Centre de Recherche en Infectiologie, Université Laval, Québec, Canada.
PLoS Negl Trop Dis. 2012;6(2):e1512. doi: 10.1371/journal.pntd.0001512. Epub 2012 Feb 14.
Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.
METHODS/PRINCIPAL FINDINGS: Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins-pyridoxal kinase and α-adaptin like protein-were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.
CONCLUSIONS/SIGNIFICANCE: Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.
米替福新（MF）是第一种用于治疗利什曼病的化疗口服化合物。由于该药物的作用机制和 MF 在利什曼原虫中的靶点尚不清楚，我们逐步生成了对 MF 高度耐药的大丽轮枝菌前鞭毛体突变体。其中两个突变体进行了短读长全基因组测序，以鉴定与 MF 耐药相关的潜在基因。
方法/主要发现：对基因组组装的分析显示，参与磷脂转运的 P 型 ATP 酶中存在几个独立的点突变。在两个独立的突变体中还观察到另两种蛋白-吡哆醛激酶和α衔接蛋白样蛋白的突变。通过基因转染和基因敲除评估这些蛋白在 MF 耐药性中的作用，发现 P 型 ATP 酶和吡哆醛激酶都与 MF 易感性有关。该研究还强调，耐药性在群体水平上可能高度异质，从该群体中衍生的单个克隆在基因型和敏感性表型方面都存在差异。
结论/意义：全基因组测序用于确定与原生动物寄生虫利什曼原虫中 MF 耐药性相关的已知和新的耐药标记。该研究还证明了耐药群体的多克隆性质，单个细胞具有不同的敏感性和基因型。
