The malaria parasite, like all organisms, must acquire nutrients from the environment and convert these nutrients to other molecules or energy (i.e., catabolism). These other molecules and the energy are then used to maintain the homeostasis of the parasite, and in the growth and reproduction of the parasite (i.e., anabolism). Both anabolic and catabolic processes are catalyzed by enzymes. Growing and reproducing organisms require high levels of macromolecules and other biochemicals for the maintenance of cellular structure and function. The malaria parasite needs to acquire these biochemicals and precursors from the host. [See also: Brief Overview of Plasmodium Biochemistry.]
The unique life cycle and resulting microenvironments of the parasite has led to the evolution of metabolic pathways which differ from the human host. It may be possible to exploit these unique pathways and enzymes in the design of therapeutic strategies. For example, many anti-malarials are known to affect the food vacuole which is a special organelle for the digestion of host of host hemoblobin.
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The malaria parasite requires amino acids for the synthesis of its proteins. The three sources of amino acids are: de novo synthesis, import from host plasma, and digestion of host hemoglobin. (See also Plasmodium Biochemistry--Proteins and Amino Acids.) Hemoglobin is an extremely abundant protein in the erythrocyte cytoplasm and serves as the major source of amino acids for the parasite (Box). Hemoglobin is broken down into peptides and amino acids within a vacuolar compartment known as the food vacuole. [See Wunderlich et al (2012) for a comprehensive review of the food vacuole and activities associated with the vacuole.]
During the early ring stage, the parasite takes up the host cell stroma by pinocytosis (Figure, right; note ppm = parasite plasma membrane) resulting in double membrane vesicles. The inner membrane, which corresponds to the PVM, rapidly disappears and the digestion of hemoglobin takes place within these small vesicles during the early trophozoite stage. As the parasite matures, it develops a special organelle, called the cytostome, for the uptake of host cytoplasm and the small pigment-containing vesicles fuse to form a large food vacuole. (Gametocytes do not form the large food vacuole and are characterized by small pigment-containing vesicles found throughout their cytoplasm.) Double-membrane vesicles pinch off from the base of the cytostome and fuse with the food vacuole. The inner membrane (originally the PVM) is lysed and the hemoglobin is released into the food vacuole.
The food vacuole is an acidic compartment (pH 5.0-5.4) that contains protease activities. In this regard the food vacuole resembles a lysosome, except other acid hydrolases (eg., glycosidases and nucleases) have not been identified. Presumably other acid hydrolases are not needed since the microenvironment of the erythrocyte is almost exclusively protein, and in particular, hemoglobin (Box). The acidic pH of the food vacuole is maintained by a H+-translocating ATPase and a H+-translocating pyrophosphatase, both of which are homologous to V-type transporters found in plants. Thus the food vacuole is probably homologous to the tonoplast found in plants and other protozoa.
|Food Vacuole Proteases|
Several distinct protease activities, representing three of the four major classes of proteases, have been identified in the food vacuole (Table). Multiple plasmepsins and falcipains have been identified. The digestion of hemoglobin probably occurs by a semi-ordered process involving the sequential action of different proteases (Goldberg, 2005). Several plasmespsin genes have been identified in the genome of P. falciparum and four of these apprear to function in the food vacuole (Banerjee, 2002). Plasmepsin-1 and plasmepsin-2 are the best characterized and both are capable of cleaving undenatured hemoglobin between phenylalanine and leucine residues located at positions 33 and 34 on the alpha-globin chains. These residues are located in a conserved domain known as the hinge region, which is believed to be crucial in stabilizing the overall structure of hemoglobin. Cleavage at this site presumably causes the globin subunits to dissociate and partially unfold. This unfolding will expose additional protease sites within the globin polypeptide chains. The other plasmepsins, as well plasmepsin-1 and plasmepsin-2, and the falcipains are then able to further degrade these large globin fragments. It has been suggested that falcipain-2 (Shenai, 2000), and possibly falcipain-3 (Sijwali, 2001), are capable of digesting either native hemoglobin and therefore may also participate in the initial cleavage of hemoglobin.
Falcilysin cannot digest either native hemoglobin or denatured globin, but readily cleaves the small polypeptide fragments (up to 20 amino acids) generated by the action of falcipain and plasmepsin. The site specifity of falcilysin complements the plasmepsins and falcipains and leads to the formation of peptides 6-8 amino acids in length. Therefore, the digestion of hemoglobin is a semi-ordered process involving the initial degradation to large fragments followed by subsequent degradation to small peptides (Figure, from Wunderlich et al, 2012). The proposed pathway of hemoglobin digestion involves an initial cleavage by plasmepsin-1 (and possibly falcipain-2) followed by the combined actions of several plasmepsins and falcipains. The peptide fragments produced by these digestions are then digested into smaller peptides by falcilysin.
Initially no food vacuole associated exopeptidase activity could be identified within the food vacuole (Kolakovich 1997). However, two amino peptidases (APP) were subsequently found in the food vacuole (Dalal and Klemba, 2007) which can convert the peptides into amino acids. In addition, a dipeptidyl aminopeptidase (DPAP) activity has been identified within the food vacuole (Kemba 2004). It is postulated that the DPAP may remove dipeptides from the N-termini of the peptides generated through the actions of the various endopeptidases in the food vacuole and then the amino peptidases can convert these to amino acids. Neutral amino peptidase activity has been identified in cytoplasm of several Plasmodium species (Curley 1994; Florent 1998).
