An apically located hybrid guanylate cyclase–ATPase is critical for the initiation of Ca2+ signaling and motility in Toxoplasma gondii

Abstract

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca 2؉ ] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca 2؉ ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K ؉ ] to activate cytosolic [Ca 2؉ ] flux. Together, the results of our work implicate TgGC as a puta-tive signal transducer that activates Ca 2؉ signaling and motility in Toxoplasma.

The Role of Potassium and Host Calcium Signaling inToxoplasma gondiiegress

“…Ionomycin, an ionophore that releases Ca 2+ from neutral stores stimulated egress at concentrations greater than 5 nM. Zaprinast, an inhibitor of cGMP-selective phosphodiesterases [45] would result in accumulation of cGMP, which will activate Protein Kinase G (PKG), a protein that plays a pivotal role in microneme secretion and tachyzoite egress [26,46,47], and results in the stimulation of cytosolic Ca 2+ and egress by presumably promoting the formation of phospholipase C substrates [48].…”

Rapid kinetics of lipid second messengers controlled by a cGMP signalling network coordinates apical complex functions inToxoplasmatachyzoites

“…Other core eukaryotic signalling elements in the cGMP pathway are absent in Apicomplexa including calcium-gated ion channels and soluble and membrane forms of guanylate cyclase. Instead, apicomplexans possess an unusual alveolate-specific dual-domain phospholipid transporter/cGMP cyclase protein (TgGC) fusion protein (Johnson and Leroux 2010;Yang et al 2019). Recently in Toxoplasma, TgGC has been shown to be critical for activation of TgPKG to trigger microneme secretion (Brown and Sibley 2018;Yang et al 2019;Bisio et al 2019).…”

“…Instead, apicomplexans possess an unusual alveolate-specific dual-domain phospholipid transporter/cGMP cyclase protein (TgGC) fusion protein (Johnson and Leroux 2010;Yang et al 2019). Recently in Toxoplasma, TgGC has been shown to be critical for activation of TgPKG to trigger microneme secretion (Brown and Sibley 2018;Yang et al 2019;Bisio et al 2019). Similarly, Plasmodium species possess two GCs, type a and type b (GCa + GCb), and the Plasmodium yoelli protein PyGCb has been shown to have an apical localization in sporozoites, critical for ookinete traversal of the mosquito midgut (Gao et al 2018).…”

“…The function of the P-Type ATPase domain of TgGC is poorly understood but is predicted to have a role in lipid trafficking across membranes, or possibly of ion transport such as K + or Ca 2+ (Yang et al 2019;Bisio et al 2019). However, several studies have concluded that both domains are essential for TgGC function and that the ATPase domain likely links a metabolic function to a signalling response (Brown and Sibley 2018;Yang et al 2019), although extensive lipidomic analysis in TgGC-depleted cells is lacking.…”

“…The function of the P-Type ATPase domain of TgGC is poorly understood but is predicted to have a role in lipid trafficking across membranes, or possibly of ion transport such as K + or Ca 2+ (Yang et al 2019;Bisio et al 2019). However, several studies have concluded that both domains are essential for TgGC function and that the ATPase domain likely links a metabolic function to a signalling response (Brown and Sibley 2018;Yang et al 2019), although extensive lipidomic analysis in TgGC-depleted cells is lacking. Exactly what the substrate of the ATPase domain might be is uncertain although it has been shown that cells with disrupted TgGC are less responsive to changes in pH while disruption of cofactors of TgGC, including TgCDC50, leads to parasites that can still egress in response to changes in pH (Bisio et al 2019), suggesting that TgGC is the primary responder to external pH signals.…”