Novel insights into the digestive vacuole biology of the malarial parasite Plasmodium falciparum

Abstract

Plasmodium falciparum is a protozoan parasite and the causative agent of the deadliest form of human malaria. The malarial parasite undergoes drastic development changes within erythrocytes and modifies them to acquire nutrients and evade host defenses. While in erythrocytes, the parasite degrades vast quantities of hemoglobin inside its digestive vacuole (DV). Four aspartic proteases, termed plasmepsins (PM) were localized to the DV and shown to degrade hemoglobin, an essential process for parasite development. This has prompted multiple investigations to identify DV PM inhibitors as candidate antimalarials. To test their essentiality we genetically disrupted the four PM genes, and found that none was essential. Nonetheless, each individual PM knockout clone presented subtle growth or morphological defects, most notably with PM IV. These results suggest that P. falciparum may require multiple PM genes in order to achieve optimal growth rates. To investigate these phenotypes further, we tested the PM knockout clones against a panel of protease inhibitors, which included aspartic protease inhibitors designed against the crystal structure of PM II. We found that PMs are not completely overlapping in function and that absence of PM III and IV demonstrated the most dramatic defects in vitro against the inhibitors tested. Our data revealed that the DV PMs are not the primary target of any of the aspartic protease inhibitors tested.;To date there are only thirteen known DV proteins and we predict that many of the biological processes in the DV remain undiscovered. In order to identify new DV proteins we developed novel computational and experimental approaches. First, we mined the P. falciparum genome database using criteria common to the known DV proteins (timing of expression, a transmembrane domain, and the presence or absence of a signal peptide). This led us to identify approximately 250 potential candidates including a new family of predicted monoglyceridelipases that target to the DV and that might help in the degradation of parasitophorous vacuolar membranes within the DV. Additionally, we developed an approach to investigate DV targeting as an alternative strategy to aid in the discovery of new DV proteins. We tested serial truncations of several DV proteins to decipher the minimal length of sequences required for proper targeting. These minimal amino acid sequences were then blasted using the MEME search engine to test for a common motif. A highly conserved motif, LXXKE, was identified and validated as a targeting motif through site-directed mutagenesis and deletions. This motif was BLAST against PlasmoDB and generated a list of 91 potential DV proteins. This enabled us to identify 2 new proteins (falcipain 1 and an amino acid transporter) that target to the DV. These findings have advanced our understanding of protein trafficking to the DV and have led to the identification of novel DV proteins. Further work on these proteins should provide new insights into the biology of the DV, a unique target for therapeutic intervention.