(A) Diagram of P12 and P41 showing regions (black lines) that were expressed in E. coli. Secretion signal sequences are in white, protein spacer regions in yellow, 6-cys domains are in red and the glycosylphosphatidyl inositol signal sequence is in black. (B) A western blot of recombinant reduced and non-reduced P12 and P41 expressed in E. coli probed with pooled malaria immune human serum. Molecular mass protein markers are indicated on the left. (C) P12 and P41 expressed recombinantly C-terminally fused to domain 3 and 4 of rat Cd4 (green) followed by 6×His (blue) (left) in HEK293E cells. Recombinant proteins were expressed as soluble proteins of expected sizes (Coomassie-stained gel, right).

(A) Equal amounts of total protein from parasites harvested at different times throughout the asexual blood-stage cell cycle were subjected to SDS-PAGE in non-reducing and reducing conditions. Blots of these proteins were probed with rabbit IgGs raised to recombinant P12 and P41 (10 µg/mL) expressed in E. coli and demonstrated maximum expression of P12 and P41 in late stage parasites. Blots were also probed with rabbit anti-GAPDH (kindly supplied by Professor Leann Tilley) to ensure equal loading of parasite material from the various time points. (LR = late ring, ET = early trophozoite, T = trophozoite, ES = early schizont, Sc = schizont) (B) Localisation of P12 was explored by immunofluorescence assays. Schizonts and merozoites were fixed and labelled with mouse anti-MSP1₁₉ (10 µg/mL) and rabbit IgG to P12 (50 µg/mL) followed by Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 568 goat anti-mouse secondary antibodies IgG. P12 was found to localise to the parasitophorous vacuole in schizonts and on the merozoite surface upon comparison with MSP1₁₉. Interestingly, P12 exhibited concentrated apical localisation on the merozoite surface (white arrow) similar to the published localisation of P41 [8]. The representatives of schizonts and merozoites are shown.

(A) The recP12-Cd4d3/4-6H (blue line), recP41-Cd4d3/4-6H (red line) and the co-incubated proteins (green line) were resolved on a Superdex 200 10/300 GL column. Upon co-incubation, a single peak was observed at a lower retention volume than that observed for the individual proteins, corresponding in size to a P12/P41 heterodimer. (B) Biochemical analysis of the recombinant P12/P41 interaction by surface plasmon resonance. (a) Equilibrium dissociation constant (K_D) for P12/P41 interaction. Serial dilutions of recP41-Cd4d3/4-6H were injected through flow cells containing Cd4d3/4 (as a reference) and recP12-Cd4d3/4-bio captured on a streptavidin-coated sensor chip until equilibrium had been reached (see inset). Reference-subtracted binding data were plotted as a binding curve and an equilibrium dissociation constant calculated using non-linear regression fitting of a simple Langmuir binding isotherm to the data (solid line). (b) Kinetic data for the P12/P41 interaction. Serial two-fold dilutions of 3.6 µM purified recP41-Cd4d3/4-6H were injected at high flow rates (100 µL min⁻¹) over recP12 and a reference flow cell. Sensorgrams showed excellent fits to a simple 1∶1 binding model (shown in red) and were used to derive association (k_a) and dissociation (k_d) rate constants. (c, d) P12 and P41 do not interact homophilically. (c) Injection (solid bar) of purified 2.5 µM recP12-CD4d3/4-6H over recP41-Cd4d3/4-bio (blue line) and recP12-Cd4d3/4-bio (red dotted line) immobilised on a streptavidin-coated sensor chip. The reciprocal experiment using purified P41 is shown in (d).

