De novo mapping of the apicomplexan Ca²⁺-responsive proteome

Abstract

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the transition between the replicative and lytic phases of the infectious cycle. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

“…Gene ontology enrichment analysis was performed as described (Herneisen et al, 2022 ). Sets of gene ontology terms from the differentially regulated (SPARK-dependent) and background proteome (all proteins with quantification values in the SPARK-AID mass spectrometry experiment) were downloaded from ToxoDB.org (Molecular Function, Computed evidence, P-Value cutoff set to 1).…”

“…Samples were reduced, alkylated, and digested as described previously (Herneisen et al, 2022 ). In brief, the eluates were reduced with 5 mM TCEP for 20 minutes at 50°C.…”

“…, ME49Δku80Δhxgprt/TIR1(Licon et al, 2023 ) RHΔku80Δhxgprt/TIR1/pTUB1-GCaMP6f(Smith et al, 2022 ), RHΔku80Δhxgprt/TIR1/pMIC2-MIC2-Gluc-myc-P2A-GCaMP6f(Herneisen et al, 2022 ), PKG-mAID-HA/RHΔku80/TIR1(Brown et al, 2017 …”

SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

“…We also observed downregulation of phosphosites belonging to proteins involved in ubiquitin transfer, such as an E2 enzyme and a ubiquitin family protein with a C-terminal extension. The precise pathways regulated by TgPKG remain to be defined; however, recent studies triggering PKG activity in T. gondii (Chan et al, 2023 ;Herneisen et al, 2022 ) or related Plasmodium spp. (Alam et al, 2015 ;Balestra et al, 2021 ;Brochet et al, 2014 ) suggested that PKG substrates relevant for egress reside at the parasite periphery and ER, as observed for the overlapping PKG and SPARK targets here.…”

“…Several recent studies have sought to characterize the downstream targets of second messenger kinases in apicomplexans, including PKA, PKG, and calcium-dependent protein kinases (Alam et al, 2015 ;Balestra et al, 2021 ;Brochet et al, 2014 ;Chan et al, 2023 ;Herneisen et al, 2022 ;Jia et al, 2017 ;Nofal et al, 2022 ). The second messenger networks in these parasites are intricately interwoven, with documented crosstalk between parasite cAMP, cGMP, and calcium signaling cascades.…”