Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes
Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes
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DOI：
10.1371/journal.pone.0154101
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发表时间：
2016-04-28
期刊：
影响因子：
3.7
通讯作者：
Maes, Louis
中科院分区：
文献类型：
--
作者：
Mondelaers, Annelies;Sanchez-Canete, Maria P.;Maes, Louis
在过去的十年中，米替福新（MIL）已被用作一线治疗内脏利什曼病与锑抗性流行地区，但临床疗效下降，现在正在报道。虽然只有两种来自HIV合并感染患者的婴儿利什曼原虫MIL耐药株已被记录，但L。多诺万尼尚未在实验室中得到确认。因此，有必要更好地了解导致MIL治疗失败增加的因素。由于缺乏定义的抗MIL L。donovani临床分离株，本研究使用实验无鞭毛体选择的MIL抗性L。婴儿分离株（LEM 3323）。通过将基因组数据与表型数据相结合，深入探索MIL抗性表型，以深入了解基因功能和突变表型。天然抗MIL的L.包括婴儿临床分离株（LEM 5159）以比较两个数据集。表型上，通过测定细胞内无鞭毛体体外敏感性和实际MIL-摄取来评估抗性。基因组分析提供了支持性证据，即细胞内无鞭毛体的抗性选择模型可以很好地代表体内田间情况，因为两种抗性菌株在同一内向转运系统中显示突变，导致获得MIL抗性表型。与以前的文献结果在前鞭毛体，我们的数据证实了一个有缺陷的进口机械通过失活的LiMT/LiRos 3蛋白复合物的主要机制，MIL-抗性也在细胞内无鞭毛体。LEM 3323的全基因组测序分析揭示了LiMT基因中的2个碱基对缺失，这导致形成早期终止密码子和LiMT蛋白的截短。有趣的是，LEM 5159揭示了LiMT和LiRos 3基因的突变，导致LiMT蛋白的异常表达。为了验证这些突变确实是获得性抗性的原因，进行转染实验以重新建立MIL-易感性。在LEM 3323中，在LiMT野生型基因表达后恢复了易感性，而LEM 5159的MIL易感性在LiRos 3野生型基因表达后可以逆转。在LEM 5159临床分离株和Delta LiRos 3菌株中LiRos 3基因的拯救后，LiMT蛋白的异常表达谱可以恢复，表明LdMT的表达依赖于LdRos 3表达。目前的发现清楚地证实了LiMT/LiRos 3复合物在抗MIL中的关键作用。
During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a Delta LiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL.