Genetic knockout growth analysis and invasion inhibition assays suggest a non-essential role for P12/P41 <i>in vitro</i>.

<p>(A) Growth rates of Δ<i>p12</i> and Δ<i>p41</i> mutants were measured. All parasite lines, 3D7, Δ<i>p12</i> clone 1, Δ<i>p41</i> clone 1, and Δ<i>p36</i> 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, Δ<i>p12</i> clone 1, Δ<i>p41</i> clone 1, and Δ<i>p36</i> 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 <i>E. coli</i> 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.