Genetic deletion of an essential lipid storage enzyme attenuates the virulence of Toxoplasma gondii

The ability of Toxoplasma gondii to infect a wide range of hosts is greatly influenced by its capability to salvage essential nutrients from the host cell and its environment. Toxoplasma relies on lipids for its critical functions, acquiring fatty acids from the host cell, in addition to de novo synthesis, to support its growth and ensure survival. Fatty acids are important components for Toxoplasma's membrane biogenesis, energy generation, and other metabolic processes. However, excessive free fatty acids (FFA) are toxic to the parasite. To evade lipotoxicity, Toxoplasma expresses lipid-esterifying enzymes to divert surplus FFA into major storage neutral lipids, such as triacylglycerol (TAG) and cholesteryl ester (CE), stored within intracellular lipid droplets (LD). One such enzyme, the acyl-CoA:diacylglycerol acyltransferase (TgDGAT1) catalyzes the final step in the acyl-CoA-dependent synthesis of TAG. TgDGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) superfamily, shares structural and functional features with enzymes in the DGAT1 family. Interestingly, the DGAT1 inhibitor T863 arrests the growth and multiplication of Toxoplasma in cultured mammalian cells, suggesting an important role for this enzyme in storing energy-rich FAs as TAGs and protecting the parasite from FFA-induced toxicity. However, the direct effect of loss of DGAT1 enzyme activity in the parasite remains unexplored. In this study, we generated a genetically modified Toxoplasma type I strain devoid of the TgDGAT1 gene, rendering it incapable of storing FFAs as TAGs within LDs. Of interest, we note that RH ∆DGAT1 parasites can grow and multiply in vitro under lipid-depleted conditions, showing a moderate growth defect. However, in standard growth media, the mutant's inability to store fatty acids severely impairs its ability to thrive within host mammalian cells. Additionally, EM studies reveal gross morphological abnormalities, with skinny parasites disorganized in the parasitophorous vacuole. We further show that the RH ∆DGAT1 strain is avirulent in mice, even at higher infection doses. Active immunization with these genetically attenuated whole-cell tachyzoites boosts the host immune response, providing protective immunity against subsequent acute lethal challenges by wild-type parasites. Overall, our findings suggest that unraveling the mechanisms of fatty acid storage in Toxoplasma is crucial for developing strategies to disrupt the parasite's survival mechanisms.