Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii.

Expression of the 30 kDa small heat shock protein BAG1 is restricted to the latent bradyzoite 'tissue cyst' form of Toxoplasma gondii, first appearing approximately 2-3 days after the initiation of bradyzoite differentiation. Although developmental expression of small heat shock proteins has been described for many species, their precise function is unclear. In order to examine the function of BAG1 in T. gondii bradyzoites and its role during parasite differentiation, we have used homologous recombination to produce a knock-out mutant in the cyst-forming strain P(LK), a clonal derivative of ME49. Under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), the mutant was found to express several bradyzoite-specific markers with the same kinetics and frequency as the parental strain. Neither enhanced nor decreased susceptibility to stress was observed for the BAG1-deficient mutant. In vivo studies revealed that tachyzoites of the bag1 knock-out mutant were fully able to establish a chronic infection in C57BL/6 mice, producing brain cysts of a size, morphology and frequency indistinguishable from cysts formed by the parental control strain. Brain cysts of the bag1 knock-out mutant contained viable parasites capable of establishing an acute infection after oral administration, demonstrating that conversion of bradyzoites to tachyzoites is also unimpaired. We conclude that BAG1 is not essential for normal function of bradyzoite containing tissue cysts, at least in intermediate host species. This clone of P(LK) was found to be unable to produce oocysts and is therefore unsuitable for studies in cats.