Assessing the function of the aspartic proteinases of the Plasmodium falciparum digestive vacuole using gene-knockout strategies

During the intra-erythrocytic stage of infection by Plasmodium falciparum (the causative agent of malaria) the parasite consumes 70 to 80% of the host cell's hemoglobin by proteolytic digestion with enzymes located in the digestive vacuole (DV). Four DV aspartic proteinases (plasmepsins) in P. falciparum (PfPM1, PfPM2, PfHAP and PfPM4) were targeted for study using gene-knockout approaches. We tested the hypothesis that despite functional redundancy among the DV plasmepsins, at least one DV plasmepsin is necessary for the hemoglobin digestion process. Further, we postulate that PfPM4 (in the absence of PfPM1, PfPM2, and PfHAP) is the plasmepsin most likely to be capable of maintaining fitness of P. falciparum sufficient for survival during the asexual stage growth.;Creating a triple-plasmepsin knockout (with only PfPM4 remaining) established that PfPM4 alone is sufficient to fulfill any function of this set of enzymes required to maintain the normal rate of asexual growth of the parasite. However, successful creation of a quadruple-plasmepsin knockout lacking PfPM1, PfPM2, PfHAP, and PfPM4 proved that none of the DV plasmepsins are essential for asexual-stage growth. The slower growth phenotype and the reduced rate of hemozoin formation of the quadruple-plasmepsin knockout established that the function of the plasmepsins was required for efficient growth in vitro .;The quadruple-plasmepsin knockout showed an increase in susceptibility to cysteine protease inhibitors E-64 and leupeptin, suggestive of an increased dependence on the falcipain class of enzymes. Despite several reports suggesting that one or more of the DV plasmepsins is the target of HIV proteinase inhibitors, our study showed that the target of inhibition of these clinically important compounds is not found among the four DV plasmepsins. The digestive vacuole of the quadruple-plasmepsin knockout contained membranous whorls characteristic of the insufficient breakdown of autophagic vacuoles within a lysosome. Growth of the quadruple-plasmepsin knockout in amino acid-limited medium was severely affected; suggesting lack of the DV plasmepsins increases the susceptibility to starvation-induced autophagy. The DV plasmepsins play a role in the hemoglobin digestion pathway and are involved in the endosome/autophagosome processing of the digestive vacuole.