Host cell autophagy contributes to Plasmodium liver development
CEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
These authors contributed equally to this work.Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
These authors contributed equally to this work.Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorMalaria Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649‐028 Lisboa, Portugal
Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorInstitute of Ophthalmology, University College London, London, EC1V 9EL UK
Search for more papers by this authorMalaria Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649‐028 Lisboa, Portugal
Search for more papers by this authorCorresponding Author
CEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
For correspondence. E‐mail miguel.seabra@fcm.unl.pt; Tel. (+351) 218803033.
E‐mail duarte.barral@fcm.unl.pt; Tel. (+351) 218803033.
Search for more papers by this authorCorresponding Author
CEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, SW7 2AZ UK
For correspondence. E‐mail miguel.seabra@fcm.unl.pt; Tel. (+351) 218803033.
E‐mail duarte.barral@fcm.unl.pt; Tel. (+351) 218803033.
Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
These authors contributed equally to this work.Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
These authors contributed equally to this work.Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorMalaria Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649‐028 Lisboa, Portugal
Search for more papers by this authorCEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Search for more papers by this authorInstitute of Ophthalmology, University College London, London, EC1V 9EL UK
Search for more papers by this authorMalaria Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649‐028 Lisboa, Portugal
Search for more papers by this authorCorresponding Author
CEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
For correspondence. E‐mail miguel.seabra@fcm.unl.pt; Tel. (+351) 218803033.
E‐mail duarte.barral@fcm.unl.pt; Tel. (+351) 218803033.
Search for more papers by this authorCorresponding Author
CEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169‐056 Lisboa, Portugal
Instituto Gulbenkian de Ciência, 2780‐156 Oeiras, Portugal
Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, SW7 2AZ UK
For correspondence. E‐mail miguel.seabra@fcm.unl.pt; Tel. (+351) 218803033.
E‐mail duarte.barral@fcm.unl.pt; Tel. (+351) 218803033.
Search for more papers by this authorSummary
Autophagy plays an important role in the defence against intracellular pathogens. However, some microorganisms can manipulate this host cell pathway to their advantage. In this study, we addressed the role of host cell autophagy during Plasmodium berghei liver infection. We show that vesicles containing the autophagic marker LC3 surround parasites from early time‐points after invasion and throughout infection and colocalize with the parasitophorous vacuole membrane. Moreover, we show that the LC3‐positive vesicles that surround Plasmodium parasites are amphisomes that converge from the endocytic and autophagic pathways, because they contain markers of both pathways. When the host autophagic pathway was inhibited by silencing several of its key regulators such as LC3, Beclin1, Vps34 or Atg5, we observed a reduction in parasite size. We also found that LC3 surrounds parasites in vivo and that parasite load is diminished in a mouse model deficient for autophagy. Together, these results show the importance of the host autophagic pathway for parasite development during the liver stage of Plasmodium infection.
Supporting Information
Fig. S1. GFP LC3‐positive vesicles surround Plasmodium berghei parasites throughout liver infection. Hepa 1‐6 cells transduced with GFP‐LC3wt, in green, were infected with GFP‐P. berghei parasites, and infection was stopped at 1 , 2, 16 or 40 hour post‐infection (hpi). Parasites were stained for UIS4, in red, and with DAPI to detect nuclei, in blue. Representative images for each time‐point are shown. Scale bars, 10 µm.
Fig. S2. A late parasitophorous vacuole membrane marker, EXP1, co‐localizes with host LC3. Hepa 1‐6 cells expressing GFP‐LC3 were infected with RFP‐P. berghei parasites, and infection was stopped at 40 hpi. Infected cells were stained for EXP1, in red. Scale bar, 10 µm.
Fig. S3. The autophagic pathway is activated in P. berghei‐infected cells. (A) Representative images of Hepa 1‐6 cells infected with P. berghei parasites for 16 or 44 hpi. Cells were stained for Hsp70 (2E6), in green, LC3, in red, and with DAPI to detect nuclei, in blue. Scale bars, 10 µm. (B) Mean fluorescence intensity of LC3 in isolated primary hepatocytes infected with P. berghei parasites was quantified per area of infected or non‐infected cells at 16 or 44 hpi. (C) Mean fluorescence intensity of LC3 in Hepa 1‐6 cells infected with P. yoelii parasites was quantified per area of infected or non‐infected cells at 16 and 44 hpi.
