Development and regulation of tachyzoite-bradyzoite differentiation in Toxoplasma gondii

Toxoplasma gondii differentiation is a complex process in which the parasite develops from a rapidly dividing and metabolically active tachyzoite into a quiescent encysted bradyzoite within a host cell. Acute toxoplasmosis is frequently associated with recrudescence from this latent stage to the acute lytic tachyzoite stage in immunosuppressed patients. No treatment is known to clear bradyzoite cysts from an infected host therefore intervention strategies that would prevent infection and disease, are dependent on the dissection of the differentiation process. In order to better our understanding of bradyzoite biology in vitro induction protocols and various fluorescent markers available for T. gondii targeted to specific organelles were used to follow the differentiation process in parasites. This analysis has made it clear that morphological changes during in vitro differentiation including alterations in protein secretion, rearrangement of the cytoskeleton and gross ultrastructural perturbations are evident from as early as 12 hrs after induction. The high frequency of intracellular parasites lacking rhoptries and/or apicoplasts suggests reduced control over organellar segregation in bradyzoites, and indicates that these organelles are not essential for intracellular survival. To advance our understanding of what regulatory elements are important for the expression of bradyzoite genes in T. gondii, a bradyzoite-specific promoter, pt7, identified by insertional mutagenesis has been mapped and provides further evidence that bradyzoite-specific elements are not well conserved. This promoter element has been employed to drive bradyzoite-specific expression of a secreted form of GFP that is efficiently expressed in both in vitro and in vivo derived bradyzoites. Stage-specific cytosolic expression of the yellow fluorescent protein (YFP) under control of bradyzoite-specific promoter pt7 yields fluorescence after 1–2 days of in vitro bradyzoite induction. The developed bradyzoite-specific fluorescent reporter is a powerful tool for the rapid and efficient FACS-based enrichment of differentiation mutants that inappropriately express YFP as tachyzoites and identification of genes critical for this stage-specific expression by gene rescue. Regulatory mutants, the possible result of inactivation of a negative regulator, have been identified and indicate that the differentiation pathway is complex but can be dissected using the developed tools