Validation of isoleucine utilization targets in Plasmodium falciparum
Validation of isoleucine utilization targets in Plasmodium falciparum
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DOI：
10.1073/pnas.1011560108
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发表时间：
2011-01-25
影响因子：
11.1
通讯作者：
Goldberg, Daniel E.
中科院分区：
文献类型：
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作者：
Istvan, Eva S.;Dharia, Neekesh V.;Goldberg, Daniel E.
红细胞内疟原虫可以通过降解宿主细胞血红蛋白获得几乎全部的氨基酸需求。唯一的例外是异亮氨酸，它不存在于成人血红蛋白中，必须从外源获得。我们评估了两种化合物干扰异亮氨酸利用的潜力。莫匹罗星是一种临床上使用的抗菌剂，在纳摩尔浓度下杀死恶性疟原虫寄生虫。硫代异亮氨酸，一种异亮氨酸类似物，也具有抗疟活性。为了鉴定这两种化合物的靶标，我们选择了对莫匹罗星或硫代异亮氨酸具有抗性的寄生虫。通过全基因组高密度平铺微阵列、DNA测序和拷贝数变异分析来分析突变体。三个独立的莫匹罗星抗性寄生虫克隆的基因组均获得了包含染色体编码的细胞器（顶质体）异亮氨酰-tRNA合成酶的扩增或SNP。抗硫代异亮氨酸寄生虫的细胞质异亮氨酰-tRNA合成酶发生突变。该突变在硫代异亮氨酸抗性中的作用通过等位基因置换得到证实。这种方法通常可用于阐明恶性疟原虫中的新靶标。我们的研究表明，异亮氨酸的利用是一个重要的途径，可以针对抗疟药物的开发。
Intraerythrocytic malaria parasites can obtain nearly their entire amino acid requirement by degrading host cell hemoglobin. The sole exception is isoleucine, which is not present in adult human hemoglobin and must be obtained exogenously. We evaluated two compounds for their potential to interfere with isoleucine utilization. Mupirocin, a clinically used antibacterial, kills Plasmodium falciparum parasites at nanomolar concentrations. Thiaisoleucine, an isoleucine analog, also has antimalarial activity. To identify targets of the two compounds, we selected parasites resistant to either mupirocin or thiaisoleucine. Mutants were analyzed by genome-wide high-density tiling microarrays, DNA sequencing, and copy number variation analysis. The genomes of three independent mupirocin-resistant parasite clones had all acquired either amplifications encompassing or SNPs within the chromosomally encoded organellar (apicoplast) isoleucyl-tRNA synthetase. Thiaisoleucine-resistant parasites had a mutation in the cytoplasmic isoleucyl-tRNA synthetase. The role of this mutation in thiaisoleucine resistance was confirmed by allelic replacement. This approach is generally useful for elucidation of new targets in P. falciparum. Our study shows that isoleucine utilization is an essential pathway that can be targeted for antimalarial drug development.