PKAc is not required for the preerythrocytic stages of Plasmodium berghei

(A) Oocysts count per mosquito. One-way ANOVA was used for statistical analysis, and no difference in oocyst numbers was observed (P = 0.923). (B) Magnified oocyst showing sporulation. Scale bar, 25 μm. (C) Quantifications of the midgut (MG)-associated sporozoites. Data are shown as means ± SEM (n = 3). (D) PCR-based confirmation of flirted locus excision in PKAc cKO parasites using primers 1409-1073. Both excised and non-excised loci were amplified in PKAc cKO MG parasites, but only the intact PKAc locus was amplified from the blood stage (SC). (E) Quantifications of the SG-associated sporozoites. Data are shown as means ± SEM (n = 3). One-way ANOVA was used for statistical analysis for MG (P = 0.945) and SG (P = 0.806). Error bars represent SEM. (F) Flirted PKAc cKO locus excision was also monitored in SG and after infecting mice with sporozoites as described previously. The excised locus was amplified in PKAc cKO SG parasites, but only the intact PKAc locus was amplified from the blood stage (SC) parasites both before (SC) and after passing through mosquito (PPP). MG, midgut; NTC, no template control; PPP, prepatent period; SC, single clone.

(A) PKAc cKO sporozoites show normal CSP shedding and secretion in the medium. Hsp70 was used as the loading control. No difference was observed in three independent experiments. (B, C) Gliding motility of PKAc cKO sporozoites was similar to the gliding activity observed for TRAP/FlpL parasites. Scale bar, 25 μm. Representative data of three independent experiments. One-way ANOVA was used for statistical analysis for 1 (P = 0.358), 2–10 (P = 0.457), and >10 circles (P = 0.273). (D) Immunofluorescence assay showing inside/outside sporozoites by differential staining with CSP antibody before and after permeabilization in HepG2 cells. Outside sporozoites were stained before permeabilization with CSP antibody (CSP-R), and total sporozoites were stained after permeabilization (CSP-G). Scale bar, 25 μm. (E) Bar graph showing percent sporozoites inside versus outside. Data are shown as means ± SEM (n = 3) in which the total number of sporozoites counted were 2,332 (TRAP/FlpL), 3,527 (PKAc cKO c1) and 3,147 (PKAc cKO c2). One-way ANOVA was used for statistical analysis (P = 0.717).

(A) To determine if PKAc cKO parasites develop and egress from the liver, infected mice livers were harvested at the indicated time points. The parasite burden in the liver was checked by amplifying the parasite 18S rRNA. Mice were treated with a combination of chloroquine, beginning 24 h after sporozoite injection, and artesunate, beginning 39 h after sporozoite injection, to prevent the growth of blood stage parasites. Therefore, the decrease of copy number between 55 and 72 h indicates a loss of liver-specific signal. This experiment was performed twice with similar results, and data from one experiment are presented. Unpaired two-tailed t test was used for statistical analysis for 36 h (P = 0.824), 55 h (P = 0.623), and 72 h (P = 0.493). (B) Development of PKAc cKO and TRAP/FlpL EEFs in vitro. HepG2 cells were infected with SG sporozoites, and the culture was harvested at different time points. Cultures removed at 36 h p.i. were fixed and parasites were detected using anti-Hsp70 and anti-UIS4 antibodies. Scale bar, 5 μm. (C) Quantification of EEF numbers at 36 h p.i.; no significant difference was observed in three independent experiments. One-way ANOVA was used for statistical analysis (P = 0.693). (D) Cultures removed at 62 h p.i. were probed with UIS4 and MSP1 to visualize PV membrane and formation of merozoites, respectively. Scale bar, 5 μm. (E) Quantitation of EEF area at 36 and 62 h p.i. The EEF size was similar in both PKAc cKO and TRAP/FlpL parasites. Unpaired two-tailed t test was used for statistical analysis for 36 (P = 0.406), and 62 h (P = 0.977). Error bars represent SD. To determine the size of the EEFs, the perimeter was delineated using the “region of interest” tool and the area was calculated using Nikon NIS elements BR imaging software.

(A) PKAc cKO parasites released a similar number of merosomes in the culture supernatant as compared with TRAP/FlpL. Merosome experiments were performed three times with similar results, and data from one experiment are presented. Error bars represent SD. Unpaired two-tailed t test was used for statistical analysis (P = 0.104). (B) PKAc cKO merosomes show normal loss of the PV membrane and normal segregation of merozoite membranes. Scale bar, 5 μm. (C) Measurement of UIS4 intensity showing normal loss of PV membrane in PKAc cKO parasites. One-way ANOVA was used for statistical analysis (P = 0.481). (D, E) PCR-based confirmation of flirted locus excision in PKAc cKO parasites using primers 1409-1073. Genomic DNA was isolated from the TRAP/FlpL line and different stages of PKAc cKO parasites. The wild-type locus was amplified from the TRAP/FlpL line, and the excised locus was amplified in PKAc cKO SG, LS, and MS parasites, but only the intact PKAc locus was amplified from the blood stage (SC) parasites both before (SC) and after passing through mosquito (PPP). (F) Amplification of the non-excised region using internal primers 1409-1074. (G) Presence of FRT site in the amplified product was confirmed by XbaI digestion, which yielded 348- and 255-bp products.