Host PI(3,5)P2 Activity Is Required for Plasmodium berghei Growth During Liver Stage Infection
CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorDepartment of Physiology, Wayne State University School of Medicine, Detroit, MI, 48201 USA
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorDepartment of Physiology, Wayne State University School of Medicine, Detroit, MI, 48201 USA
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, SW7 2AZ UK
Search for more papers by this authorCorresponding Author
CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Corresponding author: Duarte C. Barral,E-mail address: duarte.barral@fcm.unl.pt
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorDepartment of Physiology, Wayne State University School of Medicine, Detroit, MI, 48201 USA
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorDepartment of Physiology, Wayne State University School of Medicine, Detroit, MI, 48201 USA
Search for more papers by this authorCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
IGC, Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, SW7 2AZ UK
Search for more papers by this authorCorresponding Author
CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Corresponding author: Duarte C. Barral,E-mail address: duarte.barral@fcm.unl.pt
Search for more papers by this authorAbstract
Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is dependent on the phosphoinositide 5‐kinase (PIKfyve) that converts phosphatidylinositol 3‐phosphate [PI(3)P] into phosphatidylinositol 3,5‐bisphosphate [PI(3,5)P2] in the endosomal system. We found that inhibition of PIKfyve by either pharmacological or non‐pharmacological means causes a delay in parasite growth. Moreover, we show that the PI(3,5)P2 effector protein TRPML1 that is involved in late endocytic membrane fusion, is present in vesicles closely contacting the PVM and is necessary for parasite growth. Thus, our studies suggest that the parasite PVM is able to fuse with host late endocytic vesicles in a PI(3,5)P2‐dependent manner, allowing the exchange of material between the host and the parasite, which is essential for successful infection.
Citing Literature
Supporting Information
| Filename | Description |
|---|---|
| tra12190-sup-0001-FigureS1.docWord document, 458.5 KB | Figure S1: Parasite maturation is not affected in YM201636‐treated red blood cells. A) Infected mice, at day 6 post‐infection, were bled and the blood used for in vitro culture for 18–20 h, so that parasites could develop into schizonts. Parasite cultures were performed in medium with 1 µm of YM201636 (YM), or with DMSO or only medium, as controls. Parasitemia was monitored by flow cytometry. The numbers inside the plots indicate the percentage of GFP‐iRBCs in each condition. Different lines in the same plot represent replicates of the same sample. B) Schizonts obtained from the different culture conditions were injected into mice and parasitemia was monitored by flow cytometry, starting at day 6 after infection. Parasitemia levels are not significantly different between conditions for each time point. iRBCs, infected red blood cells. |
| tra12190-sup-0002-FigureS2.docWord document, 2.4 MB | Figure S2: Kinetics of vacuolation upon addition of YM201636 to Hepa 1‐6 cells. A) Hepa 1‐6 cells were imaged immediately after treatment with 1 µm of YM201636 (YM). Images were acquired every 15 min for 95 min. Scale bar, 10 µm. B) Brighfield microscopy images of Hepa 1‐6 cells treated with 0.2 of μm YM201636 (YM), where no vacuolation is apparent, or 1 µm YM201636, showing strong vacuolation, after 24 h of treatment. Scale bar, 20 µm. C) Cell viability was measured using the MTT assay. Hepa 1‐6 cells were seeded on 48‐well plates and treated or not with 0.2 µm or 1 µm of YM201636 (YM) for 24 h (t1) or 48 h (t2) (*p < 0.05). |
