Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.
Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.
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DOI：
10.1371/journal.pntd.0010779
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发表时间：
2022-09
影响因子：
3.8
通讯作者：
中科院分区：
文献类型：
--
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两性霉素B越来越多地用于治疗利什曼病。在这里，14个独立的墨西哥利什曼原虫和一个L。婴儿品系被选择为对抗生素B或相关的多烯抗微生物剂制霉菌素具有抗性。甾醇谱显示，在每个抗性品系中，主要的野生型甾醇麦角甾-5，7，24-三烯醇被其他甾醇中间体取代。广义上，两个不同的配置文件中出现的抗性线。全基因组测序结果表明，这些不同的配置文件是由于在甾醇甲基转移酶（C24 SMT）基因座或甾醇C5去饱和酶（C5 DS）基因突变。在三个线的额外删除的米替福新转运基因被发现。在敏感性的差异，以阿替霉素B是显而易见的，这取决于细胞是否生长在HOMEM中，补充胎牛血清，或无血清的确定成分培养基（DM）。暴露于AmB后的代谢组学分析表明，在HOMEM中而不是DM中持续的细胞中，通过磷酸戊糖途径的葡萄糖通量的大幅增加先于细胞死亡，表明在HOMEM条件下更显著地诱导氧化应激。测试了几种细胞系感染巨噬细胞和作为无鞭毛体形式复制的能力，以及它们在小鼠中建立感染的能力。虽然几个AmB耐药株系显示出降低的毒力，但至少有两个株系在小鼠中显示出增强的毒力，同时保留了它们的耐药表型，强调了对这种关键药物出现耐药性的风险。两性霉素B越来越多地用于治疗利什曼病。抗生素耐药性是任何药物的持续威胁，尽管该领域很少报告对利什曼菌素B的耐药性，但这是不可忽视的，因为该药物用于利什曼病复发病例，并适用于治疗其他感染性疾病，如真菌病。在这里，我们报告了14个独立的墨西哥利什曼原虫和一个L。对阿替霉素B或其类似物制霉菌素耐药婴儿。与野生型相比，在所有抗性品系中甾醇谱都发生了改变。主要的野生型甾醇麦角甾-5，7，24-三烯醇被其他甾醇中间体取代。在一组突变体中，这可能是由于来自甾醇甲基转移酶（C24 SMT）基因位点突变的杂合性丢失，而在第二组中，甾醇C5去饱和酶（C5 DS）基因发生了变化。与含有血清的HOMEM相比，在无血清的限定培养基中生长的细胞对Amstericin B的敏感性的差异是明显的，并且代谢组学分析表明，当血清存在时，AmB促进氧化应激，但在血清不存在时，Amstericin B促进氧化应激。虽然一些细胞系在小鼠感染中失去了毒力，但其他细胞系在小鼠中显示出增强的毒力，而所有细胞系都保留了其耐药表型，强调了患者中出现耐药的风险。
Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug. Amphotericin B is increasingly used in treatment of leishmaniasis. Antimicrobial resistance is a persistent threat for any drugs and although reports of resistance to amphotericin B are rare in the field, it is something that cannot be ignored since this drug is used in leishmaniasis relapse cases and is indicated to treat other infectious diseases such as mycoses. Here, we report the selection and characterisation of fourteen independent lines of Leishmania mexicana and one of L. infantum resistant to amphotericin B or its analogue nystatin. Sterol profiles were altered in all resistant lines compared to wild-type. The predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. In one set of mutants this could be attributed to loss of heterozygosity derived from mutations in the sterol methyl transferase (C24SMT) gene locus and in a second set the sterol C5 desaturase (C5DS) gene was changed. Differences in sensitivity to amphotericin B were apparent in cells grown in serum free defined medium compared to serum containing HOMEM and metabolomics analysis indicated that AmB promotes oxidative stress when serum is present but not in its absence. While some lines lost virulence in mouse infections others displayed heightened virulence in mice whilst all cell lines retaining their resistance phenotype emphasising the risk of resistance emerging in patients.
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影响因子：
14.8
作者：
Anderson TM;Clay MC;Cioffi AG;Diaz KA;Hisao GS;Tuttle MD;Nieuwkoop AJ;Comellas G;Maryum N;Wang S;Uno BE;Wildeman EL;Gonen T;Rienstra CM;Burke MD
通讯作者：
Burke MD
影响因子：
1.2
作者：
Cingolani, Pablo;Platts, Adrian;Ruden, Douglas M.
通讯作者：
Ruden, Douglas M.
影响因子：
1.6
作者：
Chakravarty J;Sundar S
通讯作者：
Sundar S
影响因子：
3.8
作者：
Bansal, Ruby;Sen, Shib Sankar;Madhubala, Rentala
通讯作者：
Madhubala, Rentala
影响因子：
5.2
作者：
Alcazar-Fuoli L;Mellado E
通讯作者：
Mellado E