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

Abstract

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.

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Figure 1: TgCDPK1 is essential for the lytic cycle.
Figure 2: TgCDPK1 is required for phenotypes associated with microneme secretion.
Figure 3: Calcium-dependent microneme secretion requires TgCDPK1.
Figure 4: PP1 derivatives specifically inhibit TgCDPK1 and block microneme-mediated functions.

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Acknowledgements

We thank O. Billker, G. Ward, S. Moreno and V. Carruthers for discussions; F. Dzierszinski for the DsRed plasmid; D. Soldati for the Tet-transactivator system; and K. Tang for technical assistance. This work was supported by a predoctoral fellowship from the American Heart Association (S.L.) and a grant from the National Institutes of Health (L.D.S.).

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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to L. David Sibley.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-4 with legends and Supplementary Tables 1-2. (PDF 3880 kb)

Supplementary Movie 1

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. (MOV 3868 kb)

Supplementary Movie 2

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. (MOV 2858 kb)

Supplementary Movie 3

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. (MOV 561 kb)

Supplementary Movie 4

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. (MOV 524 kb)

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Lourido, S., Shuman, J., Zhang, C. et al. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465, 359–362 (2010). https://doi.org/10.1038/nature09022

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