Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions
Summary
Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.
Abstract
Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFNγ). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNγ and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNγ and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.
Protocol
Discussion
While the mechanism of bradyzoite development is not fully understood, molecular genetic analyses of T. gondii stage conversion in tissue culture has led to the discovery of genes that are involved in bradyzoite cyst formation2,3,4. The analyses also led to the observation that some bradyzoite markers are expressed in other prolonged stress conditions, including growth in activated macrophages5,6. The above methods describe how to grow T. gondii and induce development a DBA positiv…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Bovine serum albumin | Sigma | A7906 | ||
| Dulbecco’s Modified Eagle Medium (DMEM) | Gibco | 11960-051 | ||
| Fetal Bovine Serum (FBS) | Atlanta Biologicals | S11150 | heat inactivate | |
| Rhodamine Dolichos biflorus agglutinin | Vector Laboratories | RL-1032 | ||
| Formaldehyde (16%) | Polysciences | 18814 | ||
| Glycine | Fisher Scientific | BP381-5 | ||
| HEPES | Fisher Scientific | BP310-1 | ||
| IFNγ | PeproTech | 315-05 | Store in single use aliquots | |
| L-glutamine (200mM) | Gibco | 25030 | ||
| Lipopolysaccharide (LPS) | Sigma | L4391-1MG | Store in single use aliquots | |
| Microscope Cover Glass | Fisher Scientific | 12-545-80 12CIR-1 | ||
| Penicillin-Streptomycin | Gibco | 15140 | ||
| RPMI medium 1640 powder (with L-glutamine, without bicarbonate) | Gibco | 31800-022 | ||
| Triton-X-100 | Fisher Scientific | BP151-500 | ||
| Trypsin-EDTA (0.25%) | Gibco | 25200 | ||
| VectaShield mounting media with DAPI | Vector Laboratories | H-1200 |
Riferimenti
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