A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in calcium-induced Toxoplasma gondii egress

Abstract

Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis inToxoplasma

“…Lastly, signaling proteins regulating intracellular cGMP levels, such as the guanylate cyclase (GC) and the unique GC organizer (UGO), were also phosphorylated in a CDPK1-dependent manner (Bisio et al, 2019; Brown and Sibley, 2018; Yang et al, 2019). CDPK1 acts downstream of GC-mediated production of cGMP, which may suggest regulatory feedback on Ca 2+ release, as has been suggested for CDPK3 (Nofal et al, 2022). These data demonstrate that identification of CDPK1 targets can uncover proteins involved in Ca 2+ -regulated exocytosis.…”

“…The Ca 2+ that activates CDPK1 and other cellular processes is released from intracellular stores following cyclic nucleotide–mediated activation of protein kinase G (PKG) (Bisio and Soldati-Favre, 2019; Brown et al, 2017; Sidik et al, 2016). This process can be artificially induced by treating parasites with cGMP specific phosphodiesterase (PDE) inhibitors zaprinast or BIPPO that indirectly activate PKG ( Figure 1A )(Lourido et al, 2012; Nofal et al, 2022; Sidik et al, 2016; Yuasa et al, 2005). PDE inhibition activates PKG within seconds and triggers the Ca 2+ and lipid signaling nodes controlling parasite motility, including the exocytosis of micronemes ( Figure 1A )(Lourido et al, 2012; Yuasa et al, 2005).…”

“…Global phosphoproteomic studies of apicomplexans found that Ca 2+ -dependent phosphorylation encapsulated proteins involved in signal transduction, motility, exocytosis, and the cytoskeleton, as well as those with unknown functions; however, the functional relevance and organization of these proteins—especially CDPK1—in regulating exocytosis remain unclear (Herneisen et al, 2022; Invergo et al, 2017; McCoy et al, 2012; Nofal et al, 2022; Treeck et al, 2014, 2011). Here, we utilized time-resolved phosphoproteomics and chemical genetics to identify 163 proteins phosphorylated by CDPK1.…”

“…Determining CDPK1-dependent phosphorylation is challenging because the signaling pathways controlling parasite motility are rapid and integrate signals from multiple kinases. Global phosphoproteomic studies found that Ca 2+ -dependent phosphorylation included proteins involved in signal transduction, motility, exocytosis, cytoskeleton, in addition to many proteins with unknown functions; however, the functional relevance and organization of these proteinsespecially CDPK1-in regulating exocytosis remain unclear (Herneisen et al, 2022;Invergo et al, 2017;McCoy et al, 2012;Nofal et al, 2022;Treeck et al, 2014Treeck et al, , 2011. Here, we utilized timeresolved phosphoproteomics and chemical genetics to identify 163 proteins phosphorylated by CDPK1.…”

Phosphatidylinositol-4-phosphate signaling regulates dense granule biogenesis and exocytosis inToxoplasma gondii

“…This seems probable as T. gondii potentially encodes ten Sec14-like PITPs and one mammalian PITPα-like ortholog (ToxoDB (v56); http://ToxoDB.org). Moreover, time-resolved phosphoproteome analyses identified a Sec14-like protein (TGME49_254390) and a putative PtdIns 4-OH kinase (TGME49_276170) in a cohort of lipid signaling proteins phosphorylated upon induction of egress -- a process dependent on phosphatidic acid and phospholipase C signaling (Herneisen et al, 2022, Nofal et al 2022). Yet another of the T. gondii Sec14-like PITPs (TGME49_213790) is appended to a PH domain -- as is the single PITPα-like ortholog (TGME49_289570).…”