The PTEX Pore Component EXP2 Is Important for Intrahepatic Development during the Plasmodium Liver Stage

During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole, which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To traverse this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood-stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are suggested by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver-stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3' untranslated region (UTR) enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Postinvasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid- to late-liver-stage marker LISP2. As an orthogonal approach to monitor development, EXP2-glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding to merosome detachment, which marks the culmination of liver-stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal that EXP2 is important for intrahepatic parasite development, indicating that PTEX components also function at the hepatocyte-parasite interface. IMPORTANCE After the mosquito bite that initiates a Plasmodium infection, parasites first travel to the liver and develop in hepatocytes. This liver stage is asymptomatic but necessary for the parasite to transition to the merozoite form, which infects red blood cells and causes malaria. To take over their host cells, avoid immune defenses, and fuel their growth, these obligately intracellular parasites must import nutrients and export effector proteins across a vacuole membrane in which they reside. In the blood stage, these processes depend on a translocon called PTEX, but it is unclear if PTEX also functions during the liver stage. Here, we adapted the glmS ribozyme to control expression of EXP2, the membrane pore component of PTEX, during the liver stage of the rodent malaria parasite Plasmodium berghei. Our results show that EXP2 is important for intracellular development in the hepatocyte, revealing that PTEX components are also functionally important during liver-stage infection.