Toxoplasma and Plasmodium resistance to dinitroanilines

描述(由申请人提供)：人类感染Apicomplexan寄生虫会导致多种疾病的无法估量的发病率。例如，弓形虫会导致出生缺陷，在免疫功能受损的人中，还会导致危及生命的疾病。由疟原虫引起的疟疾感染每年造成多达100万人死亡。顶面复合体的侵袭期有两类MTS：(1)纺锤形MTS(负责细胞分裂)和(2)膜下MTS(负责细胞形状和极性)。亚细胞MTS的破坏与极性和运动性的丧失有关，因此，无法侵袭细胞。纺锤体MTS的破坏防止了寄生虫的复制。包括弓形虫和疟原虫在内的顶体复合体对一组对植物和原生动物MTS有强烈破坏作用的二硝基苯胺类除草剂敏感。这些药物不影响脊椎动物细胞，这表明它们可能代表着一种治疗Apicomplexan感染的新的化疗方法。其中一种二硝基苯胺类化合物oryzalin已用于化学诱变后选育弓形虫抗性品系。耐药突变预计通过三种机制发生：1)P-糖蛋白样药物外排泵；2)微管蛋白突变；以及3)调节MT行为的蛋白质突变。将使用各种药理学药物和微管蛋白在反式中的表达来分析44个独立的耐药株系，以确定含有微管蛋白或MDR样位点突变的株系。其余的菌株可能含有辅助蛋白的突变，这些辅助蛋白介导了MT的相互作用(在对二硝基苯胺耐药的衣原体菌株中发现了这一点)。弓形虫中的非微管蛋白抗性等位基因将被克隆和鉴定。这些蛋白质的同源物将在恶性疟原虫中被鉴定出来，类似的突变将被引入以评估这些等位基因是否赋予了对疟原虫裂殖子的抵抗力。

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.