title: Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum.
abstract: Erythrocytic malaria parasites degrade hemoglobin in an acidic food vacuole to acquire free amino acids and maintain parasite homeostasis. Hemoglobin hydrolysis appears to be a cooperative process requiring cysteine proteases (falcipains) and aspartic proteases (plasmepsins), but the specific roles of different enzymes in this process are unknown. We previously showed that falcipain-2 is a major trophozoite food vacuole cysteine protease. To characterize the specific role of falcipain-2, we disrupted the falcipain-2 gene and assessed the effect of this alteration. Falcipain-2-knockout trophozoites had markedly diminished cysteine protease activity and swollen, dark staining food vacuoles, consistent with a block in hemoglobin hydrolysis, as caused by cysteine protease inhibitors. However, more mature stages of knockout parasites were indistinguishable from wild-type parasites and developed normally. The knockout parasites had decreased and delayed expression of falcipain-2, which appeared to be directed by increased transcription of a second copy of the gene (falcipain-2'). Expression of other falcipains and plasmepsins was similar in wild-type and knockout parasites. Compared with wild-type, knockout parasites were about 3 times more sensitive to the cysteine protease inhibitors E-64 and leupeptin, and over 50-fold more sensitive to the aspartic protease inhibitor pepstatin. Our results assign a specific function for falcipain-2, the hydrolysis of hemoglobin in trophozoites. In addition, they highlight the cooperative action of cysteine and aspartic proteases in hemoglobin degradation by malaria parasites.
authors.affiliation: Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94143, USA.
authors.forename: Puran S
authors.initials: PS
authors.lastname: Sijwali
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Rosenthal
mesh_terms: D000818:Animals; D003546:Cysteine Endopeptidases / Q000235:genetics*; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006454:Hemoglobins / Q000378:metabolism*; D006868:Hydrolysis; D010957:Plasmids; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D006454:Hemoglobins; D012333:RNA, Messenger; D003546:Cysteine Endopeptidases; C416224:falcipain 2