Antimony resistance in Leishmania major Friedlin

Antimonials are still the mainstay of treatment against Leishmaniasis but in the past decade resistance has been a severe threat. We carried out short read next generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony resistant mutants. Copy number variations were consistently detected in both NGS and CGH where several chromosomal aneuploidies were correlated to antimony resistance. A major attribute of antimony resistance was a novel terminal deletion of variable length (67kb-204kb) of the polyploid chromosome 31 in the three mutants and was experimentally validated. Terminal deletion in two mutants occurred at the level of inverted repeated sequences in chromosome 31. AQP1 (LmjF.31.0020), a gene encoding for an aquaglyceroporin, which facilitates uptake of trivalent metalloids, was a part of the deleted region. Transfection of AQP1 into resistant mutants rendered them hypersensitive to SbIII. CGH, NGS and Southern Blot analysis also highlighted a novel stable, intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded redox enzymes like ascorbate dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH) and overexpression of the genes coding for these enzymes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS) accumulation. Generation of G6PDH null mutant in one revertant exhibited SbIII sensitivity and protection from ROS which were rescued in the add back. Our genomic analyses and parallel functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.

抗锑剂仍是治疗利什曼病（Leishmaniasis）的主流手段，但近十年来，抗药性已成为严重威胁。本研究对三株独立的硕大利什曼原虫（Leishmania major）抗锑突变株开展了短读长下一代测序（short read next generation sequencing, NGS）与比较基因组杂交（comparative genomic hybridization, CGH）分析。研究在NGS与CGH检测中均稳定检出拷贝数变异（copy number variations），且多条染色体的非整倍性（chromosomal aneuploidies）与抗锑药性显著相关。其中，抗锑药性的核心特征为三株突变株中多倍体31号染色体出现新型长度可变的末端缺失，缺失片段长度介于67kb至204kb，该变异经实验验证。两株突变株的末端缺失发生于31号染色体的反向重复序列（inverted repeated sequences）区域。编码水甘油通道蛋白（aquaglyceroporin）的AQP1（LmjF.31.0020）基因可介导三价类金属摄取，该基因恰好位于上述缺失区域内。将AQP1转染至抗锑突变株后，突变株对三价锑（SbIII）的敏感性显著升高。CGH、NGS与Southern印迹（Southern Blot）分析还在一株突变株中发现了34号染色体亚端粒位点的新型稳定染色体内扩增（intrachromosomal amplification）。该区域编码抗坏血酸过氧化物酶（ascorbate dependent peroxidase, APX）、葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase, G6PDH）等氧化还原酶；在回复突变背景中过表达这些编码基因，可使菌株获得SbIII抗性并抵御活性氧（reactive oxygen species, ROS）积累。在一株回复突变株中构建G6PDH敲除突变株（null mutant）后，菌株恢复对SbIII的敏感性且无法抵御ROS积累，而通过回补实验（add back）可挽救该表型。本研究通过基因组分析与平行功能验证，揭示了利什曼原虫抗锑药性相关的新型基因组重排、功能关键抗性位点以及新的抗性相关基因。