The cysteine protease dipeptidyl aminopeptidase 3 does not contribute to egress of Plasmodium falciparum from host red blood cells

The ability of Plasmodium parasites to egress from their host red blood cell is critical for the amplification of these parasites in the blood. Previous forward chemical genetic approaches have implicated the subtilisin-like protease (SUB1) and the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) as key players in egress, with the final step of SUB1 maturation thought to be due to the activity of DPAP3. In this study, we have utilized a reverse genetics approach to engineer transgenic Plasmodium falciparum parasites in which dpap3 expression can be conditionally regulated using the glmS ribozyme based RNA-degrading system. We show that DPAP3, which is expressed in schizont stages and merozoites and localizes to organelles distinct from the micronemes, rhoptries and dense granules, is not required for the trafficking of apical proteins or processing of SUB1 substrates, nor for parasite maturation and egress from red blood cells. Thus, our findings argue against a role for DPAP3 in parasite egress and indicate that the phenotypes observed with DPAP3 inhibitors are due to off-target effects.疟原虫（Plasmodium）从宿主红细胞中逸出的能力，对于其在血液中的增殖至关重要。既往正向化学遗传学研究已将类枯草杆菌蛋白酶（SUB1）与半胱氨酸蛋白酶二肽基肽酶3（DPAP3）确定为疟原虫逸出过程的关键调控因子，学界普遍认为SUB1的成熟终末步骤依赖于DPAP3的催化活性。本研究采用反向遗传学策略，构建了可通过基于glmS核酶（glmS ribozyme）的RNA降解系统条件性调控dpap3基因表达的转基因恶性疟原虫（Plasmodium falciparum）。研究结果显示，DPAP3在裂殖体阶段与裂殖子中表达，定位于与微线体、棒状体及致密颗粒不同的细胞器中；其既不参与顶膜蛋白的转运过程，也不参与SUB1底物的加工，同时对疟原虫的成熟以及从红细胞中逸出并非必需。综上，本研究的发现不支持DPAP3在疟原虫逸出过程中发挥功能，并表明此前观测到的DPAP3抑制剂相关表型源于脱靶效应。