Abstract
Toxoplasma gondii establishes a lifelong chronic infection in humans and animals1. Host cell entry and egress are key steps in the lytic cycle of this obligate intracellular parasite, ensuring its survival and dissemination. Egress is temporally orchestrated, underpinned by the exocytosis of secretory organelles called micronemes. At any point during intracellular replication, deleterious environmental changes such as the loss of host cell integrity can trigger egress2 through the activation of the cyclic guanosine monophosphate-dependent protein kinase G3. Notably, even in the absence of extrinsic signals, the parasites egress from infected cells in a coordinated manner after five to six cycles of endodyogeny multiplication. Here we show that diacylglycerol kinase 2 is secreted into the parasitophorous vacuole, where it produces phosphatidic acid. Phosphatidic acid acts as an intrinsic signal that elicits natural egress upstream of an atypical guanylate cyclase (GC), which is uniquely conserved in alveolates4 and ciliates5, and composed of a P4-ATPase and two GC catalytic domains. Assembly of GC at the plasma membrane depends on two associated cofactors — the cell division control 50.1 and a unique GC organizer. This study reveals the existence of a signalling platform that responds to an intrinsic lipid mediator and extrinsic signals to control programmed and induced egress.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
References
Blader, I. J., Coleman, B. I., Chen, C. T. & Gubbels, M. J. Lytic cycle of Toxoplasma gondii: 15 years later. Annu. Rev. Microbiol. 69, 463–485 (2015).
Moudy, R., Manning, T. J. & Beckers, C. J. The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii. J. Biol. Chem. 276, 41492–41501 (2001).
Brown, K. M., Long, S. & Sibley, L. D. Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii. MBio 8, e00375-17 (2017).
Baker, D. A. et al. Cyclic nucleotide signalling in malaria parasites. Open Biol. 7, 170213 (2017).
Linder, J. U., Hoffmann, T., Kurz, U. & Schultz, J. E. A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases. J. Biol. Chem. 275, 11235–11240 (2000).
Roiko, M. S. & Carruthers, V. B. New roles for perforins and proteases in apicomplexan egress. Cell. Microbiol. 11, 1444–1452 (2009).
Brochet, M. et al. Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca2+ signals at key decision points in the life cycle of malaria parasites. PLoS Biol. 12, e1001806 (2014).
Bullen, H. E. et al. Phosphatidic acid-mediated Signaling regulates microneme secretion in Toxoplasma. Cell Host Microbe 19, 349–360 (2016).
Farrell, A. et al. A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis. Science 335, 218–221 (2012).
Roiko, M. S., Svezhova, N. & Carruthers, V. B. Acidification activates Toxoplasma gondii motility and egress by enhancing protein secretion and cytolytic activity. PLoS Pathog. 10, e1004488 (2014).
Nagamune, K. et al. Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii. Nature 451, 207–210 (2008).
Bullen, H. E. & Soldati-Favre, D. A central role for phosphatidic acid as a lipid mediator of regulated exocytosis in apicomplexa. FEBS Lett. 590, 2469–2481 (2016).
Jia, Y. et al. Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii. EMBO J. 36, 3250–3267 (2017).
Schaap, D., van der Wal, J. & van Blitterswijk, W. J. Consensus sequences for ATP-binding sites in protein kinases do not apply to diacylglycerol kinases. Biochem. J. 304, 661–662 (1994).
Mercier, C. et al. Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins. Mol. Biol. Cell 13, 2397–2409 (2002).
Kuhn, M. Molecular physiology of membrane guanylyl cyclase receptors. Physiol. Rev. 96, 751–804 (2016).
Takada, N. et al. Phospholipid-flipping activity of P4-ATPase drives membrane curvature. EMBO J. 37, e97705 (2018).
Sidik, S. M. et al. Using a genetically encoded Sensor to identify inhibitors of Toxoplasma gondii Ca2+ signaling. J. Biol. Chem. 291, 9566–9580 (2016).
Frénal, K. et al. Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii. Nat. Commun. 8, 15710 (2017).
Takatsu, H. et al. Phospholipid flippase activities and substrate specificities of human type IV P-type ATPases localized to the plasma membrane. J. Biol. Chem. 289, 33543–33556 (2014).
Kühlbrandt, W. Biology, structure and mechanism of P-type ATPases. Nat. Rev. Mol. Cell Biol. 5, 282–295 (2004).
Segawa, K., Kurata, S. & Nagata, S. The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane. J. Biol. Chem. 293, 2172–2182 (2018).
Caldas, L. A., Attias, M. & de Souza, W. A structural analysis of the natural egress of Toxoplasma gondii. Microbes Infect. 20, 57–62 (2018).
