Supplemental material for mBio00630-22

Supplemental material for mBio00630-22

SUPPLEMENTAL FIGURES

Autophagy Underlies the Proteostasis Mechanisms of Artemisinin Resistance in P. falciparum Malaria

                                                                                                         

Ananya Ray,a Miti Mathur,a Deepak Choubey,b Krishanpal Karmodiya,c Namita Suroliaa,#

a. Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India

b. Life Science Research unit, Persistent Systems Limited, Pune, India

c. Department of Biology, Indian Institute of Science Education and Research, Pune, India

Running Head: Autophagy underlies mechanisms of ART resistance

# Address correspondence to Namita Surolia (N.S.), surolia@jncasr.ac.in

Fig S1 ART induced ER stress activates the UPR pathway. To visualize the expansion and morphology of the parasite ER, PfSec62, a translocon complex component present on the ER membrane was labelled with GFP. (A) Live cell confocal microscopy images of P. falciparum 3D7 parasites overexpressing PfSec62-GFP. ER expansion is observed after 1.5h of incubation with DHA. PfSec62-GFP signal extends into the parasite cytoplasm whereas it localizes to the nuclear periphery in control (untreated) parasites. ER expansion is analysed using GFP fluorescence intensities measured along the white line in the enlarged panel, and were plotted as the line scan graphs. Nucleus was stained using Hoechst. Scale bar: 5 μm. Scatter plot represents the extent of ER expansion as measured by the distance of PfSec62-GFP signal (obtained from line scan graphs of GFP fluorescence intensities) in control and parasites incubated with DHA. N = 25 parasites, n = 3 independent experiments. The data points are expressed as mean ± standard error of the means (SEM). Statistical significance is quantified using unpaired Student’s t-test, **** = P < 0.0001. (B) 3D reconstruction with Z-stacks obtained from confocal images of control and of parasites incubated with DHA overexpressing PfSec62-GFP. Volume of ER is measured through the surface function in Imaris 3.0. (C) Effect of DHA on the phosphorylation status of PfeIF2𝛼.Control and parasites incubated with DHA were harvested. Parasite lysates were subjected to immunoblot analysis and blots were probed with phosphorylated-PfeIF2𝛼 and total PfeIF2𝛼 antibodies. β-Actin was used as the loading control. Fold difference, normalized with respect to control is shown below each blot.

Fig S2 ART increases expression of the autophagy proteins. P. falciparum 3D7 parasites were incubated with either 700nM DHA or starvation media for 1.5h and harvested. 3D7 parasite lysates were subjected to immunoblot analysis and blots were probed with PfATG8 and PfATG18 antibodies as shown in Fig. 1B. Graphs showing the fold changes of PfATG8 (left) and PfATG18 (right) protein expression levels upon incubation with DHA and starvation media. n = 3 independent experiments. The data points are expressed as mean ± SEM. Statistical significance is quantified using unpaired Student’s t-test, *** = P < 0.0005, * = P < 0.05, ns = nonsignificant.

Fig S3 Expression levels and colocalization of the autophagy proteins in ART resistant and its isogenic parasites. (A) K13WT and K13C580Y (0-3 hpi) ring stage parasite lysates were subjected to immunoblot analysis and blots were probed with PfATG8 and PfATG18 antibodies as shown in Fig. 2C. Graphs showing fold changes of PfATG8 (left) and PfATG18 (right) protein expression levels in K13C580Y parasites relative to K13WT. n = 3 independent experiments. (B) K13WT and K13C580Y young trophozoite stage parasite lysates were subjected to immunoblot analysis and blots were probed with PfATG8 and PfATG18 antibodies as shown in Fig. 2D. Graphs showing fold changes of PfATG8 (left) and PfATG18 (right) protein expression levels in K13C580Y parasites relative to K13WT n = 3 independent experiments. The data points are expressed as mean ± SEM. Statistical significance is quantified using unpaired Student’s t-test, ** = P < 0.005, ns = non-significant. (C) Immunofluorescence analysis of K13WT and K13C580Y early rings stained with PfATG8 and PfATG18 antibodies using the Zenon antibody labelling system. (D) Immunofluorescence analysis of K13WT and K13C580Y young trophozoites stained with PfATG8 and PfATG18 antibodies using the Zenon antibody labelling system. Regions within the dashed white lines are enlarged and placed next to the merged panel to represent colocalization. Extent of colocalization between PfATG8 and PfATG18 labelled puncta is represented by the Pearson’s coefficient value (R) and evaluated from the PfATG8 (red) and PfATG18 (green) fluorescent signals within the white square region in the enlarged panel. Scatter plot representing the number of PfATG8 and PfATG18 colocalizing puncta. N = 10 parasites, n = 3 experiments, Scale bar = 5 μm. The data points are expressed as mean ± SEM. Statistical significance is quantified using unpaired Student’s t-test, * = P < 0.05.

Fig S4 Starvation induces parasite autophagy in ART resistant as well as its isogenic isolate. (A) Immunofluorescence analysis of K13WT (top two panels) and K13C580Y (bottom two panels) parasites stained with anti-PfATG18 antibodies showing PfATG18 labelled puncta upon starvation. (B) Immunofluorescence analysis of K13WT (top two panels) and K13C580Y (bottom two panels) parasites stained with anti-PI3P antibodies showing PI3P labelled puncta upon starvation. Nucleus was stained using Hoechst. N = 15 parasites, n = 3 experiments, Scale bar = 5 μm. (C) Scatter plots representing the number of PfATG18 labelled puncta in K13WT and K13C580Y parasites. (D) Scatter plots representing the number of PI3P labelled puncta in K13WT and K13C580Y parasites. The data points are expressed as mean ± SEM. Statistical significance is quantified using unpaired Student’s t-test, **** = P < 0.0001, *** = P < 0.0005; ** = P < 0.005. (E and F) Effect of starvation (1.5h) on PfATG8 and PfATG18 protein expression levels. K13WT and K13C580Y parasite lysates were subjected to immunoblot analysis and blots were probed with PfATG8 and PfATG18 antibodies as shown in Fig. 4C. Graphs representing fold changes of PfATG8 (E) and PfATG18 (F) protein expression levels  upon starvation. n = 3 (K13WT) and 2 (K13C580Y) independent experiments. The data points are expressed as mean ± SEM. Statistical significance is quantified using unpaired Student’s t-test, * = P < 0.05.

Fig S5 PfK13 and PfATG18 colocalize on HCv in young trophozoites and localize near the parasite periphery in rings of ART resistant isolates. (A) Immunofluorescence analysis of K13C580Y parasites stained with anti-PfFP2, anti-PfATG18 and anti-PfK13 antibodies using the Zenon antibody labelling system. Representative images showing colocalization of PfFP2 with PfATG18 (top panel) and PfK13 (bottom panel) labelled puncta. (B) Immunofluorescence analysis of K13C580Y parasites stained with anti-PfPTEX-150, anti-PfATG18 and anti-PfK13 antibodies using the Zenon antibody labelling system. Representative images showing colocalization of PfPTEX-150 with PfATG18 (top panel) and PfK13 (bottom panel) labelled puncta. Scale bar = 5 μm. Regions within the dashed white lines are enlarged and placed next to the merged panel to represent colocalization. Extent of colocalization is represented using the Pearson’s coefficient value (R) evaluated from the PfFP2 and PfPTEX-150 (red) and PfATG18 and PfK13 (green) fluorescent signals within the white square region in the enlarged panel. Nucleus was stained using Hoechst.