Abstract
Toxoplasma gondii, like all apicomplexan parasites, uses Ca2+ signaling pathways to activate gliding motility to power tissue dissemination and host cell invasion and egress. A group of "plant-like" Ca2+-dependent protein kinases (CDPKs) transduces cytosolic Ca2+ flux into enzymatic activity, but how they function is poorly understood. To investigate how Ca2+ signaling activates egress through CDPKs, we performed a forward genetic screen to isolate gain-of-function mutants from an egress-deficient cdpk3 knockout strain. We recovered mutants that regained the ability to egress from host cells that harbored mutations in the gene Suppressor of Ca2+-dependent Egress 1 (SCE1). Global phosphoproteomic analysis showed that SCE1 deletion restored many Δcdpk3-dependent phosphorylation events to near wild-type levels. We also show that CDPK3-dependent SCE1 phosphorylation is required to relieve its suppressive activity to potentiate egress. In summary, our work has uncovered a novel component and suppressor of Ca2+-dependent cell egress during Toxoplasma lytic growth.
Highlights
Introduction of phosphomimeticSuppressor of Ca2؉-dependent Egress 1 (SCE1) mutants into ⌬cdpk3⌬sce1 resulted in intermediate levels of egress (ϳ50%) (Fig. 6Diii)
Egress-deficient ⌬cdpk3 parasites were split into 10 independent populations and mutagenized using the chemical mutagen ethyl methane sulfonate (EMS)
We have shown previously that CDPK3 in Toxoplasma is critical for host cell egress [11], but how this kinase regulates egress remains unexplained
Summary
Toxoplasma gondii, like all apicomplexan parasites, uses Ca2؉ signaling pathways to activate gliding motility to power tissue dissemination and host cell invasion and egress. We show that CDPK3-dependent SCE1 phosphorylation is required to relieve its suppressive activity to potentiate egress. Toxoplasma initiates motility, invasion, and egress via an intracellular signaling cascade that activates exocytosis of adhesins from the microneme organelles and stimulates the glideosome, an actomyosin-based motor, required to drive motility. Ca2ϩ, cGMP, and phospholipid signaling pathways all play important and interconnected roles in activating microneme release and parasite motility (4 – 6). 1 To whom correspondence should be addressed: The Walter and Eliza Hall We show that the functions of CDPK3 and SCE1 are interconnected and that phosphorylation of SCE1 by CDPK3 controls its suppressive activity during host cell egress
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