The parasite Plasmodium falciparum is the causative agent of the most severe form of human malaria. During its intraerythocytic life cycle, P. falciparum transports red blood cell contents to its acidic organelle, known as the food vacuole, where a series of proteases degrade a majority of the host hemoglobin. Two metalloaminopeptidases, PfAPP and PfA-M1, have been previously localized to the food vacuole (in addition to distinct secondary locations for each), implicating them in the final stages of hemoglobin catabolism. Prior genetic work has determined these enzymes are necessary for efficient parasite proliferation, highlighting them as potential anti-malarial drug targets. This study presents the biochemical basis for the catalytic roles of these two enzymes in the hemoglobin degradation pathway. PfAPP, an aminopeptidase P homolog, is specific for hydrolyzing the N-termini of peptides containing penultimate prolines. PfA-M1 is a member of the expansive M1 family of proteases and exhibits a broad specificity towards substrates. The two enzymes are ubiquitous, found in organisms across all kingdoms of life. Their presence in an acidic environment is unique for aminopeptidase P proteins and rare for M1 homologs. Our immunolocalization results have confirmed the dual distribution of these two enzymes in the parasite. Vacuolar targeting was found to be associated with the Plasmodium specific N-terminal extension found in the PfA-M1 sequence by yellow fluorescent protein fusion studies. Kinetic analysis of recombinant forms of PfAPP and PfA-M1 revealed both enzymes are stable and catalytically efficient in the substrate rich, acidic environment of the parasite food vacuole. In addition, mutagenic exploration of the PfA-M1 active site has determined a residue important in dictating substrate specificity among homologs of the same family. These results provide insight into the parasite's functional recruitment of these enzymes to deal with the final stages of hemoglobin catabolism and necessary considerations for inhibitor design. / Ph. D.

The parasite Plasmodium falciparum causes the most virulent form of human malaria and is responsible for the vast majority of malaria-related deaths. During the asexual intraerythrocytic stage, the parasite must transport newly synthesized proteins and endocytosed cargo to a variety of organelles, many of which are formed de novo and have no human equivalent. This process in mammalian cells would utilize an endosomal protein trafficking system, but no endosomal structures or proteins have been described in the parasite. Prior work on the parasite genome indicated that several proteins, which could potentially coordinate an endosomal network, were encoded in the genome and expressed during the asexual parasite stages. In this study, we have localized and attempted to further characterize these proteins in the context of the endosomal system. Two well-conserved protein components of the late endosome, the retromer cargo-selective complex and Rab7, were found on a previously un-described inherited structure adjacent to the parasite Golgi apparatus and in close opposition to nascent rhoptries (specialized secretory organelles required for invasion). The retromer cargo-selective complex was also in close proximity to its putative cargo, a P. falciparum homolog of the sortilin family of protein sorting receptors, PfSortilin. Another protein, PfFCP, the sole FYVE domain-containing protein in the P. falciparum genome, was localized to the membrane of a specialized acidic organelle, known as the food vacuole, where the parasite catabolizes the majority of its host cell hemoglobin. We analyzed the effects of a PfFCP dominant negative mutant and found that it altered food vacuole morphology and trafficking. A previous report localized the early endosome phosphoinositide, phosphatidylinositol 3-phosphate, to the food vacuole membrane, and in conjunction with our studies on PfFCP, this has raised doubts about the food vacuole as a lysosome equivalent in the parasite. The combination of both early and late endosome protein homologs in the parasite, and their potential function, has led to a new model of protein trafficking within the parasite that includes the food vacuole as a terminal early endosome and the apical organelles as lysosome equivalents. / Ph. D.