Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii

Abstract

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.

A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation inToxoplasma gondii

“…1B and C and data not shown). Although nine Tbd Ϫ mutants have consistent differentiation defects with both stress stimuli, these three mutants stand in contrast to previously published data in which Tbd Ϫ mutants, although obtained by high-pH selection, were also resistant to other inducers of bradyzoite formation (25,33).…”

“…Calculations performed between the 12-hour and 24-hour time points or the 24-hour and 48-hour time points yield similar doubling rates and trends, indicating a consistently lower rate of replication in PUS1 mutants rather than just late invasion or a late start in replication upon invasion. Tbd Ϫ mutants have thus been characterized as having higher, equivalent, and, with these data, lower replication rates relative to WT (25,38).…”

“…Chemical mutagenesis has been used to produce tachyzoite-to-bradyzoite differentiation (Tbd Ϫ ) mutants with consistent, reproducible phenotypes, but the genes responsible have not been identified (25,33). These Tbd Ϫ mutants have a 50% to 85% drop in differentiation efficiency compared to the wild type (WT) and produce fewer cysts in mice during chronic infection, implying that the lower cyst burden may be due to an inability to differentiate and consequent clearance by the immune system.…”

Histone-Modifying Complexes Regulate Gene Expression Pertinent to the Differentiation of the Protozoan Parasite Toxoplasma gondii

“…Stage conversion is associated with dramatic morphological and physiological changes, including alteration of the parasite surface, changes in parasite metabolism, and induction of genes associated with stress response (12,17,34,57). Differentiation is clearly regulated in part at the transcriptional level (37,40,49), but the control of gene expression in apicomplexan parasites is profoundly understudied.…”

What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

“…However, in this approach only non-essential genes for tachyzoite growth in vitro can be identified. Hence this strategy has been mainly employed to identify genes required for differentiation from tachyzoites to bradyzoites (Matrajt et al 2002, Vanchinathan et al 2005). In the future, a combination of ectopic gene regulation systems with random insertional mutagenesis might be possible.…”