Abstract
Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.
Highlights
Apicomplexan parasites like Toxoplasma gondii and Plasmodium species contain a number of plant-like calcium-dependent protein kinases (CDPKs) [1]
We hypothesized that the signaling pathways controlled by TgCDPK3 might be most detected during ionophoreinduced egress
We have not further investigated these events here, but they are consistent with the fact that breakdown of the PVM, normally an early event in egress, is defective in the TgCDPK3 mutant parasites [6,7,8,13]
Summary
Apicomplexan parasites like Toxoplasma gondii and Plasmodium species contain a number of plant-like calcium-dependent protein kinases (CDPKs) [1]. These have been shown to be druggable targets that are distinct from their mammalian hosts. TgCDPK3 mutants show retarded microneme-secretion and ionophore-induced egress they still extrude the conoid, a complex structure in the apical part of the parasites, with the usual kinetics [13] [6,7]. These results show that the mutant parasites can sense the calcium signal induced by the ionophore to some extent but fail to transduce that signal in the normal way. The fact that egress does still occur in the TgCDPK3 mutants, albeit with a delay, indicates that other signaling pathways can operate in the absence of this enzyme [6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
