The Association of the Stress Response and Toxoplasma gondii Bradyzoite Development

Toxoplasma gondii is a well described ubiquitous Apicomplexan protozoan parasite of mammals and birds which presents as an often fatal necrotizing encephalitis in patients with A I D S . The development of Toxoplasma encephalitis is believed to be due to the transition of bradyzoites to tachyzoites [ 121. Several studies have demonstrated that bradyzoites can develop in vitro using bradyzoite specific monoclonal antibodies [mAbs] [2,9-111. Those factors affecting the transition of bradyzoites to tachyzoites remain to be defined. Stress conditions are, however, associated with the induction of bradyzoite development. It has been found that temperature stress (43°C [9]), pH stress (pH 6.8 or 8.2 [2,9-111) or chemical stress (Na arsenite [9], mitochondrial inhibitors [2], nitric oxide [2]) results in an increase in cyst-like structures and bradyzoite antigen expression by T. gondii in virro. These conditions are also associated with the induction of heat shock proteins (hsps) [5]. The hsps fall into several subfamilies, namely, the low molecular weight hsps (16-35 m a ) , the hsp60 family, the hsp70 family (68-78 ma): and the high molecular weight hsps (89-110 kDa) [6]. It has become clear that hsps are not just limited to stress responses. Hsps are developmentally regulated [4-61 and have been associated with differentiation [4]. In this regard, we have recently cloned the gene for BAG5 [7] (also known as BAGllhsp30 [l]), a bradyzoite specific antigen, that displays homology at the carboxy terminus to a number of small heat shock proteins of plants with molecular sizes of 17-22 kDa. Given the association of the induction of bradyzoite development in virro with temperature, pH, mitochondrial inhibitors, sodium arsenite and many of the other stressors associated with hsp induction as well as evidence that hsps are developmentally regulated, we examined if hsp induction is an early event in bradyzoite differentiation. MATERIALS AND METHODS. 5000 ME49 T. gondii were used to infect human fibroblasts [ATCC CRL 14751 in a two chamber slide culture system (Nunc) and induction of bradyzoites was done in pH 7.1 or 8.1 Dulbecco's modified Eagle's Medium (DME) supplemented with lOmM Hepes @H 7.1 or 8.1), 10% fetal calf serum (Hyclone) and 1 % Penicillin-Streptomycin as previously described [ 1 11. Quercetin (Sigma) 100 pg/ml was also added to some cultures. To study the effect of extracellular pH exposure, T. gondii were incubated in pH 8.1 or 7.1 DME for 1 hour before they were used to infect a human fibroblast monolayer at pH 7.1. Development and quantifkation of T. gondii bradyzoites was monitored in vitro as previously described [ 1 I], following futation with 2% buffered formalin and permeabilization with 0.2% Triton X-100, by humnofluorescence employing IgG2b mAb 74.1. 8 (anti-BAGS) [7,11], rabbit anti-recombinant MAG1 (anti-matrix) serum, [8] or rabbit anti-Toxoplasma serum. In order to examine hsp expression, mAb C92F3A-5 (StressGen), specific to the inducible form of hsp70, was employed in immunofluorescence and immunoelectronmicroscopy [ l l ] . C92F3A-5 was also used for western blotting [ll] on T. gondii incubated for one hour at pH 8.1 and pH 7.1 as well as on extracellular pH shocked parasites after culture for 3 days at pH 7.1 in human fibroblasts. For western blotting, samples of cell culture supernatant (i.e. free cysts) or T. gondii infected human fibroblast monolayers were assayed for protein concentration and equal amounts of protein loaded onto SDS-PAGE gels, electrophoresed, transferred to nitrocellulose and western blotted as previously described [I I]. We found that exposure of extracellular tachyzoites of ME49 strain T. gondii to pH 8.1 media for one hour followed by culture of these organisms in human fibroblasts at pH 7.1 resulted in a 2 to 4 fold increase in bradyzoite development as compared to tachyzoites treated for one hour at pH 7.1 before cell culture. Thus, short term exposure of tachyzoites to alkaline pH should facilitate the study of early genes in bradyzoite development which may act as triggers for stage conversion. Extracellular and intracellular T. gondii after exposure to pH 8.1 versus pH 7.1 were analyzed for the expression of inducible hsp7O using mAb C92F3A-5. Western blotting demonstrated that a 72 kDa protein reactive to C92F3A-5, which we believe is part of the hsp70 family, is induced during bradyzoite development demonstrating that at least one hsp is associated with bradyzoite formation. This 72 kDa band was specific to T. gondii and was not seen in human fibroblasts without infection. There was a 2 to 4 fold increase in this 72 kDa protein in T. gondii pretreated at pH 8.1 compared to those pretreated at pH 7.1. Immunofluorescence on human fibroblasts infected with ME49 T. gondii that had been treated at pH 8.1 demonstrated that mAb C92F3A-5 reactivity was localized to T. gondii expressing bradyzoite specific antigens. Similarly, by immuno-elec&on microscgpy, we were able to demonstrate reactivity of mAb C92F3A-5 with bradyzoites isolated from tissue culture and from mouse brain. Quercetin, a bioflavonoid which inhibits the synthesis of hsp90, hsp7O and hsp27 [12]. suppressed the induction of bradyzoite development in vitro due to pH shock [pH 7.1 49 cysts/slide, pH 8.1 119 cystslslide, quercetin 100 pglml pH 7.1 46 cystslslide, quercetin 100 pglml pH 8.1 54' cystslslide ('p<.O5)]. Thus, we believe our evidence suggests that heat shock proteins are important in the process of bradyzoite differentiation. [Supported by NIH-AI394541