Tejera Nevado Paloma, Bifeld Eugenia, Höhn Katharina, Clos Joachim
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Antimicrob Agents Chemother. 2016 Aug 22;60(9):5262-75. doi: 10.1128/AAC.00544-16. Print 2016 Sep.
The mechanisms underlying the drug resistance of Leishmania spp. are manifold and not completely identified. Apart from the highly conserved multidrug resistance gene family known from higher eukaryotes, Leishmania spp. also possess genus-specific resistance marker genes. One of them, ARM58, was first identified in Leishmania braziliensis using a functional cloning approach, and its domain structure was characterized in L. infantum Here we report that L. infantum ARM58 is part of a gene cluster at the telomeric end of chromosome 34 also comprising the neighboring genes ARM56 and HSP23. We show that overexpression of all three genes can confer antimony resistance to intracellular amastigotes. Upon overexpression in L. donovani, ARM58 and ARM56 are secreted via exosomes, suggesting a scavenger/secretion mechanism of action. Using a combination of functional cloning and next-generation sequencing, we found that the gene cluster was selected only under antimonyl tartrate challenge and weakly under Cu(2+) challenge but not under sodium arsenite, Cd(2+), or miltefosine challenge. The selective advantage is less pronounced in intracellular amastigotes treated with the sodium stibogluconate, possibly due to the known macrophage-stimulatory activity of this drug, against which these resistance markers may not be active. Our data point to the specificity of these three genes for antimony resistance.
利什曼原虫属耐药性的潜在机制是多方面的，尚未完全明确。除了从高等真核生物中已知的高度保守的多药耐药基因家族外，利什曼原虫属还拥有属特异性耐药标记基因。其中一个基因ARM58，最初是在巴西利什曼原虫中通过功能克隆方法鉴定出来的，其结构域结构在婴儿利什曼原虫中得到了表征。在此我们报告，婴儿利什曼原虫的ARM58是34号染色体端粒末端一个基因簇的一部分，该基因簇还包括相邻基因ARM56和HSP23。我们表明，这三个基因的过表达均可赋予细胞内无鞭毛体抗锑性。在杜氏利什曼原虫中过表达时，ARM58和ARM56通过外泌体分泌，提示其作用机制为清除/分泌机制。通过功能克隆和新一代测序相结合的方法，我们发现该基因簇仅在酒石酸锑钾攻击下被选择，在铜离子攻击下选择较弱，但在亚砷酸钠、镉离子或米托蒽醌攻击下未被选择。在用葡萄糖酸锑钠处理的细胞内无鞭毛体中，这种选择优势不太明显，这可能是由于该药物已知的巨噬细胞刺激活性，而这些耐药标记物可能对此无活性。我们的数据表明这三个基因对锑耐药具有特异性。
