Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma

Journal name:
Nature
Volume:
465,
Pages:
359–362
Date published:
DOI:
doi:10.1038/nature09022
Received
Accepted

Calcium-regulated exocytosis is a ubiquitous process in eukaryotes, whereby secretory vesicles fuse with the plasma membrane and release their contents in response to an intracellular calcium surge1. This process regulates various cellular functions such as plasma membrane repair in plants and animals2, 3, the discharge of defensive spikes in Paramecium4, and the secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons5. In animal cells, serine/threonine kinases including cAMP-dependent protein kinase, protein kinase C and calmodulin kinases have been implicated in calcium-signal transduction leading to regulated secretion1, 6, 7. Although plants and protozoa also regulate secretion by means of intracellular calcium, the method by which these signals are relayed has not been explained. Here we show that the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) is an essential regulator of calcium-dependent exocytosis in this opportunistic human pathogen. Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress. These phenotypes were recapitulated by using a chemical biology approach in which pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite’s life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, because expression of a resistant mutant kinase reversed sensitivity to the inhibitor. TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates8, suggesting that related CDPKs may have a function in calcium-regulated secretion in other organisms. Because this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T.gondii and related apicomplexans.

At a glance

Figures

  1. TgCDPK1 is essential for the lytic cycle.
    Figure 1: TgCDPK1 is essential for the lytic cycle.

    a, Regulatable, HA9-tagged TgCDPK1 was added to the wild type (WT) to create a merodiploid (mDip). Endogenous TgCDPK1 was replaced with phleomycin resistance (bleR) to generate the cKO. Complementation with c-Myc-tagged mutant alleles (denoted by cKO/‘allele’). UTR, untranslated region; YFP, yellow fluorescent protein. b, Multiplexed PCR analysis of TgCDPK1. bp, base pairs. c, Immunofluorescence analysis of the cKO in the presence or absence of ATc; green, endogenous MIC2; red, HA9 tag; blue, DNA. Scale bar, 5μm. d, Immunoblot of HA9-tagged regulatable and c-Myc-tagged constitutive TgCDPK1 in cKO and complemented strains in the presence or absence of ATc. Aldolase, loading control. e, Plaque formation on fibroblast monolayers, in the presence or absence of ATc for 7 days.

  2. TgCDPK1 is required for phenotypes associated with microneme secretion.
    Figure 2: TgCDPK1 is required for phenotypes associated with microneme secretion.

    a, Types of gliding motility as quantified by videomicroscopy. Student’s t-test; asterisk, P<0.05; means±s.e.m. for n = 4 experiments. b, Invasion of fibroblasts by wild-type, cKO and complemented strains. Extracellular and intracellular parasites were stained differentially and enumerated as described in Supplementary Information. Student’s t-test; three asterisks, P<0.0005; two asterisks, P<0.005; means±s.e.m. for n = 3 experiments. c, Ionophore-induced egress of the cKO in the presence or absence of ATc. The time stamps are coded as minutes:seconds after the addition of calcium ionophore. See Supplementary Movies 1 and 2.

  3. Calcium-dependent microneme secretion requires TgCDPK1.
    Figure 3: Calcium-dependent microneme secretion requires TgCDPK1.

    a, Western blot analysis of microneme protein MIC2 secretion after induction with ethanol for 15min. Dense granule protein-1 (GRA1) shows constitutive secretion of dense granules. Student’s t-test; two asterisks, P<0.005; means±s.e.m. for n = 3 experiments. b, Ionophore-induced permeabilization detected by vacuolar DsRed leakage monitored by fluorescence videomicroscopy of strains after treatment with ATc for 90h. The time stamps are coded as minutes:seconds after the addition of calcium ionophore. CytochalasinD was added to prevent egress. c, d, Quantification of maximal rate (c) and timing (d) of fluorescence loss from rupturing vacuoles. Mann–Whitney test; three asterisks, P<0.0005; two asterisks, P<0.005; n = 3 experiments.

  4. PP1 derivatives specifically inhibit TgCDPK1 and block microneme-mediated functions.
    Figure 4: PP1 derivatives specifically inhibit TgCDPK1 and block microneme-mediated functions.

    a, Alignment of the kinase subdomain V, highlighting the gatekeeper residue. b, Structures of 3-MB-PP1 and 3-BrB-PP1. c, Effect of 5μM 3-MB-PP1 on host cell invasion. Student’s t-test; asterisk, P<0.05; means±s.e.m. for n = 3 experiments. d, Effect of 5μM 3-MB-PP1 on MIC2 secretion. Student’s t-test; asterisk, P<0.05; means±s.e.m. for n = 3 experiments. e, f, Effect of PP1 derivatives on host lysis by T.gondii in the presence or absence of 3-MB-PP1 (e) and 3-Br-PP1 (f). Means±s.e.m. for n = 3 experiments.

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Author information

Affiliations

  1. Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, Missouri 63110, USA

    • Sebastian Lourido,
    • Joel Shuman &
    • L. David Sibley
  2. Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, California 94158, USA

    • Chao Zhang &
    • Kevan M. Shokat
  3. Structural Genomics Consortium, University Toronto, MaRS South Tower, Suite 732, 101 College Street, Toronto, Canada M5G 1L7

    • Raymond Hui

Contributions

S.L. designed and performed the majority of experiments, analysed the data, generated the figures and wrote the manuscript. J.S. performed the video miscopy measurements of motility and analysed the data. R.H. provided key insight into the regulation of CDPKs by calcium. C.Z. and K.M.S. provided inhibitors and insight into the strategy for chemical biology experiments. L.D.S. supervised the project, assisted with experimental design and analyses, and contributed to writing the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

PDF files

  1. Supplementary Information (3.7M)

    This file contains Supplementary Figures 1-4 with legends and Supplementary Tables 1-2.

Movies

  1. Supplementary Movie 1 (3.7M)

    This movie shows ionophore-tiggered egress of cKO parasites grown in the absence of ATc. Groups of intracellular parasites are observed in round vacuoles. The time lapse covers a period of about 5 min during which time parasites start moving and egress normally from the host cells. Time stamp is given as h:min:sec.

  2. Supplementary Movie 2 (2.7M)

    This movie shows cKO vacuoles grown in the presence of ATc fail to egress upon ionophore treatment. Independent parasite vacuoles in five host cells can be observed. The time lapse covers a period of about 5 min, during which time wild type parasites would have normally egressed from the host cells, but the TgCDPK1 depleted parasites remain intracellular. Time stamp is given as h:min:sec.

  3. Supplementary Movie 3 (561K)

    This movie shows PVM permeabilization by DsRed-expressing WT parasites grown in the presence of ATc. Six round parasite vacuoles are shown in three independent host cells. Parasites were treated with ionophore immediately prior to recording, and immobilization with cytochalasin D prevented mechanical rupture by the PVM. The time lapse covers a period of about 9 min during which time DsRed is released from all vacuoles into their respective host cells. Time stamp is given as h:min:sec.

  4. Supplementary Movie 4 (524K)

    This movie shows PVM permeabilization by DsRed-expressing cKO parasites grown in the presence of ATc. The time lapse covers a period of about 9 min during which time only one of the two of vacuoles releases DsRed at a much slower rate than WT. Time stamp is given as h:min:sec.

Additional data