Purpose of Study Toxoplasma gondii causes significant morbidity and mortality in immunocompromised patients such as those co-infected with HIV. We have cloned and sequenced two mitogen-activated protein kinase (MAPK) genes which encode novel MAP kinases that are predicted to have distinctly different properties. These predictions are based on examining differences in their primary amino acid sequences and differential gene expression in context with available functional data. Both MAPKs are druggable targets, and we predict that reducing or abolishing their expression in T. gondii will prove to be detrimental to the parasite's ability to proliferate and/or differentiate. Current experiments are underway in this regard.
Methods The T. gondii strain Me-49 genome has been completely sequenced, but individual genes from this avirulent strain are still in the process of being annotated. Using information available at this genomic database (www.toxodb.org), along with our nucleotide sequencing data obtained from the virulent RH strain (acquired using an ABI-7700 automated sequencer), we have identified two MAPK genes, which we have designated tgMAPK-1 and tgMAPK-2. Expression, purification, and functional assays performed on TgMAPK-1 have been previously described (Brumlik et al, 2004). Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out in order to examine the differential expression of both these genes.
Results Based on the deduced amino acid sequences encoded by these genes (which share only 33% identity), each MAP kinase has unique characteristics. RT-PCR profiles suggest that although both genes encode stress-response MAPKs, tgMAPK-1 appears to be expressed in the bradyzoite (latent) form while tgMAPK-2 seems to be more constitutively expressed. ClustalW alignment of these MAPKs has been performed, along with a phylogenetic analysis. Strain variations between both MAPKs are also discussed.
Conclusions Pyridinylimidazoles designed to block the human stress-response p38 MAPK also inhibit TgMAPK-1 in vitro, block T. gondii replication, and protect T. gondii-infected mice. Both TgMAPK-1 and TgMAPK-2 are druggable targets, although it remains to be seen the effect that pyridinylimidazoles have on TgMAPK-2. Understanding the relationship between TgMAPK-1 and TgMAPK-2 and their ability to mediate stress-response and/or stage-differentiation will be important to advancing treatment strategies for T. gondii-infected patients.
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