Journal: bioRxiv

Article Title: Plasmodium falciparum Niemann-Pick Type C1-Related Protein is a Druggable Target Required for Parasite Membrane Homeostasis

doi: 10.1101/371484

Figure Lengend Snippet: A) Live fluorescence microscopy with C-terminally GFP-tagged wild-type PfNCR1-expressing parasites (clone Wt-GFP1 from ) localizes PfNCR1 to the parasite surface. Scale bar 5μm. B-C) Immuno-electron-micrographs of trophozoite-stage parasites using α-GFP antibody. Arrows mark gold particles, RBC=infected red blood cell, DV=digestive vacuole, N=nucleus. The close-up in C) shows gold particles clustered at the parasite-delimiting membranes. EM= erythrocyte membrane; PVM=parasitophorous vacuolar membrane; PPM=parasite plasma membrane. D-G) Live fluorescence microscopy on split-GFP expressing parasites. D) Co-expression of PfNCR1-GFP11 with cytoplasmic GFP1-10. E) Co-expression of PfNCR1-GFP11 with vacuolar GFP1-10. F) Cytoplasmic GFP1-10 without expression of PfNCR1-GFP11. F) Vacuolar GFP-1-10 without expression of PfNCR1-GFP11. Scale bar: 1μm. H) Cartoon of the proposed orientation of PfNCR1 in the PPM (parasite plasma membrane). PV = parasitophorous vacuole; PVM = parasitophorous vacuolar membrane.

Article Snippet: Primary antibodies were: rabbit α-HAD1 (a gift from Dr. Audrey Odom John, WU) (1:1000), rabbit α-Hsp60 (1:500) (a gift from Dr. Sabine Rospert, University of Freiburg), mouse α-PM-V (1:20) , rabbit α-RFP (1:1000) (Thermofisher, Cat. No. R10367), mouse α-GFP (Living Colors JL-8, Clontech, Cat. No. 632380) (1:1000), HRP-conjugated α-aldolase (Abcam, Cat. No. ab38905) (1:10000), α-EXP2 antibody (gift from Professor James Burns, Drexel University) (1:10000) .

Techniques: Fluorescence, Microscopy, Expressing, Infection

Journal: bioRxiv

Article Title: Plasmodium falciparum Niemann-Pick Type C1-Related Protein is a Druggable Target Required for Parasite Membrane Homeostasis

doi: 10.1101/371484

Figure Lengend Snippet: A) Cartoon of trafficking route to the DV in an infected red blood cell. DV = digestive vacuole; PVM = parasitophorous vacuolar membrane; PPM = parasite plasma membrane. B) Live microscopy of PMII-GFP parasites after incubation with MMV009108 (1μM, 3 hrs) or with vehicle (DMSO). Scale = 1μm. C) Quantitation of abnormal DVs from parasites in (B) after incubation with MMV009108, or MMV019662 or vehicle (DMSO) (1μM, 3 hrs). P<0.0001, Fisher’s exact test. D) Live microscopy of PfNCR1 K/D parasites expressing PMII-GFP, after removal of aTc. Scale = 1μm. E) Quantitation of abnormal DVs from parasites in (D) after aTc washout. P<0.0001, Fisher’s exact test. D and E): cycle 0 = trophozoites after removal of aTc within the same replication cycle (27hrs post washout), cycle 1 = trophozoites after removal of aTc in the preceding replication cycle (68 hrs post washout). F) Transmission electron micrographs of PfNCR1 K/D parasites (clone 2) after aTc removal (68 hrs post washout). Scale = 0.5μm. G) Transmission electron micrographs of PfNCR1 K/D parasites maintained with aTc and incubated with 500nM MMV009180 for 1 hr. Scale = 0.5μm. H) Western blot analysis of PMII-GFP parasites after treatment with 1μM compounds for 2 hrs. Parasites were released from RBCs with low (L) (0.009%) or high (H) (0.035%) saponin. Top blot was probed with α-GFP antibody, bottom blot (loading control) was probed with α-Hsp60 antibody, an organellar marker. This experiment was done two times. I) Western blot analysis of PMII-GFP, PfNCR1 K/D parasites after aTc washout for 22 hrs. Parasites were released from RBCs with 0.009%, 0.0175%, 0.035%, 0.07% or 0.14% saponin. Top blot was probed with α-GFP antibody, bottom blot (loading control) was probed with α-Hsp60 antibody. This experiment was done two times. Expected size of pro-PMII-GFP=79kDa, free GFP=27kDa.

