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.