Toxoplasma gondii: Bradyzoite Differentiation In Vitro and In Vivo

Abstract

Toxoplasma gondii, a member of the Apicomplexa, is known for its ability to infect an impressive range of host species. It is a common human infection that causes significant morbidity in congenitally infected children and immunocompromised patients. This parasite can be transmitted by bradyzoites, a slowly-replicating life stage found within intracellular tissue cysts, and oocysts, the sexual life cycle stage that develops in domestic cats and other Felidae. T. gondii bradyzoites retain the capacity to revert back to the quickly-replicating tachyzoite life stage, and when the host is immune compromised unrestricted replication can lead to significant tissue destruction. Bradyzoites are refractory to currently available Toxoplasma treatments. Improving our understanding of bradyzoite biology is critical for the development of therapeutic strategies to eliminate latent infection. This chapter describes a commonly used protocol for the differentiation of T. gondii tachyzoites into bradyzoites using human foreskin fibroblast cultures and a CO 2limited alkaline cell media, which results in a high proportion of differentiated bradyzoites for further study. Also described are methods for purifying tissue cysts from chronically infected mouse brain using isopycnic centrifugation and a recently developed approach for measuring bradyzoite viability.

Acquisition of Host Cytosolic Protein byToxoplasma gondiiBradyzoites

“…Eight-week old male CBA/J (Jackson Laboratories) mice were infected with ME49 WT tachyzoites and humanely sacrificed 5 weeks post-infection following protocols approved by the University of Michigan's Animal Care and Use Committee. Brains from infected mice were homogenized in 1 mL of sterile Hanks Buffered Salt Solution (HBSS) and bradyzoites were subsequently harvested using pepsin treatment (19). Purified bradyzoites invaded iCHO under conversion conditions in the presence of DOX, LHVS, and DMSO for either 4 h (10 µM LHVS) or overnight (3 µM LHVS).…”

Proximity biotinylation reveals novel secreted dense granule proteins of Toxoplasma gondii bradyzoites

“…The fusion protein migrated at a molecular weight of~105 kDa as predicted [42] In vivo biotinylation of the PV using MAG1-BirA � parasites After producing switch-efficient MAG1-BirA � parasites, we assessed whether the fusion protein was active in the PV. To do this, we switched MAG1-BirA � parasites in vitro using high pH media for 5 days and supplemented biotin to the media on the final day [43]. Transition from the tachyzoite to bradyzoite stage was confirmed by the presence of GFP in the parasite cytosol ( Fig 1C).…”

“…[35,72]. Parasites were incubated at 37˚C in ambient air (0.05% CO2) for 5 days to induce switch to tissue cysts, exchanging media every 2 days to maintain alkaline pH [33,43]. 150 μM biotin was supplemented in the media on the final 24 hrs.…”

“…Media was changed to bradyzoite "switch" media (DMEM with 1% FBS, 50mM HEPES, 1%PSG, pH 8.1). [35,72] and parasites were incubated at 37˚C in ambient air (0.05% CO 2 ) for 3 days to induce switch to tissue cysts [33,43]. The Coverslips were then PLOS ONE fixed for IFA analysis.…”

A conserved coccidian gene is involved in Toxoplasma sensitivity to the anti-apicomplexan compound, tartrolon E

“…This observation appears to be specific to trtE-treatment and not related to a general inhibition of the parasites since treatment with the anti- Toxoplasma compound pyrimethamine did not induce an increase in trg transcripts. Stress can cause T. gondii parasites to form bradyzoites in vitro ( Mayoral et al, 2020 ). However, trtE treatment reduced expression of the bradyzoite-specific gene bag1 after 4 h of treatment, suggesting that the increase in expression of trg is not related to stress-associated bradyzoite formation.…”