Malaria parasites replicate within a parasitophorous vacuole in red blood cells (RBCs). Progeny merozoites egress upon rupture of first the parasitophorous vacuole membrane (PVM), then poration and rupture of the RBC membrane (RBCM). Egress is protease-dependent1, but none of the effector molecules that mediate membrane rupture have been identified and it is unknown how sequential rupture of the two membranes is controlled. Minutes before egress, the parasite serine protease SUB1 is discharged into the parasitophorous vacuole2,3,4,5,6 where it cleaves multiple substrates2,5,7,8,9 including SERA6, a putative cysteine protease10,11,12. Here, we show that Plasmodium falciparum parasites lacking SUB1 undergo none of the morphological transformations that precede egress and fail to rupture the PVM. In contrast, PVM rupture and RBCM poration occur normally in SERA6-null parasites but RBCM rupture does not occur. Complementation studies show that SERA6 is an enzyme that requires processing by SUB1 to function. RBCM rupture is associated with SERA6-dependent proteolytic cleavage within the actin-binding domain of the major RBC cytoskeletal protein β-spectrin. We conclude that SUB1 and SERA6 play distinct, essential roles in a coordinated proteolytic cascade that enables sequential rupture of the two bounding membranes and culminates in RBCM disruption through rapid, precise, SERA6-mediated disassembly of the RBC cytoskeleton.
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This work was supported by funding to M.J.B. from the Francis Crick Institute (https://www.crick.ac.uk/), which receives its core funding from Cancer Research UK (FC001043; https://www.cancerresearchuk.org), the UK Medical Research Council (FC001043; https://www.mrc.ac.uk/) and the Wellcome Trust (FC001043; https://wellcome.ac.uk/). J.A.T. and M.S.Y.T. were in receipt of Crick PhD studentships, and V.L.H. was supported by Gatan BBSRC CASE PhD studentship BB/F016948/1. The work was also supported by MRC project grants G1100013 and MR/P010288/1 (H.R.S., M.J.B. and R.A.F.), Wellcome equipment grants 101488, 079605 and 086018 (H.R.S., M.J.B. and R.A.F.) and Wellcome ISSF2 funding to the London School of Hygiene & Tropical Medicine.
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A correction to this article is available online at https://doi.org/10.1038/s41564-018-0134-6.
Composite DIC time-lapse video showing the different fates of control and RAP-treated (ΔSUB1) SUB1HA3:loxP schizonts following washing away of the PKG inhibitor C2 (elapsed time indicated). The ΔSUB1 parasites undergo none of the morphological changes associated with egress
Genetic complementation of the ΔSUB1 egress defect by a WT SUB1 transgene. Simultaneous DIC and fluorescence time-lapse video showing normal egress of RAP-treated (ΔSUB1) SUB1HA3:loxP schizonts harbouring a transgene expression construct for expression of the WT SUB1 gene and mCherry. Elapsed time following washing away of the PKG inhibitor C2 is indicated.
Composite DIC time-lapse video showing the different fates of control and RAP-treated (ΔSERA6) SERA6:loxP schizonts following washing away of the PKG inhibitor C2 (elapsed time indicated). While PVM rupture appears to take place normally in the ΔSERA6 parasites, rupture of the RBCM does not occur.
Genetic complementation of the ΔSERA6 egress defect by a WT SERA6 transgene. Simultaneous DIC and fluorescence time-lapse video showing normal egress of a RAP-treated (ΔSERA6) SERA6:loxP schizont harbouring a transgene expression construct for expression of the WT SERA6 gene and mCherry. Elapsed time following washing away of the PKG inhibitor C2 is indicated.
SUB1 is required for PVM rupture and all subsequent events leading to egress, whereas SERA6 is required for RBCM rupture but not PVM rupture or RBCM poration. Composite time-lapse video showing fates of control and RAP-treated SUB1HA3:loxP:EXP1mCherry and SERA6:loxP:EXP1mCherry schizonts labelled with fluorescent wheat germ agglutinin (WGA) and in the presence of fluorescent phalloidin. PVM rupture occurs in control and ΔSERA6 parasites, followed by RBCM poration as indicated by phalloidin-labelling of the RBC cytoskeleton. The RBCM then ruptures and vesiculates in control parasites. Indicated, elapsed time following washing away the PKG inhibitor C2.
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Thomas, J.A., Tan, M.S.Y., Bisson, C. et al. A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells. Nat Microbiol 3, 447–455 (2018). https://doi.org/10.1038/s41564-018-0111-0
A malaria parasite subtilisin propeptide-like protein is a potent inhibitor of the egress protease SUB1
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