Toxoplasma and Plasmodium resistance to dinitroanilines

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DESCRIPTION (provided by applicant): Human infection with Apicomplexan parasites causes incalculable morbidity from a wide variety of diseases. For example, Toxoplasma gondii causes birth defects and, in immunocompromised individuals, life-threatening illness. Malarial infections caused by Plasmodium species account for as much as a million deaths a year. The invasive stages of Apicomplexans have two populations of MTs (1) spindle MTs (which mediate cell division) and (2) subpellicular MTs (responsible for cell shape and polarity). Disruption of the subpellular MTs is associated with a loss of polarity and motility and, consequently, an inability to invade cells. Disruption of the spindle MTs prevents parasite replication. Apicomplexans including Toxoplasms and Plasmodium spp., are susceptible to a group of dinitroaniline herbicides that are potent destabilizers of plant and protozoan MTs. These drugs do not affect vertebrate cells, suggesting that they may represent a novel chemotherapeutic treatment for Apicomplexan infections. One of the dinitroanilines, oryzalin, has been used to select resistant Toxoplasma gondii lines after chemical mutagenesis. Resistance mutations are predicted to occur by three mechanisms: 1) a P-glycoprotein-like drug efflux pump; 2) mutations to tubulin; and, 3) mutations to proteins that modulate MT behavior. The 44 independent resistant lines will be analyzed using a variety of pharmacological agents and expression of tubulin in trans to identify lines containing mutations to tubulin or MDR-like loci. The remaining lines potentially contain mutations to accessory proteins that mediate MT interactions (a finding in dinitroaniline resistant Chlamydomonas lines). The non-tubulin resistance alleles will be cloned and characterized in Toxoplasma gondii. The homologs of these proteins will be identified in Plasmodium falciparum, and analogous mutations will be introduced to assess whether these alleles confer resistance to Plasmodium merozoites.