Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress Victoria L. Halea, Jean M. Watermeyera,1, Fiona Hackettb, Gema Vizcay-Barrenac, Christiaan van Ooijb, James A. Thomasb, Matthew C. Spinkd, Maria Harkiolakid, Elizabeth Duked, Roland A. Fleckc, Michael J. Blackmanb,e, and Helen R. Saibila,2 aCrystallography, Institute of Structural and Molecular Biology, Birkbeck College, London, WC1E 7HX, United Kingdom; bFrancis Crick Institute, London, NW1 1AT, United Kingdom; cCentre for Ultrastructural Imaging, Kings College London, London, SE1 9RT, United Kingdom; dDiamond Light Source, Didcot, OX11 0DE, United Kingdom; and eFaculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom Edited by John C. Boothroyd, Stanford University Medical Center, Stanford, CA, and approved February 15, 2017 (received for review December 1, 2016) In the asexual blood stages of malarial infection, merozoites invade single pore, followed by blebbing and vesiculation at the site of erythrocytes and replicate within a parasitophorous vacuole to form release (4, 5, 7, 8, 11). High-speed video microscopy and modeling daughter cells that eventually exit (egress) by sequential rupture of of the erythrocyte membrane has suggested that the ruptured the vacuole and erythrocyte membranes. The current model is that blood cell membrane then spontaneously curls open and inverts to PKG, a malarial cGMP-dependent protein kinase, triggers egress, facilitate merozoite dispersal (7, 12). However, the single pore is activating malarial proteases and other effectors. Using selective not the only disruption to the membrane as both membrane ves- inhibitors of either PKG or cysteine proteases to separately inhibit icles and ghosts generally remain after egress (5, 13). There is the sequential steps in membrane perforation, combined with video currently no corresponding model for escape from the PVM. microscopy, electron tomography, electron energy loss spectros- The precise biochemical mechanisms mediating egress are not fully Plasmodium copy, and soft X-ray tomography of mature intracellular elucidated, but multiple factors play a role. Minutes before initiation falciparum parasites, we resolve intermediate steps in egress. We show of egress, the malarial cGMP-dependent protein kinase G (PKG) is CELL BIOLOGY that the parasitophorous vacuole membrane (PVM) is permeabilized – activatedtoregulatereleaseofaparasite serine protease called 10 30 min before its PKG-triggered breakdown into multilayered ves- SUB1 from specialized organelles called exonemes into the PV (14). icles. Just before PVM breakdown, the host red cell undergoes an SUB1 has several targets, including a multiprotein merozoite surface abrupt, dramatic shape change due to the sudden breakdown of the complex called MSP1/6/7 and a set of soluble papain-like PV proteins erythrocyte cytoskeleton, before permeabilization and eventual rup- called the SERA family (15–18) that have been implicated in egress ture of the erythrocyte membrane to release the parasites. In contrast – to the previous view of PKG-triggered initiation of egress and a grad- in the blood, liver, and mosquito stages of the parasite life cycle (19 ual dismantling of the host erythrocyte cytoskeleton over the course of 22). Processing of the MSP1/6/7 complex is necessary for efficient schizont development, our findings identify an initial step in egress and escape from the erythrocyte through interaction of SUB1-processed show that host cell cytoskeleton breakdown is restricted to a narrow MSP1 on the surface of the merozoites with spectrin of the host time window within the final stages of egress. erythrocyte cytoskeleton (10). Proteolytic processing by SUB1 of the malaria | egress | electron tomography | soft X-ray microscopy | electron Significance energy loss spectroscopy Malaria parasites develop within red blood cells inside a membrane- he major cause of severe human malaria is Plasmodium enclosed parasitophorous vacuole. An essential step in their life Tfalciparum, and its asexual blood cycle is the source of all cycle is the exit of mature parasites from the blood cell, a multistage clinical disease (1). Egress is an important step in the blood life process termed egress. To do this, the parasites orchestrate a cycle, as it allows daughter merozoites produced by intracellular highly regulated sequence of membrane permeabilization and parasite replication to escape and invade new erythrocytes, breakage steps culminating in the explosive release of parasites thereby continuing and amplifying the infection. Merozoites de- for a new round of infection. Here, we describe a previously velop within a parasitophorous vacuole (PV), a