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Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states – genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage.

Summary Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite‐specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine–xanthine–guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage‐specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6‐thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.

Signaling via the NF-κB cascade is critical for innate recognition of microbial products and immunity to infection. As a consequence, this pathway represents a strong selective pressure on infectious agents and many parasitic, bacterial and viral pathogens have evolved ways to subvert NF-κB signaling to promote their survival. Although the mechanisms utilized by microorganisms to modulate NF-κB signaling are diverse, a common theme is targeting of the steps that lead to IκB degradation, a major regulatory checkpoint of this pathway. The data presented here demonstrate that infection of mammalian cells with Toxoplasma gondii results in the activation of IKK and degradation of IκB. However, despite initiation of these hallmarks of NF-κB signaling, neither nuclear accumulation of NF-κB nor NF-κB-driven gene expression is observed in infected cells. However, this defect was not due to a parasite-mediated block in nuclear import, as general nuclear import and constitutive nuclear-cytoplasmic shuttling of NF-κB remain intact in infected cells. Rather, in T. gondii-infected cells, the termination of NF-κB signaling is associated with reduced phosphorylation of p65/RelA, an event involved in the ability of NF-κB to translocate to the nucleus and bind DNA. Thus, these studies demonstrate for the first time that the phosphorylation of p65/RelA represents an event downstream of IκB degradation that may be targeted by pathogens to subvert NF-κB signaling.