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Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms

Abstract

We established a conditional site-specific recombination system based on dimerizable Cre recombinase−mediated recombination in the apicomplexan parasite Toxoplasma gondii. Using a new single-vector strategy that allows ligand-dependent, efficient removal of a gene of interest, we generated three knockouts of apicomplexan genes considered essential for host-cell invasion. Our findings uncovered the existence of an alternative invasion pathway in apicomplexan parasites.

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Figure 1: Conditional Cre recombinase system in T. gondii.
Figure 2: Using the DiCre system to dissect the invasion machinery.

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Acknowledgements

We thank D. Soldati-Favre (University of Geneva), C.J. Beckers (University of North Carolina, Chapel Hill), A. Scherf (Pasteur Institute, Paris), J.F. Dubremetz (University of Montpellier), V. Carruthers (University of Michigan, Ann Arbor), B. Striepen (University of Georgia, Athens) and D.L. Sibley (Washington University) for sharing reagents, and members of the FACS facility of the Institute of Infection, Immunity and Inflammation at the University of Glasgow for their support. This work was supported by the Wellcome Trust. M.M. is funded by a Wellcome Trust Senior Fellowship (087582/Z/08/Z). N.A. is supported by an EviMalaR (European FP7/2007-2013, grant number 242095) PhD fellowship, and S.E. was funded via Signalling in life cycle stages of malaria parasites (MALSIG) (European FP7/2009-2012, grant number 223044). The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust (085349).

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Authors and Affiliations

Authors

Contributions

N.A. established the DiCre system and generated and analyzed the myoA knockout parasites. A.J.J. generated and analyzed the mic2 knockout. S.E. generated and analyzed the conditional act1 knockout. N.J. and J.-P.H. shared confidential information for the establishment of the DiCre system. M.M. initiated and guided this study. M.M., A.J.J., N.A. and S.E. wrote the manuscript.

Corresponding author

Correspondence to Markus Meissner.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1, Supplementary Discussion, Supplementary Note (PDF 12842 kb)

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Andenmatten, N., Egarter, S., Jackson, A. et al. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat Methods 10, 125–127 (2013). https://doi.org/10.1038/nmeth.2301

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