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Survival of Malaria Parasites in the Human Host
Summary: Daniel Goldberg is interested in the biochemistry and chemotherapy of parasitic diseases.
Parasites have evolved many clever ways to infect their hosts and develop within them. Study of these processes at a molecular level should lead to treatment or prevention of parasitic infections that afflict most of humanity. It will also shed light on general principles of biochemistry and cell biology. The organism we are studying is Plasmodium falciparum, a protozoan parasite that causes malaria.
Intraerythrocytic malaria parasites degrade vast quantities of hemoglobin to provide nutrients for their growth and maturation. This process occurs in the acidic food vacuole. My laboratory is defining the proteolytic enzymes involved, their specificities and roles in hemoglobin breakdown, and their targeting to the food vacuole. The data suggest an ordered catabolic pathway. Four aspartic proteases, a metalloprotease, three cysteine proteases, a dipeptidyl peptidase, and aminopeptidases are involved in the process. The aspartic proteases (plasmepsins) make a strategic cleavage in the hemoglobin hinge region, unraveling the molecule for further proteolysis. We are determining the molecular basis for plasmepsin recognition and cleavage of native hemoglobin.
Most species have a single food vacuole plasmepsin, but P. falciparum has four tandemly arranged plasmepsin genes on chromosome 14, encoding proteins that share 60–70 percent amino acid identity. These enzymes are unusual in their ability to cleave intact hemoglobin well. The initial cleavage is on the globin α chain between 33Phe and 34Leu. This peptide bond is buried in the B helix of native hemoglobin. We have identified a loop in the plasmepsins that appears to be critical for gaining access to the scissile bond. Cathepsin E is a mammalian ortholog that can cleave at the same site if the hemoglobin is denatured. A chimeric plasmepsin possessing the cognate loop from cathepsin E is fully active on peptides or on loosely wound hemoglobin α chains but cannot cleave native hemoglobin. We have mapped interactions of this loop with the beginning of the B helix of hemoglobin and believe that the plasmepsin loop may pry apart the helix, exposing the 33–34 bond for hydrolysis.
Since the plasmepsins are involved in the initial steps of hemoglobin degradation, they are viewed as attractive drug targets. To define their roles in catabolism, we made gene disruptions in cultured intraerythrocytic parasites. Knockout of any of the plasmepsins gave a subtle growth phenotype. It occurred to us that the standard growth medium we all use to grow parasites, RPMI 1640, has 5- to 20-fold-higher amino acid concentrations than those found in normal human blood and hugely higher concentrations than those found in malnourished children, who are the most likely victims of the disease and in whom plasma amino acid levels can be undetectable. To investigate this further, we tried growing parasites in amino acid–limited conditions and found that P. falciparum grows well in modified RPMI medium lacking all amino acids except isoleucine (which is absent from human hemoglobin). Some isolates also require exogenous methionine for optimal growth. When we tested our knockout clones in amino acid–deficient medium, we found substantial growth phenotypes. These results suggest that P. falciparum has obtained a growth advantage through plasmepsin gene duplication and explain why it has maintained four enzymes with overlapping function. The data tell us as well that hemoglobin degradation is sufficient to supply nearly all of the parasite's amino acid requirements.
We also prepared disruptants in the falcipain-2 gene, which encodes the main cysteine protease expressed during hemoglobin degradation. Previous knockouts in this gene were reported to grow normally. We found that in our amino acid-deficient medium, there was a significant growth defect. When we made the falcipain-2 knockout on a plasmepsin IV/I double-knockout background, we observed profound growth impairment. Low concentrations of the aspartic protease inhibitor pepstatin that had minimal effect on parental parasites were lethal to the falcipain-2–knockout parasites and were even more potent in the plasmepsins/falcipain triple-knockout clones (plasmepsins IV and I are the two least pepstatin-sensitive plasmepsins; in their absence, pepstatin inhibition of plasmepsin activity is achieved at even lower concentrations).
The data tell us that hemoglobin degradation is essential to the parasite. This is most dramatic in nutrient-limited media, but even in rich RPMI medium, blockade is lethal. There is extensive overlap in function between the two protease families, but disabling both arms at once is devastating to the parasite.
The biosynthesis of the aspartic proteases appears to involve targeting to the parasite surface as integral membrane proenzymes and then ingestion with their substrate hemoglobin. Once the plasmepsin precursors reach the food vacuole, they are cleaved from the membrane by the falcipains, after a conserved sequence at the plasmepsin pro-mature junction. If falcipain activity is inhibited, the plasmepsins can mature by autoprocessing, another example of redundancy between these two families of proteases.
The metalloprotease (falcilysin) involved in food vacuole catabolism cannot cleave hemoglobin or acid-denatured globin; rather, it only works on fragmented globin or small, synthetic peptides. Falcilysin is capable of working at the acidic pH of the food vacuole. However, it also functions at neutral pH, where it has very different substrate specificity. Consistent with this, we found that falcilysin is located in the apicoplast, a chloroplast remnant organelle that the malaria parasite uses to synthesize lipids and heme. Falcilysin appears to play an important role in transit peptide processing during protein import into the apicoplast.
Another protease of interest to our lab is Plasmodium calpain. P. falciparum has a single calpain gene. Phylogenetic analysis reveals that the encoded protein (Pf_calpain) has a distinct domain structure found only in Apicomplexa and some other alveolates. To evaluate the potential of Pf_calpain as a drug target, we assessed its essentiality. We were unable to achieve gene disruption by double-crossover recombination and failed to achieve gene truncation by single-crossover recombination. We were unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we obtained synonomous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are likely to be important for optimal parasite growth but give no indication of a biological role for this gene.
To gain further insight we used the FKBP degradation domain system to generate fusion proteins whose levels in transfected parasites could be modulated by a small-molecule FKBP ligand, Shld1. We made a calpain-FKBP fusion by single-crossover integration at the endogenous calpain locus. Parasite growth was normal in the presence of Shld1 but was greatly impaired in the absence of ligand. Parasites were delayed in transitioning out of the ring stage. This is the first regulated knockdown of a Plasmodium gene. Further clues to function come from localization studies showing concentration of Pf_calpain in the nucleolus and regulation of targeting by palmitoylation. We identified a small sequence capable of targeting reporters to the nucleolus of Plasmodium or mammalian cells. Pf_calpain is a novel nucleolar protease required for cell cycle progression; it appears to be an attractive drug target.
Once intraerythrocytic malaria parasites mature and replicate, they must exit the host cell to infect new erythrocytes (figure). We have found that this is a two-step process. The parasites must escape from the host erythrocyte but must also get out of their own parasitophorous vacuolar membrane. Both processes involve specific proteolytic events. Further studies focus on characterization of the implicated enzymes. Protease inhibitors block the escape and multiplication of the organism, suggesting that this is an attractive chemotherapeutic target.
Our work involves a combination of biochemical, genetic, genomic, and physiological approaches aimed at understanding the biology of this nefarious organism.
The studies of plasmepsin biosynthesis and calpain are supported by funding from the National Institute of Allergy and Infectious Diseases.
Last updated: April 9, 2008
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