Toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions.
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 (IFNgamma). 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 IFNgamma 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 IFNgamma 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.
Tobin
Crystal
C
Department of Medical Microbiology and Immunology, University of Wisconsin, USA.
Pollard
Angela
A
Knoll
Laura
L
D000818:Animals; D017077:Culture Media, Conditioned; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma; D008070:Lipopolysaccharides; D008264:Macrophages / Q000469:parasitology*; D051379:Mice; D014122:Toxoplasma / Q000254:growth & development*
D017077:Culture Media, Conditioned; D008070:Lipopolysaccharides; D007371:Interferon-gamma