Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

  1. Yong Fu
  2. Kevin M Brown
  3. Nathaniel G Jones
  4. Silvia NJ Moreno
  5. L David Sibley  Is a corresponding author
  1. Washington University in St. Louis, United States
  2. University of Georgia, United States

Abstract

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

Data availability

All of the data generated and analysed are included in the manuscript and supporting files including the meta data files.

Article and author information

Author details

  1. Yong Fu

    Department of Molecular Microbiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Kevin M Brown

    Department of Molecular Microbiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Nathaniel G Jones

    Department of Molecular Microbiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Silvia NJ Moreno

    Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2041-6295
  5. L David Sibley

    Department of Molecular Microbiology, Washington University in St. Louis, St Louis, United States
    For correspondence
    sibley@wustl.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7110-0285

Funding

National Institutes of Health (AI034036)

  • L David Sibley

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Malcolm J McConville, The University of Melbourne, Australia

Version history

  1. Preprint posted: May 17, 2021 (view preprint)
  2. Received: August 12, 2021
  3. Accepted: December 3, 2021
  4. Accepted Manuscript published: December 3, 2021 (version 1)
  5. Version of Record published: December 16, 2021 (version 2)
  6. Version of Record updated: January 27, 2022 (version 3)

Copyright

© 2021, Fu et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,411
    Page views
  • 215
    Downloads
  • 6
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Yong Fu
  2. Kevin M Brown
  3. Nathaniel G Jones
  4. Silvia NJ Moreno
  5. L David Sibley
(2021)
Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress
eLife 10:e73011.
https://doi.org/10.7554/eLife.73011

Further reading

    1. Cell Biology
    2. Developmental Biology
    Simon Schneider, Andjela Kovacevic ... Hubert Schorle
    Research Article

    Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Bronwyn A Lucas, Benjamin A Himes, Nikolaus Grigorieff
    Research Advance

    Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.