Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, University of Glasgow, Glasgow, United Kingdom.
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, Houston, Texas, United States of America.
PLoS Negl Trop Dis. 2019 Feb 4;13(2):e0007052. doi: 10.1371/journal.pntd.0007052. eCollection 2019 Feb.
Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3' untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern.
两性霉素 B 是减少利什曼原虫寄生虫造成的全球疾病负担的重要工具。由于治疗利什曼病的其他化学治疗选择很少，因此这种药物出现耐药性的可能性是一个相当大的威胁。在这里，我们描述了四种新型两性霉素 B 耐药的墨西哥利什曼原虫系。所有系均表现出生长甾醇生物合成改变和对喷他脒的超敏反应。全基因组测序表明，一种系中固醇 C5-去饱和酶的固醇生物合成基因发生了耐药相关突变。然而，在四分之三的系中，RNA-seq 显示出负责药物抗性和改变的甾醇生物合成的固醇 C24-甲基转移酶 (SMT) 表达缺失。其中一个系还伴有米替福新转运蛋白的缺失。SMT 由两个串联的基因拷贝编码，我们发现它们的表达水平有很大差异。在所有情况下，由于该基因座的结构变异，主导基因拷贝的 3'非翻译区缺失导致整体表达减少。基因拷贝本身之间以及存在促进多基因扩增的 SIDER1 反转座子元件的局部区域序列同源性与这些结构变异相关。此外，在至少一种情况下，SMT 表达缺失与原发性巨噬细胞或体内毒力丧失无关。虽然这种重复序列介导的不稳定性在利什曼原虫基因组中是已知的，但它与主要抗利什曼原虫药物耐药性相关，且没有证据表明存在相关的适应性成本，这是一个重大问题。
