BRADYZOITE DEVELOPMENT IN TOXOPLASMA-GONDII AND THE HSP70 STRESS-RESPONSE

Toxoplasma gondii is a well described ubiquitous Apicomplexan protozoa n parasite that is an important opportunistic pathogen. The factors af fecting the transition of tachyzoites to the latent bradyzoite stage r emain to be defined. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium a rsenite, and many of the other stressors associated with heat shock pr otein (hsp) induction. There is evidence for other organisms that hsps are developmentally regulated. Therefore, we examined whether hsp ind uction is an early event in bradyzoite differentiation. Extracellular and intracellular T. gondii cells, after exposure to pH 8.1 or 7.1, we re analyzed for the expression of inducible hsp70 by using monoclonal antibody C92F3A-5 (specific to hsp70). Western blotting demonstrated t hat a 72-kDa protein reactive with C92F3A-5 (hsp70), which we believe is part of the hsp70 family, is induced during bradyzoite development. By immunofluorescence and immunoelectron microscopy, we were able to demonstrate that hsp70 staining colocalized to T.gondii expressing bra dyzoite-specific antigens and the presence of hsp70 in bradyzoites iso lated from mouse brain. Quercetin, a bioflavonoid which inhibits the s ynthesis of hsp90, hsp70, and hsp27, suppresses the induction of brady zoite development in vitro. Reverse transcription-PCR with conserved h sp70 primers demonstrated an increase in hsp70 in T.gondii on exposure to conditions which induce bradyzoite formation. A T. gondii hsp70 wa s subsequently cloned and sequenced by using this amplified fragment. We believe our evidence suggests that hsps are important in the proces s of bradyzoite differentiation.