Figure 3. Recombinant EBA-175 RII causes RBC clustering in parasite culture and enhances parasite growth.

(A) Effects of picomolar to low nanomolar RII on parasite growth assessed by [³H]-hypoxanthine uptake for 3D7, Dd2, FVO/FCR1, HB3 strains, and the luciferase activity for NF54-GFP-luc strain (far right). Data shown are mean ± SD of three biological replicates for all strains. Significance (*, p<0.05; ***, p<0.001) (B) Uptake of [³H]-hypoxanthine into parasite nucleic acids by 3D7, Dd2, FVO/FCR1, HB3 strains, and the luciferase activity in NF54-GFP-luc strain (far right), in the presence or absence of RII, F1 or F2 at varying concentrations (10 nM – 1 μM range) for 96 hr. Results are shown as mean ± SD of three biological replicates. (C) Ab-217 (purple line) inhibition of RII-mediated growth enhancement, control Ab Ab-DBP (black line). Results are shown as mean ± SD of three biological replicates. (D) Microscopy analysis of NF54-GFP parasites (green) in RII-induced clusters (red).

Figure 3—figure supplement 1. RII enhances parasite growth.

(A) The distribution of the three biological replicates of growth curves for P. falciparum strains 3D7, Dd2, FVO/FCR1, HB3, and NF54-GFP-luc incubated with increasing concentrations of F1, F2, or RII. In accordance with ALARA for the use and disposal of radioactive waste, two technical replicates in each biological replicate were performed for [³H]-hypoxanthine incorporation assays. Three technical replicates for each biological replicate were assessed in the luciferase activity analysis. (B) The proliferation/growth of parasites was assessed by measuring [³H]-hypoxanthine uptake or luciferase activity (NF54-GFP-luc strain) in the presence or absence of 30 nM F1, F2 or RII. Data shown are mean ± SD of ten replicates and statistical analysis was performed as described in Materials and methods.

Figure 3—figure supplement 2. Parasite growth enhancement is due to the specific interaction of EBA-175 with GpA.

(A) The distribution of the three biological replicates of various P. falciparum cultures left untreated (red circle) or treated with 30 nM RII for 96 hr in the presence of various concentrations Ab-DBP (black line) or Ab-217 (purple). Growth of 3D7, Dd2, FVO/FCR1 and HB3 cultures were assessed by measuring [³H]-hypoxanthine uptake and NF54-GFP-luc culture by measuring luciferase activity. In accordance with ALARA for the use and disposal of radioactive waste, two technical replicates in each biological replicate were performed for [³H]-hypoxanthine incorporation assays. Three technical replicates for each biological replicate were assessed in the luciferase activity analysis. (B) P. falciparum 3D7, Dd2, FVO/FCR1, HB3, and NF54-GFP-luc cultures were left untreated (control) or treated with 30 nM RII for 96 hr in the presence of 1 μM Ab-DBP or Ab-217. The proliferation/growth of parasites was assessed by measuring [³H]-hypoxanthine uptake or, in the case of NF54-GFP-Luc, luciferase activity. Ab-217 significantly blocked the RII-mediated growth enhancement while Ab-DBP had no effect. Results are shown as mean ± SD of six technical replicates and statistical analysis was performed as described in Materials and methods.