Control of both human and canine leishmaniasis is based on a very short list of chemotherapeutic agents, headed by antimonial derivatives (Sb). The utility of these molecules is severely threatened by high rates of drug resistance. The ABC transporter MRPA is one of the few key Sb resistance proteins described to date, whose role in detoxification has been thoroughly studied in Leishmania parasites. Nonetheless, its rapid amplification during drug selection complicates the discovery of other mechanisms potentially involved in Sb resistance. In this study, stepwise drug-resistance selection and next-generation sequencing were combined in the search for novel Sb-resistance mechanisms deployed by parasites when MRPA is abolished by targeted gene disruption. The gene mrpA is not essential in L. infantum, and its disruption leads to an Sb hypersensitive phenotype in both promastigotes and amastigotes. Five independent mrpA^−/- mutants were selected for antimony resistance. These mutants displayed major changes in their ploidy, as well as extrachromosomal linear amplifications of the subtelomeric region of chromosome 23, which includes the genes coding for ABCC1 and ABCC2. Overexpression of ABCC2, but not of ABCC1, resulted in increased Sb tolerance in the mrpA^−/- mutant. SNP analyses revealed three different heterozygous mutations in the gene coding for a serine acetyltransferase (SAT) involved in de novo cysteine synthesis in Leishmania. Overexpression of sat^Q390K, sat^G321R and sat^G325R variants led to a 2–3.2 -fold increase in Sb resistance in mrpA^−/- parasites. Only sat^G321R and sat^G325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites.

人和犬利什曼病的控制均基于以锑衍生物（Sb）为首的非常短的化学治疗药物清单。这些分子的实用性受到高耐药率的严重威胁。ABC转运蛋白MRPA是迄今为止描述的少数几个关键的Sb抗性蛋白之一，其在解毒作用中已在利什曼原虫寄生虫中进行了深入研究。但是，它在药物选择过程中的快速扩增使发现可能与Sb耐药有关的其他机制变得复杂。在这项研究中，逐步耐药性选择和下一代测序相结合，以寻找通过靶向基因破坏取消MRPA时由寄生虫利用的新型Sb耐药机制。基因mrpA在婴儿乳杆菌中不是必需的，它的破坏导致前鞭毛体和变形虫中的Sb超敏表型。选择五个独立的mrpA ^-/-突变体用于抗锑。这些突变体显示了其倍性的重大变化，以及染色体23的亚端粒区域的染色体外线性扩增，其中包括编码ABCC1和ABCC2的基因。ABCC2的过表达，而不是ABCC1的过表达，导致mrpA ^-/-突变体的Sb耐受性增加。SNP分析显示，利什曼原虫从头半胱氨酸合成涉及的丝氨酸乙酰基转移酶（SAT）编码基因中的三个不同的杂合突变。饱和^Q390K，饱和^G321R和饱和^G325R的过表达导致mrpA ^-/-寄生虫的Sb抗性提高了2–3.2倍。只有坐在^G321R和SAT ^G325R诱导野生型寄生虫增加SB抵抗。这些结果加强和扩展了对利什曼原虫寄生虫对Sb抵抗的复杂性的认识。