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

  1. Alice L Herneisen
  2. Zhu-Hong Li
  3. Alex W Chan
  4. Silvia NJ Moreno
  5. Sebastian Lourido

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    Herneisen et al provide a comprehensive and thorough exploration of Ca2+ responsive changes in the Toxoplasma proteome and the resulting phosphorylation events during the transition from intracellular residing parasites to egress from the host cell. Furthermore, a novel temperature stability profiling method of all proteins responding to Ca2+ concentration with a change in stability is a novel applicable tool that here is used to map Ca2+-responsive proteins in the parasites. They provide a compelling analysis of the complex data and carefully validate their findings using genetics and cell biology. This work is of the highest quality in the field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca 2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca 2+ -responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca 2+ -responsive proteome of the model apicomplexan Taxoplasma gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca 2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca 2+ -responsive proteins, such as parasite Ca 2+ -dependent protein kinases. Our approach also identified numerous Ca 2+ -responsive proteins that are not predicted to bind Ca 2+ , yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca 2+ -responsive enzyme that relocalized to the parasite apex upon Ca 2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca 2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca 2+ -regulated serine/threonine phosphatase activity in apicomplexan parasites.

Article activity feed

  1. Version published to 10.7554/elife.80336 on eLife
  2. eLife

    Evaluation Summary:

    Herneisen et al provide a comprehensive and thorough exploration of Ca2+ responsive changes in the Toxoplasma proteome and the resulting phosphorylation events during the transition from intracellular residing parasites to egress from the host cell. Furthermore, a novel temperature stability profiling method of all proteins responding to Ca2+ concentration with a change in stability is a novel applicable tool that here is used to map Ca2+-responsive proteins in the parasites. They provide a compelling analysis of the complex data and carefully validate their findings using genetics and cell biology. This work is of the highest quality in the field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Cyclic GMP (cGMP) and Ca2+ signaling have been strongly linked to parasite motility and invasion in apicomplexan parasites. Over the last decade, researchers have pieced together an understanding of key molecules (in particular kinases) involved in regulating motility. Whilst there has been some attempt at identifying Ca2+ responsive kinases, using phosphoproteomics, this has lacked temporal analysis. Herneisen et al performed a time-resolve analysis of phosphorylation upon stimulation with the PDE inhibitor zaprinast (which stimulates cGMP signaling upstream of Ca2+ responses). They identify well over 4000 proteins per run, which is the highest coverage yet seen in Toxoplasma and must be close to the full proteome at this lifecycle stage. Their careful analysis (which I find the most compelling aspect of this work) clusters groups of phosphorylated proteins based on their temporal pattern and confirms and extends what is understood about the order of events that occurs during signaling that activates motility and invasion across the apicomplexan parasites.

    Hernesisen et al then combine thermal proteome profiling, to understand how proteins respond to changes in Ca2+ concentration. The aim of this is to identify effectors of phosphorylation patterns over time and is an ingenious way of getting an answer to the problem. It will not only identify proteins that directly bind Ca2+ ( a change in thermal stability in the presence of this ion) but potentially other proteins/complexes that change in structure also. They are careful in their analysis of the resulting dataset to not overinterpret their findings. Furthermore, they validate their findings on several proteins that likely do not directly bind Ca2+ (but likely change in other ways upon a Ca2+ signal). Pleasingly, these 5 candidates validate the approach. Across all datasets, the analysis of this data is robust, insightful, and concise and will be of great value to the apicomplexan research community.

    What is nice to see, and something that has not been explored much in Apicomplexa is a focus on proteins that become dephosphorylated upon signaling. They then go on to functionally characterize a PP1 orthologue, which also changes thermal stability upon increasing Ca2+ concentration and likely mediates downstream dephosphorylation. The phenotype is a little messy, likely because, as pointed out by the authors, that PP1s localisation and activity is mediated by partner proteins. They, however, clearly show a change in localisation upon stimulation of motility with zaprinast, but not the Ca2+ ionophore A23187 and that PP1 depleted parasites likely have a defect in the invasion. The level of cytosolic PP1-depleted parasites (as measured by GCaMP) only differs when stimulation with zaprinast not with A23187.

    The authors then finish by applying their phosphoproteomic approach to PP1-depleted parasites and reveal changes. The results are in line with a recent Plasmodium publication (Paul et al, Nat Comms, 2021)(which they appropriately cite).

    Overall, this paper was a pleasure to read, its conclusions were valid and not over-interpreted (which can be the case when performing these types of experiments). They have managed to extract meaningful data from these large data sets into easily interpretable graphical representations and carefully validate their results. The work is of the highest quality and sets a benchmark for the field.

  4. eLife

    Reviewer #2 (Public Review):

    The large-scale proteomic experiments shown in this work are impressive, nicely designed, and analysed to high standards. The phosphoproteomics survey provides an informative repertoire of differentially phosphorylated proteins downstream of cGMP. Similarly, the remarkable repertoire of proteins that likely bind Ca2+ will be of interest to better map pathways involved in Toxoplasma egress. Finally, the addition of PP1 in the regulation of timely egress is a significant contribution to our understanding of the regulation network underlying egress in Toxoplasma.

    One of the main weaknesses of this work is the lack of functional relationships between the three main sections. This particularly holds true for the functional characterisation of PP1 does not address the regulation of PP1 by Ca2+ as suggested by the TPP profiling. Finally, I think that more experiments would be required to support a role of PP1 in calcium entry.

  5. eLife

    Reviewer #3 (Public Review):

    The report is a major leap in understanding the Ca2+-central pathways underlying egress and invasion of Apicomplexa, using T. gondii as a model organism. Temporal phosphoproteomics is novel, yet even more innovative is to apply temperature stability profiling using various Ca2+ concentrations and temperatures. This provides a really unprecedented depth in the Ca2+ protein network, revealing several dynamic trends in the responses, reveals many new proteins with stability shifts in absence of apparent Ca2+-binding, and ties together many previous observations on putative channels and transporters and signaling pathways. The dynamics of PP1 are intriguing, first accumulating apical of the nucleus (secretory pathway compartment?) and then transitioning apically and to the cortex. Although this is characterized as 'pleiotrophic' I am not sure that is a correct term if this is a PKG-dependent trajectory (but can be bypassed by Ca ionophore) - all of which are somewhat artificial stimulations and therefore could present pleiomorphic under these conditions: some more caution in the results/discussion would be warranted.

  10. Version published to 10.1101/2022.05.25.493445v1 on bioRxiv