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A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells

Nature Microbiology volume 3, pages 447–455 (2018)Cite this article

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A Publisher Correction to this article was published on 06 March 2018

This article has been updated

Abstract

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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Fig. 1: SUB1 and SERA6 are essential for asexual blood stage P. falciparum growth.
Fig. 2: SUB1 and SERA6 play distinct, sequential roles at egress.
Fig. 3: SUB1 is required for PVM disruption and RBCM poration, whereas the ΔSERA6 phenotype mimics egress arrest with the cysteine protease inhibitor E64.
Fig. 4: RBCM rupture is associated with rapid, SERA6-dependent cleavage of host RBC cytoskeleton β-spectrin within its actin-binding domain (ABD).

Change history

  • 06 March 2018

    In the version of this Letter originally published, Michele S. Y. Tan was incorrectly listed as Michele Y. S. Tan due to a technical error. This has now been amended in all online versions of the Letter.

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Acknowledgements

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.

Author information

Author notes

  1. Victoria L. Hale

    Present address: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK

  2. These authors contributed equally: James A. Thomas and Michele S.Y. Tan.

Authors and Affiliations

  1. Malaria Biochemistry Laboratory, The Francis Crick Institute, London, UK

    James A. Thomas, Michele S. Y. Tan, Fiona Hackett & Michael J. Blackman

  2. Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, London, UK

    Claudine Bisson, Trishant R. Umrekar, Victoria L. Hale & Helen R. Saibil

  3. Protein Analysis and Proteomics Platform, The Francis Crick Institute, London, UK

    Aaron Borg & Ambrosius P. Snijders

  4. Centre for Ultrastructural Imaging, Kings College London, London, UK

    Gema Vizcay-Barrena & Roland A. Fleck

  5. Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK

    Michael J. Blackman

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Contributions

J.A.T. performed all P. falciparum genetic manipulations and phenotype analysis. M.S.Y.T. performed phenotype analysis and parasite manipulation. F.H. performed parasite manipulation. G.V.B. and R.A.F. performed SEM. C.B., T.R.U. and V.L.H. performed and interpreted TEM. A.B., M.S.Y.T. and B.S. performed and interpreted proteomic analysis. J.A.T., M.S.Y.T., B.S., H.R.S. and M.J.B. conceived the study, designed experiments, interpreted results and wrote the manuscript.

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Correspondence to Michael J. Blackman.

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A correction to this article is available online at https://doi.org/10.1038/s41564-018-0134-6.

Supplementary information

Supplementary Information

Supplementary Figures 1–16, Supplementary Table 1, Supplementary References.

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Videos

Supplementary Video 1

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

Supplementary Video 2

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.

Supplementary Video 3

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.

Supplementary Video 4

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.

Supplementary Video 5

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

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