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Updated: May 4, 2026
A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants
Published on: February 8, 2012
A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma
Erin Garrison1, Moritz Treeck, Emma Ehret
1University of Idaho, Department of Biological Sciences, Moscow, Idaho, United States of America.
Abstract:
Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.
Insights
Toxoplasma gondii egress relies on TgCDPK3, a calcium-dependent protein kinase. This kinase is essential for parasite escape from host cells and forming latent brain stages in mice.
Area of Science:
- Molecular Parasitology
- Cell Biology
- Host-Pathogen Interactions
Background:
- Egress from host cells is a critical, regulated process for obligate intracellular parasites like Toxoplasma gondii.
- This egress is calcium-dependent and vital for immune evasion and host cell entry, including brain invasion.
- Previous studies identified mutants defective in ionophore-induced egress and death.
Purpose of the Study:
- To identify the genetic basis of defects in Toxoplasma gondii egress.
- To elucidate the role of TgCDPK3 in parasite egress and latent stage formation.
Main Methods:
- Whole genome sequencing of egress-defective mutants.
- Analysis of mutations in identified genes, including TgCDPK3.
- Biochemical studies of recombinant TgCDPK3 variants.
- Complementation assays and genetic knockout of TgCDPK3.
Main Results:
- Four independent mutants shared missense mutations in the TgCDPK3 gene, encoding a calcium-dependent protein kinase.
- Mutations were predicted and biochemically confirmed to affect key regions of TgCDPK3.
- Complementation and knockout studies confirmed TgCDPK3's crucial role in rapid parasite egress and formation of latent brain stages in mice.
- TgCDPK3 was found to be non-essential for the lytic cycle but critical for egress.
Conclusions:
- TgCDPK3 is a critical regulator of Toxoplasma gondii egress from host cells.
- TgCDPK3 plays a vital role in the development of latent parasitic stages in the host brain.
- The kinase is essential for egress but not for the parasite's lytic cycle.
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