(A) A schizont lysate of 3D7 parasites in 1% Triton X-100 in PBS was subjected to immunoprecipitation using rabbit anti-P12 IgG crosslinked to Protein G agarose. Bound proteins were serially eluted off the beads by 0.1 M glycine, 0.1 M glycine with 1 M NaCl, and 2% SDS. Eluates, along with the unbound material were analysed by SDS-PAGE followed by colloidal Coomassie staining. Proteins immunoprecipitated by anti-P12 IgG were excised from the polyacrylamide gel and evaluated by LC-MS/MS sequencing. P41, HSP70, 60 s ribosomal protein, and BSA were identified, however given its co-elution in glycine, P41 was the most likely binding partner of P12. (B) The P12 and P41 interaction was also confirmed by western blotting of immunoprecipitated material using pre-immune (PI), anti-P12, and anti-P41 rabbit IgG coupled Protein G agarose. The 1% Triton X-100 soluble (S) and insoluble (I) parasite extracts were included as controls. Blots were also probed with anti-EB175 and anti-MSP1₁₉ sera to demonstrate the specificity of the P12/P41 interaction.

(A) Invasion supernatant (S/N) containing P12 and P41 was incubated with uninfected erythrocytes and bound proteins were eluted from the erythrocytes once centrifuged through oil. EBA175, a known erythrocyte binding protein, bound to the erythrocytes as expected; however, P12 and P41 did not. (B) Lack of binding of P12 and P41 to erythrocytes was also confirmed via an erythrocyte binding protein depletion assay to test for weak interactions between P12/P41 and RBCs. The relative amounts of target protein in starting material and in mock incubation (i.e. no erythrocytes) are shown in lanes marked a & b, respectively.

(A) Diagram of plasmid and strategy for disrupting p12 and p41. A positive drug selection gene encoding a human dihydrofolate reductase (hDFHR) gene is flanked by sequences derived from the P12/P41 genes that will facilitate integration into the locus by double homologous recombination. To eliminate parasites in which integration has not occurred, a negative drug selection cytosine deaminase gene is included to remove the plasmid backbone from the transfected parasite population. (B) Western blots indicate that Δp12 and Δp41 lines do not express proteins corresponding to their respective disrupted genes. (C) Immunofluorescence microscopy of Δp12 and Δp41 and parental 3D7 parasites probed with rabbit anti-P12 and anti-P41 IgGs in addition to the merozoite surface marker MSP1 mAb indicate absence of protein expression in the mutants aside from minor cross-reactivity.

(A) Growth rates of Δp12 and Δp41 mutants were measured. All parasite lines, 3D7, Δp12 clone 1, Δp41 clone 1, and Δp36 clone 4.9, were adjusted to 1% parasitemia and were subsequently diluted 100,000 fold in duplicate. Parasites were cultured for 6 cycles, until 1% parasitemia was achieved for one of the four parasite lines, then parasites were counted to calculate their rate of amplification. As shown in the graph (mean with SD), 3D7, Δp12 clone 1, Δp41 clone 1, and Δp36 clone 4.9 had similar growth rates of 6.77, 6.67, 6.56, and 7.02 fold per cycle, respectively, which is not statistically different (p = 0.4753, one-way ANOVA analysis). (B) Invasion inhibition assay indicated that antibodies to E. coli recombinant P12 and P41 only weakly inhibited merozoite invasion. Purified merozoites were incubated with rabbit polyclonal anti-P12 or anti-P41 IgGs at the final concentration of 2 mg/mL in the presence of uninfected erythrocytes. New infections were measured by counting parasitemia 40 hrs later by flow cytometry. Using rabbit pre-immune IgG as a control, the results indicated that anti-P12 and anti-P41 antibodies could inhibit parasite invasion by 15–20%. Total parasitemias and schizont-stage parasite populations were counted. (C) Invasion inhibition assays using antibodies generated against the HEK293E expressed P12 and P41 failed to demonstrate invasion inhibitory activity. Late trophozoite stage parasites were incubated with rabbit polyclonal anti-P12 or anti-P41 antibodies either independently or in combination. Final concentrations are shown. Parasites were cultured for 24 hours, and re-invasion was measured by flow cytometry. All assays were performed in triplicate, and invasion efficiency calculated comparatively with parasites incubated in the absence of antibodies. No significant difference in invasion efficiency was observed at any concentration of IgG.