title: Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse inPlasmodium falciparumegress.
abstract: In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellularPlasmodium falciparumparasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.
authors.affiliation: Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, London, WC1E 7HX, United Kingdom.
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Hale
authors.affiliation: Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, London, WC1E 7HX, United Kingdom.
authors.forename: Jean M
authors.initials: JM
authors.lastname: Watermeyer
authors.affiliation: Francis Crick Institute, London, NW1 1AT, United Kingdom.
authors.forename: Fiona
authors.initials: F
authors.lastname: Hackett
authors.affiliation: Centre for Ultrastructural Imaging, Kings College London, London, SE1 9RT, United Kingdom.
authors.forename: Gema
authors.initials: G
authors.lastname: Vizcay-Barrena
authors.affiliation: Francis Crick Institute, London, NW1 1AT, United Kingdom.
authors.forename: Christiaan
authors.initials: C
authors.lastname: van Ooij
authors.affiliation: Francis Crick Institute, London, NW1 1AT, United Kingdom.
authors.forename: James A
authors.initials: JA
authors.lastname: Thomas
authors.affiliation: Diamond Light Source, Didcot, OX11 0DE, United Kingdom.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Spink
authors.affiliation: Diamond Light Source, Didcot, OX11 0DE, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Harkiolaki
authors.affiliation: Diamond Light Source, Didcot, OX11 0DE, United Kingdom.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Duke
authors.affiliation: Centre for Ultrastructural Imaging, Kings College London, London, SE1 9RT, United Kingdom.
authors.forename: Roland A
authors.initials: RA
authors.lastname: Fleck
authors.affiliation: Francis Crick Institute, London, NW1 1AT, United Kingdom.|Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Blackman
authors.affiliation: Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, London, WC1E 7HX, United Kingdom; h.saibil@mail.cryst.bbk.ac.uk.
authors.forename: Helen R
authors.initials: HR
authors.lastname: Saibil
mesh_terms: D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000469:parasitology*; D004912:Erythrocytes / Q000378:metabolism / Q000469:parasitology*; D006801:Humans; D016778:Malaria, Falciparum / Q000235:genetics / Q000378:metabolism / Q000469:parasitology*; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D017869:Cyclic GMP-Dependent Protein Kinases
keywords: egress; electron energy loss spectroscopy; electron tomography; malaria; soft X-ray microscopy