Plate-Based Quantification of Stimulated Toxoplasma Egress

Shortt, Emily; Lourido, Sebastian

doi:10.1007/978-1-4939-9857-9_10

Cited by 7 publications

(7 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We investigated the inhibitory properties of ENH1 on parasite egress using a high-content imaging assay . Briefly, the assay monitors entry of the cell-impermeant dye DAPI into lysed host cells following stimulated parasite egress.…”

Section: Resultsmentioning

confidence: 99%

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling

et al. 2020

Self Cite

View full text Add to dashboard Cite

Apicomplexan parasites include the causative agents of malaria and toxoplasmosis. Cell-based screens in Toxoplasma previously identified a chemical modulator of calcium signaling (ENH1) that blocked parasite egress from host cells and exhibited potent antiparasitic activity. To identify the targets of ENH1, we adapted thermal proteome profiling to Toxoplasma, which revealed calcium-dependent protein kinase 1 (CDPK1) as a target. We confirmed the inhibition of CDPK1 by ENH1 in vitro and in parasites by comparing alleles sensitive or resistant to ENH1. CDPK1 inhibition explained the block in egress; however, the effects of ENH1 on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets. Thermal proteome profiling of lysates from parasites expressing the resistant allele of CDPK1 identified additional candidates associated with the mitochondria and the parasite pelliclecompartments that potentially function in calcium release and homeostasis. Our findings illustrate the promise of thermal profiling to identify druggable targets that modulate calcium signaling in apicomplexan parasites.

show abstract

Section: Resultsmentioning

confidence: 99%

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Different agonists have been used to identify pathway-specific requirements for certain kinases (19, 54). Egress leads to loss of host-cell integrity, which can be assayed quantitatively and kinetically as the incorporation of the fluorescent dye 4⍰,6-diamidino-2-phenylindole (DAPI) into the nuclei of permeabilized cells (55). We compared the impact of inhibiting CDPK1, CDPK3, or CDPK2A on A23187-stimulated egress.…”

Section: Resultsmentioning

confidence: 99%

“…Egress was quantified in a plate-based manner as in (55). HFF monolayers in a clear bottomed 96-well plate were infected with parasites and allowed to incubate 24 h before exchanging media for FluoroBrite DMEM (ThermoFisher) supplemented with 10% FBS and applying pre-treatments according to experiment type.…”

Section: Methodsmentioning

confidence: 99%

CDPK2A and CDPK1 form a signaling module upstream ofToxoplasmamotility

Shortt¹,

Hackett²,

Stadler

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

The transition between parasite replication and dissemination is regulated in apicomplexan parasites by fluctuations in cytosolic calcium concentrations, effectuated by calcium-dependent protein kinases (CDPKs). We examined the role of CDPK2A in the lytic cycle of Toxoplasma, analyzing its role in the regulation of cellular processes associated with parasite motility. We used chemical-genetic approaches and conditional depletion to determine that CDPK2A contributes to the initiation of parasite motility through microneme discharge. We demonstrate that the N-terminal extension of CDPK2A is necessary for the protein’s function. Conditional depletion revealed an epistatic interaction between CDPK2A and CDPK1, suggesting that the two kinases work together to mediate motility in response to certain stimuli. This signaling module appears distinct from that of CDPK3 and PKG, which also controls egress. CDPK2A is revealed as an important regulator of the Toxoplasma kinetic phase, linked to other kinases that govern this critical transition. Our work uncovers extensive interconnectedness between the signaling pathways that govern parasite motility.IMPORTANCEThis work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive (AS) kinase alleles and conditionally-depleted alleles uncovered epistasis between CDPK2A and CDPK1 implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting parasite kinases for anti-parasitic therapies.

show abstract

“…Automated, plate-based egress assays were performed as previously described (Shortt and Lourido, 2020). In brief, HFF monolayers in a clear-bottomed 96-well plate were infected with 7.5 × 10 4 or 1 × 10 5 parasites of the TIR1 or PP 1 -AID strains, respectively.…”

Section: Methodsmentioning

confidence: 99%

De novo mapping of the apicomplexan Ca²⁺-responsive proteome

Herneisen

Chan

et al. 2022

Preprint

View full text Add to dashboard Cite

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.

show abstract

Plate-Based Quantification of Stimulated Toxoplasma Egress

Cited by 7 publications

References 16 publications

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling

CDPK2A and CDPK1 form a signaling module upstream ofToxoplasmamotility

De novo mapping of the apicomplexan Ca²⁺-responsive proteome

Contact Info

Product

Resources

About

Plate-Based Quantification of Stimulated Toxoplasma Egress

Cited by 7 publications

References 16 publications

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling

CDPK2A and CDPK1 form a signaling module upstream ofToxoplasmamotility

De novo mapping of the apicomplexan Ca2+-responsive proteome

Contact Info

Product

Resources

About

De novo mapping of the apicomplexan Ca²⁺-responsive proteome