We recently cloned the first T. gondii MAP kinase, MAPK1-Tg, which acts as a stress MAPK and has 40% homology to human p38 MAPK. MAPK1-Tg is expressed as the full-length 58 kDa protein in tachyzoites, and is the first member of a novel MAPK family. T. gondii tachyzoites expressing a dominant-negative MAPK1-Tg replicated significantly more slowly than parental parasites in vitro, and expressed significantly more bradyzoite antigens. Most strikingly, these tachyzoites were remarkably attenuated in mouse virulence. These data implicate MAPK1-Tg in control of tachyzoite proliferation, stage differentiation and virulence. We previously showed that pyridinylimidazole drugs designed to block human p38 MAPK activation also blocked T. gondii replication in vitro, cured T. gondii-infected mice, and induced stage differentiation in vitro. Human p38 MAPK inhibitors block MAPK1-Tg activity in vitro. We hypothesize that p38 MAPK inhibitors treat T. gondii infection through inhibition of MAPK1-Tg. This proposal will study MAPK1-Tg with two fundamental objectives: i) to exploit MAPK1-Tg as a novel drug discovery target, and ii) to use MAPK1-Tg as a tool to increase our understanding of T. gondii biology. 67657 is the prototypical p38 MAPK inhibitor used. It was safe in human Phase I trials in arthritis, and could be translated into a human clinical trial as an anti-parasitic agent rapidly. MAPK homologues with structural features of MAPK1-Tg were identified in genomic databases for Plasmodium, Leishmania and Trypanosoma. We hypothesize that parasite MAPKs represent novel, broad spectrum drug development target. Finally, T. gondii is a category B bioterror agent. Thus, these discoveries could lead to novel treatment approaches to combating bioterror.
Our Specific aims are: 1 Test hypotheses regarding how MAPK1-Tg regulates replication and stage differentiation. The development of reagents to detect endogenously produced total and active MAPK1-Tg in wild-type parasites, and the development of recombinant parasites with dominant-negative MAPK1-Tg now permit definitive testing of hypotheses regarding how MAPK1-Tg activation regulates tachyzoite replication and stage differentiation, and effects on the cell cycle regulation. Test the hypothesis that 67657 blocks MAPK1-Tg activation. These studies help elucidate factors controlling parasite virulence. 2 Test the hypothesis that blocking MAPK1-Tg activation is a mechanism of therapeutic action of p38 MAPK inhibitors. 3 Test the hypothesis that blocking MAPK1-Tg augments efficacy of anti-Toxoplasma therapies in vivo. ? ? ? ? ?