membrane-bound unidentified permeabilization of the vacuolar membrane at the compartment that forms during invasion (2–4), so the daughter start of egress, preceding membrane rupture, suggesting a new parasites have two compartments to escape (5, 6). initiation step in egress. We also show that, in the final minutes Blood-stage malaria parasites replicate by schizogony, in which of egress, the blood cell membrane abruptly loses its structural several rounds of nuclear division form a multinucleated syncy- rigidity and collapses around the parasites, showing a precise tium called a schizont. Individual merozoites are then produced by timing for cytoskeletal breakdown. an unusual form of cytokinesis called budding or segmentation, Author contributions: R.A.F., M.J.B., and H.R.S. designed research; V.L.H., J.M.W., F.H., which involves invagination of the single plasma membrane of the G.V.-B., C.v.O., J.A.T., M.C.S., and M.H. performed research; E.D. contributed new schizont. Minutes before egress, the segmented schizont suddenly reagents/analytic tools; V.L.H. analyzed data; and V.L.H., M.J.B., and H.R.S. wrote the paper. transforms from an irregular to a relatively symmetrical structure The authors declare no conflict of interest. with the merozoites arranged around the central digestive vacuole This article is a PNAS Direct Submission. “ ” (5). This process, referred to as flower formation or rounding Freely available online through the PNAS open access option. up, is usually accompanied by noticeable swelling of the PV and – Data deposition: The data reported in this paper have been deposited in the EM Database apparent shrinkage of the host cell (4, 5, 7 9). The first membrane for EM tomograms (accession nos. EMD-3586, EMD-3587, EMD-3606, and EMD-3610) and to rupture at egress is the parasitophorous vacuole membrane EMPIAR for X-ray tomograms (accession no. EMPIAR-10087). (PVM) (5, 6, 8). When the PV does not occupy the entire infected 1Present address: Pinelands High School, Pinelands, 7405, South Africa. cell, the individual merozoites can be seen to be expelled into the 2To whom correspondence should be addressed. Email: [email protected]. blood cell cytosol seconds before they escape from the erythrocyte This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (8–10). Erythrocyte membrane rupture involves formation of a 1073/pnas.1619441114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1619441114 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 SERA proteins is temporally associated with egress (20, 23–27), and daughter merozoites. Importantly, all of the C1-blocked schizonts at least one P. falciparum SERA family member, SERA6, is likely a examined displayed an intact PVM, in accord with previous evi- cysteine protease that is activated by SUB1 (16). As well as PKG, a dence that PKG inhibitors potently stall egress at a stage before parasite calcium-dependent kinase called CDPK5 has been impli- rounding up and PVM rupture (10, 14, 28, 41). Consistent with cated in egress (28). However, in CDPK5-deficient parasites, SUB1 this, in all partially segmented schizonts we observed a clear con- discharge and MSP1/6/7 and SERA processing are unaffected, trast difference between the material in the vacuole and the ma- suggesting that CDPK5 action is either independent from or terial in the blood cell cytosol, with the red cell cytosol displaying a downstream of the PKG/SUB1/MSP/SERA pathway (28). darker, more electron-dense appearance than the contents of the Events downstream of SUB1 activation and MSP/SERA processing PV (Fig. 1B, Top). This difference in staining indicated that the are less well understood. SUB1 has multiple substrates in addition to contents of these two compartments differed markedly in compo- the SERAs and MSP1/6/7 (29), and other proteases have been sition. Strikingly, many tomograms of fully segmented schizonts proposed to play a role in egress, including the erythrocyte protease from these same C1-stalled preparations showed an equalization of calpain-1 (30–35). Proteomic studies based on analysis of parasite contrast across the PVM (Fig. 1B, Bottom). This contrast equal- populations synchronized by physical techniques have indicated that ization suggested that—despite the apparently intact PVM in these erythrocyte membrane and cytoskeletal proteins are proteolysed cells—the contents of the blood cell cytosol and the vacuole had during or before egress (13, 36). It has been suggested on the basis of undergone mixing. Further examples are shown in Fig. S1. that work and atomic force microscopy (AFM) that the host cell To directly assess the composition of the two compartments, we cytoskeleton