title: Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development.
abstract: PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under in vitro conditions, a fusion protein comprising a J-GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J-GFP-DD). We demonstrate that J-GFP-DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.
authors.affiliation: Centre for Immunology, Macfarlane Burnet Institute of Medical Research and Public Health, Melbourne, Victoria 3004, Australia.
authors.forename: Mauro F
authors.initials: MF
authors.lastname: Azevedo
authors.forename: Paul R
authors.initials: PR
authors.lastname: Sanders
authors.forename: Efrosinia
authors.initials: E
authors.lastname: Krejany
authors.forename: Catherine Q
authors.initials: CQ
authors.lastname: Nie
authors.forename: Ping
authors.initials: P
authors.lastname: Fu
authors.forename: Leon A
authors.initials: LA
authors.lastname: Bach
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wunderlich
authors.forename: Brendan S
authors.initials: BS
authors.lastname: Crabb
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gilson
mesh_terms: D002478:Cells, Cultured; D010963:Plasmodium falciparum / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism*; D011494:Protein Kinases / Q000096:biosynthesis*; D017434:Protein Structure, Tertiary / Q000235:genetics; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics; D052939:Schizonts / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D011494:Protein Kinases; C531238:calcium-dependent protein kinase-1, Plasmodium falciparum