PfPK2 is critical for asexual phase life cycle of <i>P</i>. <i>falciparum</i>.

A. Schematic representation of the strategy used to modify the PfPK2 locus through single-crossover homologous recombination and selection-linked integration (SLI). A 330bp region homologous to the 5’-end of PfPK2 gene was cloned in pSLI-N-sandwich-loxP vector. In addition, recodonized PfPK2 (rPK2) was cloned in frame with GFP (green). Yellow- FKBP, black- linker, triangles -loxP, *-stop codon, arrows- location of annealing site of PCR primers used for genotyping and assessing excision, which was achieved after rapamycin addition. B. Genotyping of a clone obtained after limited dilution (PfPK2-loxP). PCR amplification was carried out for both the unmodified and modified loci, using primers targeting the indicated sites in Panel A. PCR products of expected size (indicated in the figure) were amplified confirming 5’- and 3’-integration in the transgenic PfPK2-loxP parasite. While amplification for the unmodified wild type (WT) locus was observed in 1G5DC parasites, it was absent in PfPK2-loxP parasites. The amplification for episome (E) was not detectable under the same conditions. The amplicons were sequenced to further confirm the integration at the desired locus. C. PfPK2-loxP parasites were treated with DMSO or 200 nM RAP for 4 or 24h. Subsequently, genomic DNA was isolated ~44 hpi and PCR was performed using indicated primer sets (panel A). A ~0.8kb product was obtained upon RAP treatment indicating successful excision. D. Western blot examination of PfPK2-loxP parasite lysates treated with DMSO and RAP and using anti-GFP antibody revealed that the RAP-treatment resulted in effective depletion of GFP-PfPK2. As a loading control, a β-actin antibody was also used to probe the blots. E. PfPK2-loxP parasites were synchronized and ring stage parasites were used for setting up growth rate assay in the presence of DMSO or RAP. After 6h, RAP was washed and parasite growth was assessed after each cycle by performing flow cytometry (SEM ± SE, n = 3, ANOVA, ***, pF. PfPK2-loxP parasites were treated with DMSO or RAP as described in panel A and Giemsa-stained blood smears at indicated time post invasion (h.p.i) were examined. PfPK2-depleted parasites showed significant parasites attached to the erythrocytes were suggestive of failed invasion. A few parasites that entered erythrocytes exhibited abnormal morphology and/or were pyknotic. G. The number of abnormal/pyknotic parasites -indicated in panel F-were counted after RAP addition (72 h.p.i, ~40 parasites were counted for each replicate), which revealed a significant increase in number of abnormal/pyknotic RAP-treated parasites when compared to DMSO-treated controls (SEM ± SE, n = 3, * P<0.05, paired t test).