Knockdown of PTEX150-HAglmS and HSP101-HAglmS results in build-up of full-length Hb inside the parasite and reduced haemozoin crystal formation.
(A) Simple linear regression analysis was performed on protein expression and Hb build-up from western blots presented in Fig 2B for PTEX150-HAglmS, HSP101-HAglmS and FP2a-HAglmS. All parasite lines showed significant regression slope, where P values are shown in each graph along with R2. The blue dots on the x-axis are log10 of mean of the fold difference in protein expression for 6 biological replicates for 0.15 and 1 mM GlcN and 3 biological replicates for 2.5 mM GlcN plotted against log2 of the fold difference for individual biological replicates for Hb build-up (y-axis). The SD for the x-axis shown is as follows, PTEX150-HAglmS (X = 2, SD = 0; X = 1.82, SD = 0.06, X = 1.75, SD = 0.12, X = 1.67, SD = 0.21), HSP101-HAglmS (X = 2, SD = 0; X = 1.73, SD = 0.14, X = 1.69, SD = 0.08, X = 1.71, SD = 0.21), FP2a-HAglmS (X = 2, SD = 0; X = 0.99, SD = 0.19; X = 0.71, SD = 0.52, X = 0.74, SD = 0.61). (B) Highly-synchronous 3D7 WT, PTEX150-HAglmS, HSP101-HAglmS and FP2a-HAglmS trophozoite stage parasites were treated ± 2.5 mM GlcN for one cell cycle and harvested for IFA. Haemozoin crystals in the DIC channel were counted (present or absent). Images are representative of 3 (3D7 WT, PTEX150-HAglmS, FP2a-HAglmS) or 2 (HSP101-HAglmS) biological replicates. (C) Both PTEX150-HAglmS and HSP101-HAglmS knockdown experiments shown in panel B resulted in significantly less crystal formation compared to untreated cells when using Student’s t test with Welch correction. No significant difference in haemozoin crystal count was observed for 3D7 WT or FP2a-HAglmS parasites, although FP2a-HAglmS parasites often appeared to have smaller crystals. (*) Indicates P = 0.0247 (PTEX150-HAglmS) and P = 0.0277 (HSP101-HAglmS). Error bars = SD from 2 individuals counting. (D) Area of the parasites analysed in panel C (completed as described in Fig 2E) showed significant difference in size for GlcN treated PTEX150-HAglmS parasites compared with untreated indicating parasite growth was affected but no significant difference was observed for HSP101-HAglmS knockdown for the cells used in the analysis. Middle line represents mean and error bars = SD. (****) Indicates P <0.0001. Each dot on the graph represents one cell analysed. (E) FP2a-HAglmS parasites sometimes showed expansion of the food vacuole as previously observed in Giemsa-stained smears (S4 Fig), indicated with an arrow. When stained with rabbit anti-human haemoglobin antibody, a build-up of Hb was observed inside the food vacuole as previously observed in IFA treated with saponin (Fig 2C).