Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum

Abstract

During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.With an estimated 2 million deaths annually to its name (1), the human malaria parasite Plasmodium falciparum continues to present an enormous public health challenge. Clinical manifestations of infection appear as the parasite replicates within host erythrocytes. During this replication cycle, the parasite endocytoses and catabolizes large amounts (up to 75%) of host hemoglobin to constituent amino acids (2, 3). This process makes available large quantities of free amino acids for parasite protein synthesis (4) and may also modulate the osmotic environment of the host cell (5) and/or create space inside the red cell for parasite growth (6). Amino acids derived from hemoglobin catabolism are also important for the uptake of isoleucine from the extracellular environment (7). As hemoglobin catabolism is essential for parasite replication and is currently a major focus of anti-malarial drug development efforts, a comprehensive understanding of the biochemistry of this pathway is an urgent priority.Hemoglobin catabolism occurs in an acidic, degradative organelle termed the food vacuole (FV) 2 or digestive vacuole. In the lumen of the FV, endopeptidases of diverse catalytic mechanism and specificity (plasmepsin (PM) I, II, and IV, histo-aspartic protease, falcipain-2, -2Ј, and -3, and falcilysin) contribute to the hydrolysis of the ␣-and ␤-globin chains to oligopeptides (8, 9). These ol...

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

“…Whereas the precise functions of Pf A-M1 and Pf A-M17 are still speculative it is suggested that these enzymes function in the final stages of haemoglobin digestion and degrade the dipeptides produced by the action of various enzymes within the parasite's acidic digestive vacuole (4,5,7,8). This process is essential for the provision of free amino acids for parasite protein synthesis.…”

“…The consensus is that the Pf A-M17 is located within the cytosol of the parasite. However, while we (4) and others (11,12) have also located the Pf A-M1 within the cytosolic compartment by immunocytochemistry, Dalal and Klemba (2007), using immunocytochemistry, light microscopy and YFP-tagging, observed the enzyme in the acidic digestive vacuole and nucleus (8).…”

“…The concerted functions of these enzymes are essential for parasite viability as targeted gene-knock-outs have been unsuccessful (4,7,8). Both enzymes function in the blood stage of the infection and are responsible for regulating the intracellular amino acid pool within malaria cells, most likely by releasing amino acids from host-derived haemoglobin.…”

“…Malaria parasites express two such enzymes, a 122 kDa M1-family alanyl aminopeptidase and a 67.8 kDa M17-family leucyl aminopeptidase, termed Pf A-M1 (MAL13P1.56, plasmodb.org) and Pf A-M17 (Pf14_0439, plasmodb.org), respectively (4)(5)(6)(7)(8).…”

On the location of the aminopeptidase N homolog Pf A-M1 in Plasmodium falciparum

“…1) that has been proposed to play an important role in the parasite's catabolism of host red blood cell hemoglobin in its food vacuole (2). In their report on PfA-M1, McGowan et al (3) claim to provide evidence that this enzyme is located in the parasite cytosol.…”