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

1. Preparation of human foreskin fibroblasts (HFF)-coated coverslips

1. Place a sterile round glass coverslip on the bottom of the wells of a 24-well tissue culture plate.
2. To harvest HFFs from a confluent 150cm² flask, rinse the flask twice with 1X PBS and add 2.5 ml of 0.025% trypsin-EDTA. Incubate flask at 37°C for 5-10 minutes.
3. Tapping the side of the flask against the palm of your hand can aid in detaching the cells from the flask. Once cells have released from the flask, add 150 ml of HFF medium (Dulbecco's Modified Eagle Medium [DMEM] with 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin) and use the medium to....

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 formation^2,3,4. The analyses also led to the observation that some bradyzoite markers are expressed in other prolonged stress conditions, including growth in activated macrophages^5,6. The above methods describe how to grow T. gondii and induce development a DBA positiv.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine serum albumin	Sigma-Aldrich	A7906
Dulbecco’s Modified Eagle Medium (DMEM)	GIBCO, by Life Technologies	11960-051
Fetal Bovine Serum (FBS)	Atlanta Biologicals	S11150	heat inactivate
Rhodamine Dolichos biflorus agglutinin	Vector Laboratories	RL-1032
Formaldehyde (16%)	Polysciences, Inc.	18814
Glycine	Fisher Scientific	BP381-5
HEPES	Fisher Scientific	BP310-1
IFNγ	PeproTech Inc	315-05	Store in single use aliquots
L-glutamine (200mM)	GIBCO, by Life Technologies	25030
Lipopolysaccharide (LPS)	Sigma-Aldrich	L4391-1MG	Store in single use aliquots
Microscope Cover Glass	Fisher Scientific	12-545-80 12CIR-1
Penicillin-Streptomycin	GIBCO, by Life Technologies	15140
RPMI medium 1640 powder (with L-glutamine, without bicarbonate)	GIBCO, by Life Technologies	31800-022
Triton-X-100	Fisher Scientific	BP151-500
Trypsin-EDTA (0.25%)	GIBCO, by Life Technologies	25200
VectaShield mounting media with DAPI	Vector Laboratories	H-1200