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The dense granule protein 8 (GRA8) is a component of the sub-pellicular cytoskeleton in Toxoplasma gondii

  • Rubén Darío Díaz-Martín
  • Corinne Mercier
  • Carmen T. Gómez de León
  • Ricardo Mondragón González
  • Sirenia González Pozos
  • Emmanuel Ríos-Castro
  • Raúl Arguello García
  • Barbara A. Fox
  • David J. Bzik
  • Ricardo Mondragón FloresEmail author
Protozoology - Original Paper
  • 136 Downloads

Abstract

After host cell invasion, Toxoplasma secretes a variety of dense granule proteins (GRA proteins) from its secretory dense granules, which are involved in the biogenesis of the parasitophorous vacuole (PV). TgGRA8I is predicted to contain proline-rich domains, which are structural features of some cytoskeleton-related proteins. In agreement with this observation, previous proteomic analyses revealed the presence of TgGRA8I in the Toxoplasma sub-pellicular cytoskeleton. In the present study, we show (1) by docking analyses that TgGRA8I may interact with both Toxoplasma β-tubulin and actin; (2) by immunoelectron microscopy, proteomic, biochemical, and cellular approaches that TgGRA8I associates with sub-pellicular microtubules and actin at the parasite sub-pellicular cytoskeleton; (3) that type I parasites (RH strain) lacking the GRA8 gene (RHΔku80Δgra8) exhibit loss of conoid extrusion, diminished cell infection, and egress capabilities, and that these motility impairments were likely due to important alterations in their sub-pellicular cytoskeleton, in particular their sub-pellicular microtubules and meshwork. Parasites lacking the GRA4 gene (RHΔku80Δgra4) did not show modifications in the organization of the sub-pellicular cytoskeleton. Collectively, these results demonstrated that TgGRA8I is a dense granule protein that, besides its role in the formation of the PV, contributes to the organization of the parasite sub-pellicular cytoskeleton and motility. This is the first proline-rich protein described in the Toxoplasma cytoskeleton, which is a key organelle for both the parasite motility and the invasion process. Knowledge about the function of cytoskeleton components in Toxoplasma is fundamental to understand the motility process and the host cell invasion mechanism. Refining this knowledge should lead to the design of novel pharmacological strategies for the treatment against toxoplasmosis.

Keywords

Dense granules Microtubules Proline-rich proteins Toxoplasma cytoskeleton 

Notes

Acknowledgements

The authors are indebted to C.J. Beckers (Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA); D. Soldati-Favre (Department of Microbiology and Molecular Medicine, CMU, Faculty of Medicine, University of Geneva, Switzerland); L.D. Sibley (Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA); and M.F. Delauw (CNRS UMR 5525, Université Grenoble Alpes, France) for sharing reagents. RDDM, CTGL, and RMG were supported by the doctoral fellowships #324969, #324983, and #394378 from CONACyT-México, respectively. We thank Mónica Mondragón Castelán and Carlos J. Ramírez Flores for her technical support. Micrographs were obtained at the Electron Microscopy Unit (LANSE), CINVESTAV-IPN, México. Tandem Mass Spectrometry analysis was performed at the Proteomics, Genomics and Metabolomic Facility, LANSE-CINVESTAV-IPN, México.

Supplementary material

436_2019_6298_MOESM1_ESM.pdf (213 kb)
Supplementary Figure 1 Titration of the rabbit serum anti-GRA8 used in immunoblot- and indirect immunofluorescence assays. A.20 μg per lane of tachyzoites whole cell extract were separated by SDS-PAGE, transferred to nitrocellulose, probed with the rabbit serum anti-GRA8 used at the dilutions 1/500, 1/1000 or 1/5000, and revealed with goat anti-rabbit IgG (H + L) coupled to peroxidase and enhanced chemiluminescence. B. Extracellular parasites were fixed, permeabilized and incubated with rabbit serum anti-GRA8 at the dilution 1/1000, followed by goat anti-rabbit IgG (H + L) coupled to TRITC. The parasite nucleus was revealed with DAPI. Scale bar = 5 μm (PDF 213 kb)
436_2019_6298_MOESM2_ESM.pdf (61.5 mb)
Supplementary Figure 2 TEM micrographs of the 21 serial sections from a RH WT tachyzoite (A) versus the 22 serial sections from a Δgra8 parasite (B), all used to count the dense granules present in the apical end versus the posterior end of each parasite. See the sections N° 8 in both series as examples of the quantified zones. Numbering of the serial sections is indicated in the upper right corner of each micrograph. The parasites, the serial sections of which were observed and photographed are indicated by white asterisks. The number of dense granules counted in the parasites here shown is indicated at the bottom of each micrograph composition. Scale bars = 2 μm. The same type of quantification was realized in about 10 parasites for each strain. (PDF 44930 kb)
436_2019_6298_MOESM3_ESM.pdf (17.6 mb)
Supplementary Figure 3 (PDF 17.6 mb)
436_2019_6298_MOESM4_ESM.pdf (1.2 mb)
Supplementary Figure 4 Structural organization of the subpellicular cytoskeleton of the Δgra4strain. TEM micrographs of a cytoskeleton isolated from tachyzoites of the Δgra4 strain at low magnification (A), at the apical end (B), at the level of the subpellicular microtubules and the network (C) and at the posterior end (D) respectively. Frames in A correspond to the respective magnifications in C-D. Scale bar = 0.3 μm. (PDF 1260 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Rubén Darío Díaz-Martín
    • 1
  • Corinne Mercier
    • 2
  • Carmen T. Gómez de León
    • 1
  • Ricardo Mondragón González
    • 3
  • Sirenia González Pozos
    • 4
  • Emmanuel Ríos-Castro
    • 5
  • Raúl Arguello García
    • 3
  • Barbara A. Fox
    • 6
  • David J. Bzik
    • 6
  • Ricardo Mondragón Flores
    • 1
    Email author
  1. 1.Departamento de BioquímicaCentro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN)Mexico CityMexico
  2. 2.Laboratoire Techniques de l’Imagerie Médicale et de la Complexité – Informatique, Mathématiques et Applications Grenoble (TIMC-IMAG), CNRS UMR 5525Université Grenoble AlpesGrenobleFrance
  3. 3.Departamento de Genética y Biología MolecularCINVESTAV-IPNMexico CityMexico
  4. 4.Unidad de Microscopía Electrónica (LaNSE)CINVESTAV-IPNMexico CityMexico
  5. 5.Unidad de Proteómica, Genómica y Metabolómica (LaNSE)CINVESTAV-IPNMexico CityMexico
  6. 6.Department of Microbiology and ImmunologyGeisel School of Medicine at Dartmouth LebanonUSA

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