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. 2008 Feb;7(2):279-85.
doi: 10.1128/EC.00245-07. Epub 2007 Dec 14.

Disruption of the PfPK7 gene impairs schizogony and sporogony in the human malaria parasite Plasmodium falciparum

Dominique Dorin-Semblat  1 , Audrey Sicard, Caroline Doerig, Lisa Ranford-Cartwright, Christian Doerig
Affiliations

Disruption of the PfPK7 gene impairs schizogony and sporogony in the human malaria parasite Plasmodium falciparum

Dominique Dorin-Semblat et al. Eukaryot Cell. 2008 Feb.

Abstract

PfPK7 is an orphan protein kinase of Plasmodium falciparum with maximal homology to MEK3/6 and to fungal protein kinase A proteins in its C-terminal and N-terminal regions, respectively. We showed previously that recombinant PfPK7 is active on various substrates but is unable to phosphorylate the Plasmodium falciparum mitogen-activated protein kinase homologues, suggesting that it is not a MEK functional homologue. Using a reverse genetics approach to investigate the function of this enzyme in live parasites, we now show that PfPK7(-) parasite clones display phenotypes at two stages of their life cycle: first, a decrease in the rate of asexual growth in erythrocytes associated with a lower number of daughter merozoites generated per schizont, and second, a dramatic reduction in the ability to produce oocysts in the mosquito vector. A normal asexual growth rate and the ability to produce oocysts are restored if a functional copy of the PfPK7 gene is reintroduced into the PfPK7(-) parasites. Hence, PfPK7 is involved in a pathway that regulates parasite proliferation and development.

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Figures

FIG. 1.
FIG. 1.
Gene disruption and genotyping. (A) Disruption of the PfPK7 locus. Primers used for the detection of integration events are indicated by numbered arrows (see Materials and Methods for the primer sequences). ATP, the glycine triad in subdomain I of the catalytic domain that is required for the proper orientation of the ATP molecule; PE, the proline-glutamate dipeptide in subdomain VIII in which the E residue is required for the structural stability of the enzyme; BSD, blasticidin resistance cassette. (B) PCR analysis of the PfPK7 locus. Genomic DNA from wild-type 3D7, from the uncloned blasticidin-resistant population, from the PfPK7− clone B9, and from B9 parasites complemented with pCHD-PK7 was isolated and subjected to PCR analysis. The following primer pairs were used (see panel A): primers 1 and 3 to detect integration at the 5′ end of the insert (lane 1); primers 2 and 4 to detect integration at the 3′ end of the insert (lane 2); primers 2 and 3 to detect the episome (lane 3); and primers 1 and 4 to detect the wild-type locus (lane 4). The expected sizes of the amplicons are indicated on the right. (C) Southern blot analysis of the PfPK7 locus in 3D7 and PfPK7− (clone B9) parasites. Genomic DNA was cleaved with EcoRV and NcoI, transferred, and probed with a PfPK7 probe (see Materials and Methods). The sizes of the bands are indicated on the right (see panel A for their identities). WT, wild type.
FIG. 2.
FIG. 2.
Phenotype of PfPK7− parasites during the erythrocyte asexual cycle. (A) Western blot analysis. Top panels, anti-PfPK7 antibodies were used; bottom panels, anti-Pfcyc-4 antibodies (loading control) were used. Two experiments are depicted, using two PfPK7− clones (B9 and C4). The right panel also shows the reexpression of PfPK7 in clone B9 complemented with a plasmid driving the expression of PfPK7 cDNA. The sizes of the comigrating markers are indicated in kDa on the left. WT, wild type. (B) Growth rate of the PfPK7− B9 clone compared to that of wild-type 3D7 and of the B9 clone complemented with pCHD-PK7. Parasitemia was measured for three independent cultures over 5 days. Ten different fields from Giemsa-stained smears were counted for each time point, and the mean parasitemia was determined. Standard errors of the means are indicated by error bars. (C) Numbers of nuclei per segmenter in populations (200 segmenters) of the PfPK7− clone, wild-type 3D7, and the B9 clone complemented with pCHD-PK7. The data are from one experiment, for which 100 segmenters were examined independently by two different observers. Standard errors of the means are indicated by error bars. The panel at the right provides a more visual depiction of the differences between the clones. This experiment was repeated twice with similar results. (D) Giemsa-stained schizonts from the B9 clone and from B9 parasites complemented with pCHD-PK7.
FIG. 3.
FIG. 3.
Evidence that PfPK7− parasites cross the mosquito midgut. Parasite DNA from the midguts of mosquitoes infected with clone B9 (lanes 1 and 2; two different mosquitoes), clone C4 (lane 3), and H4, a PfPK7+ clone that produces wild-type levels of oocysts (lane 5), was amplified 10 days postfeeding. Lane 4, negative control (midgut from an uninfected mosquito). Nested-PCR amplification of the Pfmdr1 gene was performed as described previously (10); the expected amplicon size in kb is indicated on the right. Wild-type 3D7 gave a signal similar to that yielded by the H4 clone (lane 5; not shown).
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