Synergy between upregulated small GTPase immunity-associated proteins and lysosomal ATP6V1D in restricting intracellular Toxoplasma growth

Toxoplasma gondii is an obligate intracellular zoonotic protozoan parasite that can cause severe neonatal disease in congenitally acquired infections and severe opportunistic illnesses in immunocompromised individuals. Intracellularly, T. gondii evades host cell defense mechanisms by residing within a specialized non-fusogenic membranous compartment known as the parasitophorous vacuole (PV). Notwithstanding this, we have previously shown that upregulation of small GTPase immunity-associated proteins (GIMAPs) during T. gondii infection promotes fusion of lysosomes with the PV membrane (PVM), resulting in restriction of parasite replication in the T. gondii-refractory Lewis (LEW) rat. Herein, we found that, in addition to GIMAPs, the LEW rat upregulates ATP6V1D, a subunit of the lysosomal vacuolar-type H+-ATPase (v-ATPase) proton pump, in response to T. gondii infection. Disruption of ATP6V1D by CRISPR/Cas9 in a rat macrophage (NR8383) cell line impaired v-ATPase function, leading to defective lysosomal acidification and a concomitant increase in intracellular T. gondii growth. Intriguingly, overexpression of GIMAP 5 or 6 transgene in the ∆ATP6V1D mutant cell line reduced parasite growth by 2.5- and 5-fold, respectively. Comparatively, overexpression of GIMAP 5 or 6 transgene in the wild-type NR8383 cell line had at least threefold more robust effect of reducing parasite growth than in the ∆ATP6V1D mutant cell. Together, our findings imply that while GIMAP-induced activation of lysosome translocation to the PVM is important for restricting parasite growth, concomitant upregulation of the ATP6V1D plays an additively significant role through enhanced acidification of lysosomes to activate hydrolytic enzymes that degrade the parasites following PVM fusion.IMPORTANCEToxoplasma gondii is a highly prevalent parasite that causes severe illnesses in congenitally infected neonates and in immunocompromised individuals. No completely effective drugs nor vaccines exist against T. gondii. Thus, understanding innate mechanisms that resistant hosts employ to orchestrate defenses against the parasite could unveil new strategies for control of toxoplasmosis. Herein, we analyzed transcriptomic data of the T. gondii-resistant rat (Lewis) and identified small GTPase immunity-associated proteins (GIMAPs), and lysosome-associated proteins (including ATP6V1D) that are upregulated in response to infection. We investigated the interplay between those proteins and found that upregulated expression of GIMAPs drives lysosomes to fuse with the parasite vacuole, while upregulated ATP6V1D facilitates lysosomal acidification, which is important for activation of degradative lysosomal enzymes. Those events culminated in the restriction of intracellular T. gondii growth. Collectively, our findings indicate that upregulation of GIMAPs and ATP6V1D orchestrates synergistic mechanisms that contribute to inhibition of intracellular T. gondii growth.