Discovery of a novel T. gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates

摘要

T. gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors including nitric oxide. Mechanisms used by of T. gondii to modulate activation of its infected host cell likely underlies its ability to hijack monocytes and dendritic cells (DC) during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naïve macrophages. We identified fourteen independent T. gondii insertional mutants out of over 8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune (CIM) mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild type parasites recapitulated the RNI-resistance defect in the CIM mutant confirming the role of the protein in resistance to macrophage RNI.