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Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum

Nature Chemical Biology volume 4, pages 203–213 (2008)Cite this article

Abstract

Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.

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Figure 1: Assembly and screening of the protease inhibitor library.
Figure 2: Identification of PfSUB1 as the target of JCP104.
Figure 3: Inhibition of PfSUB1 correlates with a block in rupture.
Figure 4: Identification of DPAP1 and DPAP3 as targets of JCP405 and JCP410.
Figure 5: Design of selective DPAP3 and DPAP1 inhibitors.
Figure 6: PfSUB1 and DPAP3 mediate SERA5 processing.
Figure 7: The protease pathway involved in schizont rupture.

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Acknowledgements

The authors thank M. Blackman (UK National Institute for Medical Research) for sharing his data on SERA5 processing before its publication and for providing reagents for PfSUB1. We also thank M. Klemba (Virginia Tech) for valuable information about the DPAPs and the DPAP1-specific antibodies. We thank J. Powers (University of Georgia) and W. Roush (The Scripps Research Institute) for the directed irreversible inhibitors of serine and cysteine proteases. We thank J.-F. Dubremetz (Université de Montpellier 2) for providing the SERA5-specific antibody. We thank C. Yang for generating compound toxicity data, S. Verhelst for analysis of NMR data and A. Guzzetta for high-resolution mass spectrometry analysis of compounds. This work was supported by funding from the Kinship Foundation as part of the Searle Scholars program (to M.B.), from a Burroughs Wellcome Trust New Investigators in Pathogenesis Award (to M.B.) and by the US National Institutes of Health National Technology Center for Networks and Pathways grants U54 RR020843 and R01 EB005011 (to M.B.). S.A.K. is supported by the US National Institutes of Health Research Fellowship Award F32 AI069728-01A1. E.L.P. is supported by the US National Science Foundation Graduate Research Fellowship 2005021299. C.I.P. is supported by an American Society for Microbiology Watkins Fellowship.

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Author notes

  1. Fang Yuan

    Present address: Present address: EnzMed (Nanjing) Co., Ltd., 28 Hengjing Road, Nanjing, China 210046.,

Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, 94305, California, USA

    Shirin Arastu-Kapur, Urša Pečar Fonović, Fang Yuan, Marko Fonović & Matthew Bogyo

  2. Department of Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, 94305, California, USA

    Elizabeth L Ponder & Carolyn I Phillips

  3. Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

    Sharon Yeoh & Munira Grainger

  4. Department of Biochemistry, Molecular and Structural Biology, Jozef Stefan Institute, Jamova 39, Ljubljana, SI-1000, Slovenia

    Marko Fonović

  5. Department of Chemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, 30332, Georgia, USA

    James C Powers

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Arastu-Kapur, S., Ponder, E., Fonović, U. et al. Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum. Nat Chem Biol 4, 203–213 (2008). https://doi.org/10.1038/nchembio.70

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