核苷二磷酸激酶b和延伸因子2在巴西利什曼原虫抗锑表型中的作用
Involvement of nucleoside diphosphate kinase b and elongation factor 2 in Leishmania braziliensis antimony resistance phenotype.
作者信息
Moreira Douglas S, Murta Silvane M F
机构信息
Centro de Pesquisas René Rachou CPqRR, Fundação Oswaldo Cruz - FIOCRUZ, Avenida Augusto de Lima 1715, Belo Horizonte, MG, Brazil.
出版信息
Parasit Vectors. 2016 Dec 13;9(1):641. doi: 10.1186/s13071-016-1930-6.
BACKGROUND
Nucleoside diphosphate kinase b (NDKb) is responsible for nucleoside triphosphates synthesis and it has key role in the purine metabolism in trypanosomatid protozoans. Elongation factor 2 (EF2) is an important factor for protein synthesis. Recently, our phosphoproteomic analysis demonstrated that NDKb and EF2 proteins were phosphorylated and dephosphorylated in antimony (Sb)-resistant L. braziliensis line compared to its Sb-susceptible pair, respectively.
METHODS
In this study, the overexpression of NDKb and EF2 genes in L. braziliensis and L. infantum was performed to investigate the contribution of these proteins in the Sb-resistance phenotype. Furthermore, we examined the role of lamivudine on Sb susceptibility in clones that overexpress the NDKb gene, and the effect of EF2 kinase (EF2K) inhibitor on the growth of EF2-overexpressing parasites.
RESULTS
Western blot analysis demonstrated that NDKb and EF2 proteins are more and less expressed, respectively, in Sb-resistant line of L. braziliensis than its wild-type (WTS) counterpart, corroborating our previous phosphoproteomic data. NDKb or EF2-overexpressing L. braziliensis lines were 1.6 to 2.1-fold more resistant to Sb than the untransfected WTS line. In contrast, no difference in Sb susceptibility was observed in L. infantum parasites overexpressing NDKb or EF2. Susceptibility assays showed that NDKb-overexpressing L. braziliensis lines presented elevated resistance to lamivudine, an antiviral agent, but it did not alter the leishmanicidal activity in association with Sb. EF2-overexpressing L. braziliensis clone was slightly more resistant to EF2K inhibitor than the WTS line. Surprisingly, this inhibitor increased the antileishmanial effect of Sb, suggesting that this association might be a valuable strategy for leishmaniasis chemotherapy.
CONCLUSION
Our findings represent the first study of NDKb and EF2 genes overexpression that demonstrates an increase of Sb resistance in L. braziliensis which can contribute to develop new strategies for leishmaniasis treatment.
背景
核苷二磷酸激酶b（NDKb）负责三磷酸核苷的合成，在锥虫原生动物的嘌呤代谢中起关键作用。延伸因子2（EF2）是蛋白质合成的重要因子。最近，我们的磷酸化蛋白质组分析表明，与锑敏感株相比，抗锑的巴西利什曼原虫株中NDKb和EF2蛋白分别发生了磷酸化和去磷酸化。
方法
在本研究中，对巴西利什曼原虫和婴儿利什曼原虫中的NDKb和EF2基因进行过表达，以研究这些蛋白在抗锑表型中的作用。此外，我们检测了拉米夫定对过表达NDKb基因的克隆株中锑敏感性的影响，以及EF2激酶（EF2K）抑制剂对过表达EF2的寄生虫生长的影响。
结果
蛋白质免疫印迹分析表明，与野生型（WTS）相比，抗锑的巴西利什曼原虫株中NDKb蛋白表达增多，EF2蛋白表达减少，这与我们之前的磷酸化蛋白质组数据一致。过表达NDKb或EF2的巴西利什曼原虫株对锑的抗性比未转染的WTS株高1.6至2.1倍。相比之下，过表达NDKb或EF2的婴儿利什曼原虫株在锑敏感性方面未观察到差异。药敏试验表明，过表达NDKb的巴西利什曼原虫株对抗病毒药物拉米夫定的抗性增强，但它并未改变与锑联合使用时的杀利什曼活性。过表达EF2的巴西利什曼原虫克隆株对EF2K抑制剂的抗性略高于WTS株。令人惊讶的是，这种抑制剂增强了锑的抗利什曼作用，表明这种联合用药可能是利什曼病化疗的一种有价值的策略。
结论
我们的研究结果首次对NDKb和EF2基因过表达进行了研究，结果表明巴西利什曼原虫对锑的抗性增加，这有助于开发利什曼病治疗的新策略。
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