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.

Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting Plasmodium -secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple Plasmodium species to interrogate the trafficking, topology, and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules, and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal that vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a unique tool for dissecting secreted protein function with subcompartmental resolution that should be widely amenable to genetically tractable eukaryotes.

Obligate intracellular malaria parasites depend on protein secretion to penetrate, subvert, and exit their host cells. Despite this key reliance on the secretory pathway, none of the conditional mutagenesis tools available in Plasmodium spp. exploit the unique trafficking features of secreted proteins. Here we report knockER, a novel DiCre-mediated knock sideways approach that sequesters secreted proteins in the ER via conditional fusion of a KDEL ER-retrieval sequence to the target protein C-terminus. We applied knockER to a diverse set of reporters and endogenous P. falciparum and P. berghei proteins targeted to the rhoptries, dense granules, parasite vacuole and apicoplast and show conditional ER sequestration is not generally toxic, enabling loss-of-function studies. Taking advantage of the unique ability to redistribute secreted protein pools from their terminal destination to the ER, we employed knockER to study components of the PTEX translocon in an attempt to separate HSP101 vacuolar function in protein export from a recently proposed role in cargo recognition at the ER. Strikingly, while KDEL-fusion produced similar levels of ER retrieval for both HSP101 and the translocon adaptor PTEX150, parasite growth and effector export were completely unaffected by HSP101 retention while ER retrieval of PTEX150 produced a lethal export defect, indicating vacuolar HSP101 levels are uniquely maintained in excess. Intriguingly, redistribution of HSP101 to the ER did not further sensitize parasites to TetR-DOZI-mediated knockdown compared to parasites containing normal vacuolar levels of HSP101, suggesting PV-localized function does not fully account for HSP101 contribution to parasite fitness and consistent with an important role for HSP101 in the ER. Collectively, our work provides a novel tool for dissecting secreted protein function with sub-compartmental resolution that should be widely amenable to genetically tractable eukaryotes.

No abstract

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 transcend 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 indicated 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′-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. Post-invasion 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 with merosome detachment that 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 EXP2 is important for intrahepatic parasite development, indicating PTEX components also function at the hepatocyte-parasite interface.