Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Blackman, M. J. Malarial proteases and host cell egress: an ‘emerging’ cascade. Cell Microbiol. 10, 1925–1934 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Yeoh, S. et al. Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell 131, 1072–1083 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Collins, C. R. et al. Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog. 9, e1003344 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Withers-Martinez, C. et al. The malaria parasite egress protease SUB1 is a calcium-dependent redox switch subtilisin. Nat. Commun. 5, 3726 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Das, S. et al. Processing of Plasmodium falciparum merozoite surface protein MSP1 activates a spectrin-binding function enabling parasite egress from RBCs. Cell Host Microbe 18, 433–444 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hale, V. et al. Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress. Proc. Natl Acad. Sci. USA 114, 3439–3444 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Koussis, K. et al. A multifunctional serine protease primes the malaria parasite for red blood cell invasion. EMBO J. 28, 725–735 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Silmon de Monerri, N. C. et al. Global identification of multiple substrates for Plasmodium falciparum SUB1, an essential malarial processing protease. Infect. Immun. 79, 1086–1097 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Collins, C. R., Hackett, F., Atid, J., Tan, M. S. Y. & Blackman, M. J. The Plasmodium falciparum pseudoprotease SERA5 regulates the kinetics and efficiency of malaria parasite egress from host erythrocytes. PLoS Pathog. 13, e1006453 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Ruecker, A. et al. Proteolytic activation of the essential parasitophorous vacuole cysteine protease SERA6 accompanies malaria parasite egress from its host erythrocyte. J. Biol. Chem. 287, 37949–37963 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Miller, S. K. et al. A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle. J. Biol. Chem. 277, 47524–47532 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Thomas, J. A. et al. Development and application of a simple plaque assay for the human malaria parasite Plasmodium falciparum. PloS ONE 11, e0157873 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Glushakova, S., Yin, D., Li, T. & Zimmerberg, J. Membrane transformation during malaria parasite release from human red blood cells. Curr. Biol. 15, 1645–1650 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Glushakova, S. et al. New stages in the program of malaria parasite egress imaged in normal and sickle erythrocytes. Curr. Biol. 20, 1117–1121 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wickham, M. E., Culvenor, J. G. & Cowman, A. F. Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte. J. Biol. Chem. 278, 37658–37663 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Abkarian, M., Massiera, G., Berry, L., Roques, M. & Braun-Breton, C. A novel mechanism for egress of malarial parasites from red blood cells. Blood 117, 4118–4124 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Taylor, H. M. et al. The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony. Eukaryot. Cell 9, 37–45 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Glushakova, S., Mazar, J., Hohmann-Marriott, M. F., Hama, E. & Zimmerberg, J. Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes. Cell Microbiol. 11, 95–105 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Collins, C. R. et al. Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol. Microbiol. 88, 687–701 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jones, M. L. et al. A versatile strategy for rapid conditional genome engineering using loxP sites in a small synthetic intron in Plasmodium falciparum. Sci. Rep. 6, 21800 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ribacke, U. et al. Improved in vitro culture of Plasmodium falciparum permits establishment of clinical isolates with preserved multiplication, invasion and rosetting phenotypes. PloS ONE 8, e69781 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wirth, C. C. et al. Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes. Cell Microbiol. 16, 709–733 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Simmons, D., Woollett, G., Bergin-Cartwright, M., Kay, D. & Scaife, J. A malaria protein exported into a new compartment within the host erythrocyte. EMBO J. 6, 485–491 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lux, S. E. Anatomy of the red cell membrane skeleton: Unanswered questions. Blood 127, 187–199 (2016).

    Article  CAS  PubMed  Google Scholar 

  25. An, X. et al. Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: Regulation of the interactions by phosphatidylinositol-4,5-bisphosphate. Biochemistry 44, 10681–10688 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Karinch, A. M., Zimmer, W. E. & Goodman, S. R. The identification and sequence of the actin-binding domain of human red blood cell beta-spectrin. J. Biol. Chem. 265, 11833–11840 (1990).

    CAS  PubMed  Google Scholar 

  27. Deligianni, E. et al. A perforin-like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes. Cell. Microbiol. 15, 1438–1455 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Burda, P. C. et al. A Plasmodium phospholipase is involved in disruption of the liver stage parasitophorous vacuole membrane. PLoS Pathog. 11, e1004760 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Chandramohanadas, R. et al. Apicomplexan parasites co-opt host calpains to facilitate their escape from infected cells. Science 324, 794–797 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Baker, D. A. et al. A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission. Nat. Commun. 8, 430 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Holder, A. A. & Freeman, R. R. Biosynthesis and processing of a Plasmodium falciparum schizont antigen recognized by immune serum and a monoclonal antibody. J. Exp. Med. 156, 1528–1538 (1982).

    Article  CAS  PubMed  Google Scholar 

  32. Withers-Martinez, C. et al. Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells: Characterization, comparison with the parasite protease, and homology modelling. J. Biol. Chem. 277, 29698–29709 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Collins, C. R., Withers-Martinez, C., Hackett, F. & Blackman, M. J. An inhibitory antibody blocks interactions between components of the malarial invasion machinery. PLoS Pathog. 5, e1000273 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Blackman, M. J. Purification of Plasmodium falciparum merozoites for analysis of the processing of merozoite surface protein-1. Methods Cell Biol. 45, 213–220 (1994).

    Article  CAS  PubMed  Google Scholar 

  35. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  36. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  37. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. MacLean, B. et al. Skyline: An open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Michael J. Blackman.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Life Sciences Reporting Summary

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-018-0111-0

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing