Overexpression of leucyl aminopeptidase in Plasmodium falciparum parasites. Target for the antimalarial activity of bestatin.
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.
Gardiner
Donald L
DL
Malaria Biology Laboratory, The Australian Centre for International and Tropical Health and Nutrition, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane QLD 4029, Australia.
Trenholme
Katharine R
KR
Skinner-Adams
Tina S
TS
Stack
Colin M
CM
Dalton
John P
JP
D000818:Animals; D030801:Animals, Genetically Modified; D000962:Antimalarials / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers; D005091:Exons; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D015800:Protozoan Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D000962:Antimalarials; D017931:DNA Primers; D011480:Protease Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex