasite of clinical and veterinary significance, capable of infecting a wide range of hosts. Despite the availability of treatments, challenges such as chronic infection, drug efficiency, and strain-specific drug resistance persist. Targeting nucleotide metabolism, particularly the pyrimidine synthesis pathway, has shown promise in related parasites. Dihydroorotate Dehydrogenase (DHODH), a crucial enzyme involved in T. gondii's pyrimidine metabolism, has been identified as a potential drug target in related parasites, and previous knockout assays in T. gondii revealed that DHODH holds an essential non-pyrimidine-related role that remains uncharacterized. This project aims to perform the first steps to identify protein-protein interactions involving TgDHODH, and the specific domains mediating these interactions through blue native electrophoresis, pull-down assays, and yeast two-hybrid assays. First, the N-terminal domain of TgDHODH (NT-TgDHODH) was successfully cloned into the pET15b expression vector and expressed in E. coli. Purification of the recombinant protein presented challenges due to low protein yield and insolubility. Secondly, BN-PAGE coupled with DHODH activity assays revealed the presence of TgDHODH in two bands migrating at higher molecular weights than expected for TgDHODH alone. These bands were excised and will be subjected to mass spectrometry analysis to identify potential protein partners. Finally, the cloning of TgDHODH and NT-TgDHODH into the pGBKT7 bait plasmid for the yeast two-hybrid assay was successfully accomplished. The vectors containing the bait proteins were stored for future use in subsequent Y2H assays. Although progress has been made in this project, further experiments are required to identify protein partners and characterize the unknown, pyrimidine-independent function of TgDHODH. The purification of NT-TgDHODH, along with pull-down assays and mass spectrometry analysis, will provide valuable insights into the protein-protein interactions of the enzyme. Furthermore, the subsequent yeast two-hybrid assay will confirm the identified interactions. Understanding the function and protein interactions of TgDHODH will enhance our knowledge of the parasite's biology and facilitate the development of new drugs against toxoplasmosis.