ppat.1011006.s006.tif (19.25 MB)

Knockdown of PTEX150-HAglmS and HSP101-HAglmS results in build-up of full-length Hb inside the parasite and reduced haemozoin crystal formation.

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posted on 2023-07-31, 17:32 authored by Thorey K. Jonsdottir, Brendan Elsworth, Simon Cobbold, Mikha Gabriela, Ellen Ploeger, Molly Parkyn Schneider, Sarah C. Charnaud, Madeline G. Dans, Malcolm McConville, Hayley E. Bullen, Brendan S. Crabb, Paul R. Gilson

(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).