Abstract
Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.
Highlights
The phylum Apicomplexa is a large group of obligate intracellular parasites of wide medical and agricultural significance
At its core the invasion motor consists of a novel class XIV Myosin, MyoA [3] and Myosin Light Chain 1, MLC1 [4] which is anchored into the outer side of the inner membrane complex (IMC) by the Glideosome-Associated Proteins GAP40, GAP45, GAP50 and GAPM’s [5,6,7,8]
Using a range of proteomics approaches we reveal proteins in Toxoplasma that are phosphorylated upon calcium signaling, and identify phosphorylation sites on a range of proteins that may play crucial roles in regulating parasite motility and microneme secretion
Summary
The phylum Apicomplexa is a large group of obligate intracellular parasites of wide medical and agricultural significance. Toxoplasma gondii infects between 30- 80% of people worldwide and is one the most common infectious agents of humans. Despite each species having a range of host and cell types that they target the intracellular processes governing invasion appear to be largely conserved. A highly conserved actomyosin-based invasion motor that drives parasite motility and invasion was first identified and has been largely characterized in Toxoplasma. At its core the invasion motor consists of a novel class XIV Myosin, MyoA [3] and Myosin Light Chain 1, MLC1 [4] which is anchored into the outer side of the inner membrane complex (IMC) by the Glideosome-Associated Proteins GAP40, GAP45, GAP50 and GAPM’s [5,6,7,8]. The current model of invasion suggests that upon host cell contact GAP40/45/50, GAPM’s, MyoA and MLC1 complex and filamentous actin forms [9].
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