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

Abstract

Fundamental processes of obligate intracellular parasites, such as Toxoplasma gondii and Plasmodium falciparum, are controlled by a set of plant-like calcium dependent kinases (CDPKs), the conserved cAMP- and cGMP-dependent protein kinases (PKA and PKG), secondary messengers and lipid signalling. While some major components of the signalling networks have been identified, how these 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 to activate PKG or CDPKs, respectively. We show that both these inducers trigger near identical signalling pathways and provide evidence for a positive feedback loop involving CDPK3. We measure phospho- and lipid signalling in parasites treated with the Ca2+ ionophore A23187 in a sub-minute timecourse and show CDPK3-dependent regulation of diacylglycerol levels and increased phosphorylation of four phosphodiesterases (PDEs), suggesting their function in the feedback loop. Disruption of CDPK3 leads to elevated cAMP levels and inhibition of PKA signalling rescues the egress defect of ΔCDPK3 parasites treated with A23187. Biochemical analysis of the four PDEs identifies PDE2 as the only cAMP-specific PDE among these candidates, while the other PDEs are cGMP specific, two of which are inhibited by the predicted PDE inhibitor BIPPO. Conditional deletion of the four PDEs supports an important, but non-essential role for PDE1 and PDE2 in growth, with PDE2 controlling A23187-mediated egress. In summary we uncover a positive feedback loop that enhances signalling during egress and links several signalling pathways together.

Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways

“…Indeed, the third wave of phosphorylation targets Ca 2+ and other divalent cation ATPases, including Tg A1, Tg CuTP, and other P-type ATPases with uncharacterized ion specificity. Transporters belonging to these clusters are noted Tg CDPK3 substrates, including ATP4, SCE1, and ApiAT5-3 ( Treeck et al, 2014 ; McCoy et al, 2017 ; Dominicus et al, 2021 ); disruption of these transporters failed to alter cytosolic Ca 2+ levels ( McCoy et al, 2017 ; Parker et al, 2019 ; Wallbank et al, 2019 ). A cluster of dynamic dephosphorylation events highlights the need for phosphatases to reverse modifications as parasites transition from one phase to another ( Brautigan and Shenolikar, 2018 ; Yang and Arrizabalaga, 2017 ).…”

“…Prior proteomic surveys investigating the contribution of individual kinases ( Alam et al, 2015 ; Balestra et al, 2021 ; Brochet et al, 2014 ; Jia et al, 2017 ; Patel et al, 2019 ; Treeck et al, 2014 ) or phosphatases ( Paul et al, 2020 ; Yang et al, 2019 ) to global phosphoregulation lacked the temporal resolution to map transitions between the replicative and kinetic phases of the apicomplexan infection cycle, which occur in a matter of seconds. Advances in proteomics technologies, such as enhanced sensitivity and improved multiplexing methods, are now being leveraged to monitor the sub-minute processes of exflagellation and egress ( Dominicus et al, 2021 ; Invergo et al, 2017 ). Second-messenger signaling pathways underpin these transitions at distinct developmental stages in diverse apicomplexans, with many kinases and phosphatases functioning at multiple steps in the parasitic life cycle.…”

Babesia divergensegress from host cells is orchestrated by essential and druggable kinases and proteases

“…In T. gondii and P. falciparum , these initial signals converge to either activate GC or inhibit PDE and thus raise cGMP levels, which in turn activates PKG. In T. gondii , PKAc1 suppresses cGMP levels to prevent premature egress, putatively by activating a PDE, whereas in P. falciparum PKAc1 is required for invasion (Dominicus et al, 2021; Flueck et al, 2019; Jia et al, 2017; Moss et al, 2021; Patel et al, 2019; Wilde et al, 2019). Inhibition of PKA using H89 blocked B. divergens invasion, as well as induced low levels of egress.…”

CDPK2A and CDPK1 form a signaling module upstream ofToxoplasmamotility

“…PKG has been shown to be a calcium regulator in T. gondii and Plasmodium spp. (7,(15)(16)(17)68), placing it upstream of CDPK activation. In plants, CDPKs are tuned to respond to different calcium concentrations (38), raising the possibility that dependency on different parasite CDPKs may result from the magnitude of the calcium surge elicited by PKG.…”

“…These supportive activities may be nonetheless important for parasite fitness under particular conditions. Additionally, these functional modules seem to be spatially distinct, with CDPK1/CDPK2A signaling occurring in the parasite cytoplasm, while CDPK3/PKG signaling occurs at the parasite PM (68). Understanding the topology of signaling pathways underlying key events in the parasite life cycle can help identify compensatory changes and predict phenotypic plasticity as we contemplate targeting parasite kinases for anti-parasitic therapies.…”