Abstract
Malaria aminopeptidases are important in the generation and regulation of free amino acids that are used in protein anabolism and for maintaining osmotic stability within the infected erythrocyte. The intraerythrocytic development of malaria parasites is blocked when the activity of aminopeptidases is specifically inhibited by reagents such as bestatin. One of the major aminopeptidases of malaria parasites is a leucyl aminopeptidase of the M17 family. We reasoned that, when this enzyme was the target of bestatin inhibition, its overexpression in malaria cells would lead to a reduced sensitivity to the inhibitor. To address this supposition, transgenic Plasmodium falciparum parasites overexpressing the leucyl aminopeptidase were generated by transfection with a plasmid that housed the full-length gene. Transgenic parasites expressed a 65-kDa protein close to the predicted molecule size of 67.831 kDa for the introduced leucyl aminopeptidase, and immunofluorescence studies localized the protein to the cytosol, the location of the native enzyme. The product of the transgene was shown to be functionally active with cytosolic extracts of transgenic parasites exhibiting twice the leucyl aminopeptidase activity compared with wild-type parasites. In vitro inhibitor sensitivity assays demonstrated that the transgenic parasites were more resistant to bestatin (EC50 64 microM) compared with the parent parasites (EC50 25 microM). Overexpression of genes in malaria parasites would have general application in the identification and validation of targets for antimalarial drugs.
Highlights
During the development and asexual reproduction of malaria parasites within erythrocytes, as much as 65–75% of erythrocyte hemoglobin is internalized and digested [1, 2]
There is still some conjecture as to the reason why the parasite needs to degrade such a large proportion of the erythrocyte hemoglobin, but several theories have been proposed [3, 4]. It has been known for some time that malaria parasites require amino acids derived from host hemoglobin for protein synthesis and development [4], recent studies suggest that the fraction of digested hemoglobin used for this purpose is surprisingly small [5]
Leucyl aminopeptidase-like enzymes are believed to function in the terminal stages of hemoglobin digestion to generate free amino acids that are used for parasite protein synthesis [13,14,15]
Summary
There is still some conjecture as to the reason why the parasite needs to degrade such a large proportion of the erythrocyte hemoglobin, but several theories have been proposed [3, 4] It has been known for some time that malaria parasites require amino acids derived from host hemoglobin for protein synthesis and development [4], recent studies suggest that the fraction of digested hemoglobin used for this purpose is surprisingly small (in the region of 16%) [5]. Aminopeptidase activity predominates in trophozoites, and the greatest in vitro growth inhibition by bestatin and nitrobestatin occurs when parasites are at the late ring and early trophozoite stage [13] This coincides with the period of increased metabolic activity within the infected erythrocyte, increased hemoglobin digestion, the induction of new permeability pathways, and rapid growth of the parasite [4, 8, 9]
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