Six amino acid transporters have been identified in the Plasmodium genome. However, there locations are not known. Pfmdr-1 has been localized to the food vacuole membrane and is a member of the ATP-binding cassette (ABC) transporter superfamily. Some ABC transporters function to translocate polypeptides across membranes. For example, the STE6 gene of yeast transports the a-type mating factor (a 12 amino acid peptide). Pfmdr-1 can complement the STE6 gene (Volkman 1995) indicating that it could function to pump small peptides into the parasite cytoplasm. However, more recent data indicate that Pfmdr-1 likely functions to import solutes, including drugs, into the food vacuole (Rohrbach 2006).
Another transporter expressed on the food vacuole membrane is PfCRT (chloroquine resistance transproter). PfCRT was initially identified in genetic linkage studies as being associated with chloroquine resistance. PfCRT is a member of the drug/metabolite transporter (DMT) superfamily and can export chloroquine and other drugs from the food vacuole. Peptides are able to block drug export via PfCRT, thus suggesting that PfCRT may function in the translocation of peptides from the food vacuole to the parasite cytoplasm (Martin 2009). Additional studies indicate that PfCRT is a proton (H+)-coupled polyspecific nutrient exporter capable of transporting a wide range of substrates (Juge 2015).
In summary, a likely scenario for the complete digestion of hemoglobin consists of the concerted actions of plasmepsins, falcipains and falcilysin leading to the production of small peptides. The small peptides are then converted into amino acids or dipeptides which are then converted in amino acids. Small peptides, dipeptides, and amino acids are translocated into the parasite cytoplasm via PfCRT using an electrochemical proton gradient. Peptides and di-peptides are converted to amino acids in the cytoplasm with the abundant amino peptidases.
Digestion of hemoglobin also releases heme. Free heme is toxic due to its ability to destabilize and lyse membranes, as well as inhibiting the activity of several enzymes. Three, and possibly four, mechanisms by which heme is detoxified have been identified:
Both the hemozoin formation pathway and the degradative pathways probably function simultaneously with 25-50% of the free heme being converted into hemozoin and the remainder being degraded (Ginsburg 1999). However, some studies suggest that up to 95% of the free iron released during hemoglobin digestion is found in hemozoin (Egan 2008). X-ray crystallography and spectroscopic analysis indicates that hemozoin has the same structure as b-hematin (Pagola 2000). b-hematin is a heme dimer formed via reciprocal covalent bonds between carboxylic acid groups on the protoporphyrin-IX ring and the iron atoms of two heme molecules (Figure, see also larger images). These dimers interact through hydrogen bonds to form crystals of hemozoin. Therefore, pigment formation is best described as a biocrystallization process (Hempelmann 2007; Egan 2008).
The mechanism of hemazoin formation is not known, but a protein that may catalyze the formation of hemozoin has been described (Jani 2008). This heme detoxification protein (HDP) binds to two molecules of heme with high affinity and may promote the formation of the b-hematin dimer. The dimer is release and may seed the crystallization process (Nakatani 2014). Lipids may also participate in the process in that lipid bodies have been observed within the food vacuole and hemozoin is associated with lipids (Egan 2008).
|Structures of Heme and Hemozoin|
A portion of the free heme may be degraded into non-toxic metabolites. Three potential processes have been described: in the food vacuole a hydrogen peroxide mediated oxidation of the porphyrin ring leads to its opening and subsequent breakdown; some of the heme translocates across the food vacuole membrane into the host cytoplasm where it is oxidized by reduced glutathione (GSH); and a heme oxygenase activity has been identified in some non-human malaria parasites. However, the role these processes play in the degradation of heme is not known.
Chloroquine and other 4-aminoquinolines inhibit pigment formation, as well as the heme degradative processes (Ginsburg 1999), and thereby prevent the detoxification of heme. The free heme destabilizes the food vacuolar membrane and other membranes and leads to the death of the parasite. [See a more detailed discussion on the actions of chloroquine.] The fact that the biocrystallization of heme is a unique process to the parasite and not found in the host accounts for the high therapeutic index of such drugs in the absence of drug resistance. Many other anti-malarials target the food vacuole indicating the importance of this organelle and its various functions (Summary Figure) to the survival of the parasite.
The iron bound to hemoglobin is primarily in the ferrous state (Fe2+). Release of the heme results in iron being oxidized to the ferric state (Fe3+). Electrons liberated by this oxidation of iron promote the formation of reactive oxygen intermediates (ROI) such as superoxide anion radicals and hydrogen peroxide. ROI can cause cellular damage. Superoxide dismutase (SOD) and catalase are cellular enzymes that function to prevent oxidative stress by detoxifying the superoxide and hydrogen peroxide, respectively. Both of these activities are found in the food vacuole and may have been obtained from the host during ingestion of the erythrocyte cytoplasm. (See also Plasmodium Biochemistry--Redox Metabolism.) Hydrogen peroxide can also be exported into the parasite cytoplasm where it is detoxified by catalase and glutathione peroxidase. Some of the hydrogen peroxide produced as a result of the Fe2+®Fe3+conversion may also used for the peroxidative degradation of heme.