| tra12190-sup-0003-FigureS3.docWord document, 234.5 KB | Figure S3: The number of exo‐erythrocytic forms is not affected in YM201636‐treated cells. Hepa 1‐6 cells, infected with GFP‐Plasmodium berghei parasites, were treated with 1 µm of YM201636 (YM) from 16 hpi to 48 hpi. Widefield microscopy images of parasites, stained with anti‐GFP (green), were acquired and the number of parasites (exo‐erythrocytic forms – EEFs) per microscope field was counted. |
| tra12190-sup-0004-FigureS4.docWord document, 2 MB | Figure S4: YM201636 does not interfere with merosome formation. A) Confocal images of Hepa 1‐6 cells infected with GFP‐Plasmodium berghei for 61 h and treated with 1 µm of YM201636 (YM) from 16 hpi to 61 hpi. Late endosomes/lysosomes were stained with anti‐LAMP1 (red), parasites with anti‐UIS4 and anti‐GFP antibodies (green) and nuclei with DAPI (blue). B) Confocal images of GFP‐P. berghei‐infected Hepa 1‐6 cells at 61 hpi, where budding merosomes can be observed (arrows). ‘Wash’ indicates samples where the YM201636 was washed out from the culture, at 48 hpi. Scale bar, 10 µm. C) Widefield live cell imaging of Hepa 1‐6 cells in culture infected with GFP‐P. berghei for 61 hpi. Merosomes can be observed in the media. ‘Wash’ indicates samples where the YM201636 was washed out from the culture, at 48 hpi. Scale bar, 50 µm. |
| tra12190-sup-0005-FigureS5.docWord document, 2.3 MB | Figure S5: Cell vacuolation is triggered by a deficient PIKfyve function. A) Hepa 1‐6 cells were transduced with GFP‐2xfyveHrs(green) adenoviruses, and fixed after 48 h. B) Hepa 1‐6 cells were transduced with (i) GFP‐PIKfyvewt or (ii) GFP‐PIKfyveK1831E(green) adenoviruses, and fixed after 48 h. Scale bars, 10 µm. C) PIKfyveflox/flox MEFs were transduced with (i) GFP or (ii) GFP‐Cre adenoviruses and brightfield microscopy images were acquired after 120 h. In (i) ‘inset’ and (i) ‘white arrow inset’ a GFP‐expressing cell with no vacuolation is shown. In (ii) ‘inset’ and in (ii) ‘white arrow area’, a high Cre‐expressing cell with strong vacuolation is shown, and in (ii) ‘dashed arrow area’ a low Cre‐expressing cell with less vacuoles is shown. Scale bars, 100 µm. D) Cell viability was measured using the MTT assay. Hepa 1‐6 cells were seeded on 48‐well plates and transduced for 48 h with GFP‐2xfyveHrs, GFP‐PIKfyvewt or GFP‐PIKfyveK1831E. Values were normalized for the number of cells at 24 h. |
| tra12190-sup-0006-FigureS6.docWord document, 388 KB | Figure S6: Parasite size does not change in YM201636‐treated cells before replication. Hepa 1‐6 cells were infected with GFP‐Plasmodium berghei for 16 hpi and treated with 0.2 µm or 1 µm of YM201636 (YM). Widefield microscopy images of parasites, stained with anti‐UIS4 antibody, were acquired and sizes measured using ImageJ software. |
| tra12190-sup-0007-FigureS7.docWord document, 3.9 MB | Figure S7: PIKfyve perturbation interferes with the interaction between Plasmodium parasites and late endosomes/lysosomes. A and B) Hepa 1‐6 cells were transduced with GFP or GFP‐2xfyveHrs adenoviruses 24 h prior to infection with GFP‐Plasmodium berghei parasites for 16 hpi. The PVMs were stained with anti‐UIS4 antibody and late endosomes/lysosomes with anti‐LAMP1 antibody (A) or anti‐CD63 antibody (B). Images were acquired in a confocal microscope. C and D) Hepa 1‐6 cells were transduced with GFP‐PIKfyvewt or GFP‐PIKfyveK1831E adenoviruses 24 h prior to infection with GFP‐P. berghei parasites for 16 hpi. The PVMs were stained with anti‐UIS4 and late endosomes/lysosomes with anti‐LAMP1 antibody (C) or anti‐CD63 antibody (D). Images were acquired in a confocal microscope. Scale bars, 10 µm. |
| tra12190-sup-0008-VideoS1.avivideo/avi, 265.2 KB | Video S1: PI(3)P transiently accumulates around Plasmodium berghei parasites. Hepa 1‐6 cells were transduced with GFP‐2xfyveHrs adenoviruses and infected with RFP‐P. berghei parasites. Time‐lapse confocal microscopy images were acquired at 3 frames per second at 16 hpi. |
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