|Abstract:||Toxoplasma gondii (T. gondii) is an obligate intracellular apicomplexan protozoan parasite that infects about one-third of the world population, with estimated medical costs and loss of productivity in the USA alone amounting to about US$3.1 billion annually. Additionally, in the USA, T. gondii is the second leading cause of death due to food-borne pathogens. Current drugs against T. gondii are limited by hypersensitivity and toxicity and are ineffective against the encysted stage of the parasite. Coupled with the lack of T. gondii vaccine for humans, there is an urgent need to develop safe and effective therapeutic and prophylactic agents against T. gondii. Rats, like immunocompetent humans, develop a chronic infection, but vary in their susceptibilities to T. gondii infection depending on the rat strain. Compared to the T. gondii-susceptible Brown Norway (BN) rat, the Lewis (LEW) rat is extremely resistant to T. gondii: few to no parasites are found post-infection, no antibody to T. gondii is produced, and there is no congenital transmission. Importantly, the LEW rat inhibits proliferation of T. gondii within parasitophorous vacuoles (PV) in peritoneal macrophages, a phenomenon linked to rapid death of both parasites and infected host cells. The resemblance in progression of toxoplasmosis between rats and humans, and the refractoriness of the LEW rat to T. gondii infection when compared to the susceptible BN rat, provide an opportunity to use the two rat strains as models for deciphering host molecular mechanisms that mediate resistance to T. gondii. This can potentially unveil new strategies for developing effective therapies and vaccines against T. gondii infection. It is evident that the robust resistance of the LEW rat to T. gondii involves undefined mechanisms that rapidly kill the parasites within the parasitophorous vacuole very early in infection, irrespective of parasite strain, and independent of IFN-γ activation. Thus, to unravel molecular factors directing the resistance of the LEW rat to T. gondii, we performed RNA sequencing analysis of peritoneal cells extracted from the LEW and BN rats, with or without T. gondii Type I RH strain infection. We found that, compared to both the infected and uninfected BN rats, the infected and uninfected LEW rats had inherently higher transcript levels of several cytochrome enzymes. On the other hand, compared to the T. gondii-infected BN rat, the infected LEW rat had lower transcript levels for several antioxidant enzymes. When we measured levels of reactive oxygen species (ROS), we found that both infected and uninfected LEW rat peritoneal cells contained significantly higher ROS levels than the BN rat’s cells, consistent with the higher transcript levels of cytochrome enzymes (that catalyze generation of ROS), with concomitant lower transcript levels of antioxidant enzymes (that detoxify ROS). Intriguingly, we observed that scavenging of ROS from infected LEW rats’ peritoneal cells reduced the cells’ refractoriness to T. gondii, suggesting that the LEW rat maintains inherent oxidative stress that contributes to rapid killing of invading T. gondii. Additionally, we found that, compared to the T. gondii-infected BN rats, the infected LEW rats’ peritoneal cells had higher transcript levels for several GTPase of the Immunity-Associated Proteins (GIMAPs). GIMAPs are relatively recently described novel protein family of putative small GTPases that are conserved and expressed prominently in mammalian (including human) immune cells. Their functions and molecular mechanisms in mammalian host innate immunity against intracellular pathogens are yet to be deciphered. By bioinformatic analyses, we found that all those GIMAPs, contain a GTP-binding site motif A (P-loop) that is required for oligomerization of effector molecules to membranous structures. In addition, one of the GIMAPs contained an N-myristoylation motif that is important for protein–protein/lipid interaction and plays an essential role in membrane targeting. Importantly, we found that all those GIMAPs possess an LC3-interacting region (LIR) that is important for recruitment of molecules to phagosomal membranes. To elucidate the roles of GIMAPs during T. gondii infection, we engineered LEW rat-derived GIMAPs for stable inducible expression in a rat macrophage cell line, NR8383 (derived from a T. gondii-susceptible Sprague Dawley rat). We observed that over-expressed GIMAPs localized to the T. gondii parasitophorous vacuole membrane (PVM), and induced translocation of lysosomes to the PVM, with concomitant inhibition of T. gondii growth in the NR8383 cells, suggesting that GIMAPs contribute to the robust refractoriness of the LEW rat to T. gondiii infection. To determine the molecular networks of GIMAPs in inhibiting T. gondii growth, we engineered NR8383 cells for expression of either HA- or FLAG-tagged GIMAPs. Using the tagged GIMAPs as baits we performed immunoprecipitation assays coupled with mass-spectrometry analyses. Together, our findings have unveiled novel protective innate immune responses to T. gondii infection that will be crucially important in developing strategies for designing new effective therapies and vaccines against T. gondii infection in humans.