Fig. S4. NH4Cl inhibits vesicle acidification and GFP‐LC3 degradation. Hepa 1‐6 cells were transduced with GFP‐mCherry‐LC3 and treated with NH4Cl to inhibit vesicle acidification. GFP signal is shown in green and mCherry in red. Scale bar, 10 µm.
Fig. S5. Plasmodium liver forms are surrounded by amphisomes. Time‐lapse images of Hepa 1‐6 cells transduced with GFP‐mCherry‐LC3 and infected with P. berghei sporozoites were imaged at 3 frames/?s at 16 or 40 hpi. GFP signal is shown in green and mCherry in red. Arrows point to the PV. Scale bar, 10 µm.
Fig. S6. LC3 protein levels are decreased in LC3‐deficient cells. (A) mRNA expression levels of LC3 in lentiviral stable cell lines deficient for LC3. (B) Immunoblot and quantification of Hepa 1‐6 cells deficient for LC3. Cells were treated with EBSS (starvation media) to induce autophagy or treated with EBSS and NH4Cl to induce autophagy and block LC3 degradation, simultaneously. Blots were incubated with anti‐LC3 or anti‐Calnexin, used as a loading control. (C) Quantification of the endogenous LC3‐II in the immunoblot. Results are presented as the ratio of integrated density of LC3‐II normalized to Calnexin. (D) Immunoblot of Hepa 1‐6 cells deficient for LC3, non‐transduced or transduced with GFP‐LC3wt or with GFP alone. Blots were incubated with antibodies for LC3 and Calnexin, used as a loading control. (E) Quantification of the sum of the endogenous and exogenous LC3. Results are presented as the ratio of integrated density of LC3 signal normalized to Calnexin.
Fig. S7. Characterization of autophagy‐deficient cell lines. mRNA expression levels of Atg5, Beclin and Vps34 in stable cell lines deficient for (A) Atg5 (sh Atg5), (B) Beclin1 (sh Beclin1) and (C) Vps34 (sh Vps34), respectively. Immunoblot and quantification of Hepa 1‐6 cells deficient for (D) Atg5 (sh Atg5), (E) Beclin1 (sh Beclin1) (F) Vps34 (sh Vps34). Cells were treated with EBSS (starvation media), to induce autophagy or with EBSS together with NH4Cl to induce autophagy and block LC3 degradation, simultaneously. Blots were incubated with anti‐LC3 or anti‐Calnexin, used as a loading control. (G) Mean number of nuclei in each autophagy‐depleted cell line at 48 hpi shows that host cell viability is not compromised in any of the cell lines (each field covers 116 mm2). (H) Autophagy‐deficient cells lines were stained for LC3, in green. Nuclei were stained with DAPI, in blue. Scale bar, 50 µm. (I) The number of LC3 dots per cell was quantified per image, using Image J software.
Fig. S8. Plasmodium parasites form merosomes in autophagy‐deficient cell lines. Control and Hepa 1‐6 cells lines silenced for Atg5 (shAtg5), Vps34 (shVps34) or LC3 (shLC3) were infected with Plasmodium parasites (green), and infection was stopped at 63 hpi. Nuclei were stained with DAPI, in blue. Scale bar, 20 µm.
Table S1. List of shRNA sequences used for silencing the autophagy genes LC3, Atg5, Vps34 and Beclin‐1. The sequence with the best silencing efficiency was further used in our studies.
Table S2. List of primers used for qRT‐PCR experiments.
Table S3. List of primers used for cloning experiments.
Table S3. List of primers used for cloning experiments.
Video 1. Autophagic vesicles surrounding parasites, in early infection, are not highly acidic. Hepa 1‐6 cells were transduced with mCherry‐GFP‐LC3 and infected with P. berghei wt parasites. Time‐lapse confocal microscopy images were acquired at 3 frames/?s at 16 hpi.
Video 2. Autophagic vesicles surrounding parasites, in late infection, are not highly acidic. Hepa 1‐6 cells were transduced with mCherry‐GFP‐LC3 and infected with P. berghei wt parasites. Time‐lapse confocal microscopy images were acquired at 3 frames/?s, at 40 hpi.
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