Two putative protein export regulators promote Plasmodium blood stage development in vivo

Abstract

Protein export is considered an essential feature of malaria parasite blood stage development. Here, we examined five components of the candidate Plasmodium translocon of exported proteins (PTEX), a complex thought to mediate protein export across the parasitophorous vacuole membrane into the host cell. Using the murine malaria model parasite Plasmodium berghei, we succeeded in generating parasite lines lacking PTEX88 and thioredoxin 2 (TRX2). Repeated attempts to delete the remaining three translocon components failed, suggesting essential functions for EXP2, PTEX150, and heat shock protein 101 (HSP101) during blood stage development. To analyze blood infections of the null-mutants, we established a flow cytometry-assisted intravital competition assay using three novel high fluorescent lines (Bergreen, Beryellow, and Berred). Although blood stage development of parasites lacking TRX2 was affected, the deficit was much more striking in PTEX88 null-mutants. The multiplication rate of PTEX88-deficient parasites was strongly reduced resulting in out-competition by wild-type parasites. Endogenous tagging revealed that TRX2::tag resides in distinct punctate organelles of unknown identity. PTEX88::tag shows a diffuse intraparasitic pattern in blood stage parasites. In trophozoites, PTEX88::tag also localized to previously unrecognized extensions reaching from the parasite surface into the erythrocyte cytoplasm. Together, our results indicate auxiliary roles for TRX2 and PTEX88 and central roles for EXP2, PTEX150, and HSP101 during P. berghei blood infection.

Keywords: 3′; 3′ untranslated region; 5′; 5′ untranslated region; ER; Experimental genetics; Flow cytometry; GFP; HSP; Malaria; PEXEL; PEXEL/VTS negative exported proteins; PI3P; PMR; PNEPs; PTEX; PTEX translocon; PV; PVM; PfEMP1; Plasmodium berghei; Plasmodium berghei adaptable transfection plasmid; Plasmodium export element; Plasmodium falciparum erythrocyte membrane protein 1; Plasmodium translocon of exported proteins; Protein export; SIL6; TRX2; VTS; WT; YFP; endoplasmic reticulum; green fluorescent protein; heat shock protein; pBAT; parasite multiplication rate; parasitophorous vacuole; parasitophorous vacuole membrane; phosphatidylinositol 3-phosphate; silent intergenic locus on Plasmodium berghei chromosome 6; thioredoxin 2; vacuolar transport signal; wild type; yellow fluorescent protein.