A Forward-genetic Screen Identifies a Negative Regulator of Rapid Ca2+-dependent Cell Egress in the Intracellular Parasite Toxoplasma gondii

J Biol Chem. 2017 Mar 3. pii: jbc.M117.775114. doi: 10.1074/jbc.M117.775114. [Epub ahead of print]

McCoy JM¹, Stewart RJ¹, Uboldi AD¹, Li D², Schröder J¹, Scott NE³, Papenfuss AT¹, Lehane AM², Foster LJ³, Tonkin CJ⁴.

Toxoplasma gondii, like all apicomplexan parasites, uses Ca²⁺signalling pathways to activate gliding motility, which drives tissue dissemination, host cell invasion and egress. A group of plant-like Ca²⁺-dependent protein kinases (CDPKs), transduce cytosolic Ca²⁺ flux into enzymatic activity, but the molecular details of their activity are poorly understood. To investigate how Ca²⁺ signalling 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. These harboured 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 phosphorylation of SCE1 is required to relieve its suppressive activity to potentiate egress, and that CDPK3 may regulate this phosphorylation. In summary, our work has uncovered a novel component and suppressor of Ca²⁺-dependent cell egress during the T. gondii lytic growth.

Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

KEYWORDS:

Ca2+ signalling; Toxoplasma gondii; apicomplexa; forward genetic screen; host cell egress; molecular genetics; parasite; proteomics; signaling

PMID:

28258212

DOI:

10.1074/jbc.M117.775114