Article Snippet: Primary antibodies were: rabbit α-HAD1 (a gift from Dr. Audrey Odom John, WU) (1:1000), rabbit α-Hsp60 (1:500) (a gift from Dr. Sabine Rospert, University of Freiburg), mouse α-PM-V (1:20) , rabbit α-RFP (1:1000) (Thermofisher, Cat. No. R10367), mouse α-GFP (Living Colors JL-8, Clontech, Cat. No. 632380) (1:1000), HRP-conjugated α-aldolase (Abcam, Cat. No. ab38905) (1:10000), α-EXP2 antibody (gift from Professor James Burns, Drexel University) (1:10000) .

Techniques: Infection, Microscopy, Incubation, Quantitation Assay, Expressing, Transmission Assay, Western Blot, Marker

Journal: Nature Communications

Article Title: A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum

doi: 10.1038/s41467-018-04221-9

Figure Lengend Snippet: High-resolution immunofluorescent identification of Plasmodium liver stage (LS) parasites. a High-resolution images of P . vivax hypnozoites demonstrate these forms have minimal nuclear material and are negative for schizogony markers EXP1, EXP 2 and MSP1. Hypnozoites stain positive for cytosolic markers MIF, HSP70, and GAPDH and reveal a functioning apicoplast. b By day 8, P . vivax schizonts are several times larger than the host cell hepatic nucleus, feature genome replication and segmentation, and stain positive for EXP1, EXP2, ACP, and MSP1. c The LS of P . falciparum is shorter than that of P . vivax schizonts; developing parasites are correspondingly less small. By day 5 merozoite segmentation has begun (as noted by ACP staining of separate apicoplasts) but not complete (as noted by diffuse staining of MSPs). d Immunofluorescent staining of day 8 P . vivax LS schizonts with anti-Pvs16, a sexual stage-specific biomarker for immature gametocytes, showed co-localization with developing LS merozoites indicated by segmented DNA. However, anti-Pvs16 signal does not appear in every LS. e The PHH system successfully supports complete maturation of Plasmodium LS schizonts measured by breakthrough into blood stage using reticulocyte ( P . vivax , days 9–11) or RBC ( P . falciparum , days 7–8) overlays with initial giemsa staining every 6 h. P . vivax overlays show formation of merozoite packages which rupture into the reticulocyte culture leading to invasion. Alternatively, no merosomes were captured in P . falciparum overlays but early rings were present within the first 12 h and continued culture progressed to an asynchronous population at > 1% parasitemia. White scale bars represent 5 µm, gray scale bars represent 10 µm

Article Snippet: Briefly, the same immunofluorescence assay (IFA) protocol was followed above with the following fold dilutions of monoclonal antibodies obtained from The European Malaria Reagent Repository: mouse anti-GAPDH (Cat No. 7.2) at 1:50,000, mouse anti-EXP1and EXP2 (Cat No. 5.1 and 7.7) at 1:1000, mouse anti-GLURP (clones 22G6, 8B12, 2C7) at 1:200, anti-MSP1 (Cat No. 12.10) at 1:200, and anti-MSP2 (Cat No. 12.3) at 1:200 – .

Techniques: Staining, Biomarker Discovery

Journal: Cell Chemical Biology

Article Title: Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3

doi: 10.1016/j.chembiol.2020.03.009

Figure Lengend Snippet: Human AQP3 Is Recruited to P . vivax Schizonts and Hypnozoites (A) Schematic of P . vivax sporozoite infection of PHH to produce liver schizonts or hypnozoites. (B) Representative confocal images of a P . vivax liver schizont (top) day 8 post-infection into PHH. Cells were stained with DAPI (blue), Pv UIS4 (green), and Hs AQP3 (red). Colocalization analysis of Pv UIS4 and Hs AQP3 (bottom). (C) Representative confocal images of a P . vivax liver schizont on day 8 post-infection of PHH at two different focal planes. (D) Human AQP3 localization in a representative P . vivax hypnozoite day 8 post-infection of PHH. Cells were stained with DAPI (blue), Pv UIS4 (green), and Hs AQP3 (red). Scale bar, 10 μm.