Tomita, T., Yamada, T., Weiss, L. M. & Orlofsky, A. Externally triggered egress is the major fate of Toxoplasma gondii during acute infection. J. Immunol. 183, 6667–6680 (2009).
Persson, E. K. et al. Death receptor ligation or exposure to perforin trigger rapid egress of the intracellular parasite Toxoplasma gondii. J. Immunol. 179, 8357–8365 (2007).
Okada, T. et al. A novel dense granule protein, GRA22, is involved in regulating parasite egress in Toxoplasma gondii. Mol. Biochem. Parasitol. 189, 5–13 (2013).
LaFavers, K. A., Márquez-Nogueras, K. M., Coppens, I., Moreno, S. N. J. & Arrizabalaga, G. A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity in Toxoplasma gondii. Cell Microbiol. 19, e12749 (2017).
Brown, K. M., Lourido, S. & Sibley, L. D. Serum albumin stimulates Protein kinase G-dependent microneme secretion in Toxoplasma gondii. J. Biol. Chem. 291, 9554–9565 (2016).
Gao, H. et al. ISP1-anchored polarization of GCbeta/CDC50A Complex initiates malaria ookinete gliding Motility. Curr. Biol. 28, 2763–2776 (2018).
Meissner, M., Schlüter, D. & Soldati, D. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298, 837–840 (2002).
Soldati, D. & Boothroyd, J. C. Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260, 349–352 (1993).
Donald, R. G., Carter, D., Ullman, B. & Roos, D. S. Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation. J. Biol. Chem. 271, 14010–14019 (1996).
Kim, K., Soldati, D. & Boothroyd, J. C. Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker. Science 262, 911–914 (1993).
Donald, R. G. & Roos, D. S. Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria. Proc. Natl Acad. Sci. USA 90, 11703–11707 (1993).
Millholland, M. G. et al. A host GPCR signaling network required for the cytolysis of infected cells facilitates release of apicomplexan parasites. Cell Host Microbe 13, 15–28 (2013).
Sato, M. et al. Evaluations of the selectivities of the diacylglycerol kinase inhibitors R59022 and R59949 among diacylglycerol kinase isozymes using a new non-radioactive assay method. Pharmacology 92, 99–107 (2013).
Gaskins, E. et al. Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. J. Cell. Biol. 165, 383–393 (2004).
Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).
Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinform. 5, 113 (2004).
Dereeper, A. et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469 (2008).
Acknowledgements
This research was supported by Swiss National Science Foundation (FN3100A0-116722 to D.S.-F. and BSSGI0-155852 to M.B.) and H.B. is the recipient of a Swiss Government Excellence Scholarship with Uruguay. We thank N. Klages, J. B. Marq for their technical contributions to the project; H. Bullen and N. Tosetti for the preliminary investigations on DGKs; D. Sibley and K. Brown for sharing the mAID system prior to publication and for advice; members of the proteomics, bioimaging and flow-cytometry core facilities at the Faculty of Medicine of the University of Geneva; and all meBOP students of 2018 for repeating some of the presented experiments and critically challenging the proposed model.
Author information
Authors and Affiliations
Contributions
D.S.-F. and H.B. conceived the project. M.B. provided insightful discussions and constructive suggestions. H.B. and M.L. designed, performed and interpreted the experimental work. D.S.-F. supervised the research. H.B. and D.S.-F. wrote the paper with editorial support from M.L.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Information
Supplementary Figures 1–3, Supplementary Figures 5 and 6, and Supplementary Table 3.
Supplementary Figure 4
Alignment of full length UGO orthologue genes. Output of the sequence alignment obtained with MUSCLE was curated manually utilizing BioEdit.
Supplementary Table 1
Number of unique spectral counts detected for GC interactors.
Supplementary Table 2
Primers used in this study.
Rights and permissions
About this article
Cite this article
Bisio, H., Lunghi, M., Brochet, M. et al. Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform. Nat Microbiol 4, 420–428 (2019). https://doi.org/10.1038/s41564-018-0339-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41564-018-0339-8
This article is cited by
-
Lipid metabolism: the potential targets for toxoplasmosis treatment
Parasites & Vectors (2024)
-
Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii
Nature Protocols (2024)
-
Calreticulin (CALR) promotes ionophore-induced microneme secretion in Toxoplasma gondii
Parasitology Research (2024)
-
A splitCas9 phenotypic screen in Toxoplasma gondii identifies proteins involved in host cell egress and invasion
Nature Microbiology (2022)
-
Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress
Nature Microbiology (2022)