Identification of a novel mitogen-activated protein kinase in Toxoplasma gondii.

Toxoplasma gondii is an Apicomplexan parasite causing significant morbidity and mortality in immunocompromised hosts. Mitogen activated protein kinases regulate diverse biologic processes including proliferation, differentiation, survival and stress responses. We searched a new T. gondii genomic database to identify a 1.6 kilobase pair (kbp) coding region with features suggesting a mitogen activated protein kinase. This gene is predicted to encode a 58kDa protein with a threonine, aspartic acid, tyrosine (TDY) activation loop, similar to parasite and plant mitogen activated protein kinases, but distinct from mammalian mitogen activated protein kinases (with threonine, glycine, tyrosine (TGY) motifs). The predicted protein shares 45% amino acid identity with human stress-activated p38alpha mitogen activated protein kinase. Expression of the cloned gene in Escherichia coli produced a protein with an apparent molecular weight of 63kDa and which exhibited kinase activity. Following osmotic stress, the abundance of the mRNA encoding this T. gondii mitogen activated protein kinase, which we name TgMAPK-1, increased in tachyzoites. Its expression rescued hog1-deficient yeast grown under osmotic stress. These data confirm that the gene product is a stress-response mitogen activated protein kinase. Upon conversion of T. gondii tachyzoites to the latent bradyzoite form in vitro, tgMAPK-1 transcript accumulation increased, suggesting a role in parasite proliferation or stage differentiation. We previously demonstrated that pyridinylimidazole p38 mitogen activated protein kinase inhibitors block T. gondii replication. These inhibitors also blocked TgMAPK-1 autophosphorylation, suggesting that TgMAPK-1, or other parasite mitogen activated protein kinases are novel drug development targets.