Article Snippet: Primary antibodies rabbit anti- Hs AQP3 and mouse anti- Pv EXP2 (The European Malaria Reagent Repository, Cat# 7.7) were sequentially added, diluted 1:100 and 1:500, respectively.

Techniques: Infection, Staining

Journal: Cell Chemical Biology

Article Title: Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3

doi: 10.1016/j.chembiol.2020.03.009

Figure Lengend Snippet: P . vivax Schizonts and Hypnozoites Recruit Human AQP3 at Early Stages of Infection (A) Time course of human AQP3 localization during early stages of P . vivax infection of PHH. Percentage of infected cells with detectable AQP3 localization shown (right, red columns). (B) Time course of human AQP3 localization in P . vivax schizonts in PHH. (C) Time course of human AQP3 localization in P . vivax hypnozoites in PHH. Cells were stained with DAPI (blue), Pv UIS4 (green), and Hs AQP3 (red). Representative confocal images shown (n = 15–38). Scale bar, 10 μm.

Article Snippet: Primary antibodies rabbit anti- Hs AQP3 and mouse anti- Pv EXP2 (The European Malaria Reagent Repository, Cat# 7.7) were sequentially added, diluted 1:100 and 1:500, respectively.

Techniques: Infection, Staining

Journal: Cell Chemical Biology

Article Title: Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3

doi: 10.1016/j.chembiol.2020.03.009

Figure Lengend Snippet: Auphen Inhibits P . vivax Schizonts and Hypnozoites (A) Table with EC 50 values for auphen inhibition of P . berghei and P . vivax liver stages. Data are shown as average ± SD of three to four independent experiments. (B) Dose-response curve of auphen inhibition of P . vivax schizont growth area. (C) Dose-response curve of auphen inhibition of P . vivax hypnozoite quantity per well. Data are shown as the average ± SEM of all independent experiments. (D) Representative confocal images of P . vivax parasites on day 8 post-infection of PHH in the absence (top panel) or presence of 0.62 μM auphen (bottom panel). Arrow indicates parasite with AQP3 recruitment after auphen treatment. Cells were stained with DAPI (blue), Pv UIS4 (green), and Hs AQP3 (red). Scale bar, 10 μm.

Article Snippet: Primary antibodies rabbit anti- Hs AQP3 and mouse anti- Pv EXP2 (The European Malaria Reagent Repository, Cat# 7.7) were sequentially added, diluted 1:100 and 1:500, respectively.

Techniques: Inhibition, Infection, Staining

Journal: Cell Chemical Biology

Article Title: Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3

doi: 10.1016/j.chembiol.2020.03.009

Figure Lengend Snippet: Auphen Inhibits Blood-Stage P . vivax (A) Representative images of human AQP3 localization in P . vivax -infected reticulocytes during the ring (top panel) and schizont (bottom panel) stages. Cells were stained with DAPI (blue) and Hs AQP3 (red). Scale bar, 10 μm. (B) Representative dose-response curve of auphen inhibition of P . vivax schizonts in isolates SCPv331 (green circles) and SCPv280 (black triangles). Data are shown as the average ± SEM of all independent experiments. (C) Table of EC 50 values for auphen inhibition of P . vivax schizonts in patient isolates. Data are shown as average ± SD of 3 independent experiments. (D) Representative images of P . vivax -infected clinical isolate in the presence of 0 μM (left column), 0.5 μM (middle column), or 3 μM auphen (right column). Giemsa staining of blood cells revealed a delay in parasite development with auphen treatment.

Article Snippet: Primary antibodies rabbit anti- Hs AQP3 and mouse anti- Pv EXP2 (The European Malaria Reagent Repository, Cat# 7.7) were sequentially added, diluted 1:100 and 1:500, respectively.

Techniques: Infection, Staining, Inhibition

Journal: Cell Chemical Biology

Article Title: Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3

doi: 10.1016/j.chembiol.2020.03.009

Figure Lengend Snippet:

Article Snippet: Primary antibodies rabbit anti- Hs AQP3 and mouse anti- Pv EXP2 (The European Malaria Reagent Repository, Cat# 7.7) were sequentially added, diluted 1:100 and 1:500, respectively.

Techniques: Recombinant, Luciferase, Viability Assay, Control, Real-time Polymerase Chain Reaction, Software