Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles
Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles
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DOI：
10.1016/j.ijpddr.2020.08.003
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发表时间：
2020-12-01
影响因子：
4
通讯作者：
Andersen, Erik C.
中科院分区：
文献类型：
--
作者：
Dilks, Clayton M.;Hahnel, Steffen R.;Andersen, Erik C.
寄生线虫感染给全世界人类和牲畜的健康造成巨大负担。驱虫药是抵抗这些感染的第一道防线。不幸的是，对这些药物的耐药性十分猖獗，并且还在继续蔓延。为了改进治疗策略，我们必须了解耐药性背后的遗传学和分子机制。对真菌构巢曲霉和自由生活线虫秀丽隐杆线虫的研究发现，苯并咪唑 (BZ) 抗性菌株中的 β-微管蛋白基因发生突变。在寄生线虫种群中，三个 β-微管蛋白等位基因 F167Y、E198A 和 F200Y 长期以来一直与抗性相关。此外，测序技术的改进已发现新的等位基因 - E198V、E198L、E198K、E198I 和 E198Stop - 也与 BZ 抗性相关。然而，这些等位基因均未被证明会引起耐药性。为了凭经验证明这一点，我们使用 CRISPR-Cas9 系统将 Fl 67Y、El 98A 和 F200Y 等位基因以及两个新鉴定的等位基因 El 98V 和 El 98L 独立引入 BZ 易感线虫 N2 遗传背景中。这些基因组编辑菌株暴露于阿苯达唑和芬苯达唑，以定量测量动物对 BZ 的反应。我们使用每种 BZ 化合物的浓度范围来定义响应曲线，发现所有五个等位基因都赋予对 BZ 化合物的抗性，相当于整个 β-微管蛋白基因的丢失。这些结果证明寄生虫β-微管蛋白等位基因会引起耐药性。 E198V 等位基因与 E198L 等位基因一起在天然寄生虫种群中出现频率较低，表明它可能会影响健康。我们进行了竞争性适应性测定，并证明 E198V 等位基因会降低动物健康，支持该等位基因可能不太适合野外种群的假设。总体而言，我们提供了一个强大的平台来定量评估驱虫药耐药性以及特定耐药性等位基因在存在或不存在药物的情况下对机体健康的影响。
Infections by parasitic nematodes inflict a huge burden on the health of humans and livestock throughout the world. Anthelmintic drugs are the first line of defense against these infections. Unfortunately, resistance to these drugs is rampant and continues to spread. To improve treatment strategies, we must understand the genetics and molecular mechanisms that underlie resistance. Studies of the fungus Aspergillus nidulans and the free-living nematode Caenorhabditis elegans discovered that a beta-tubulin gene is mutated in benzimidazole (BZ) resistant strains. In parasitic nematode populations, three beta-tubulin alleles, F167Y, E198A, and F200Y, have long been correlated with resistance. Additionally, improvements in sequencing technologies have identified new alleles - E198V, E198L, E198K, E198I, and E198Stop - also correlated with BZ resistance. However, none of these alleles have been proven to cause resistance. To empirically demonstrate this point, we independently introduced the Fl 67Y, El 98A, and F200Y alleles as well as two of the newly identified alleles, El 98V and El 98L, into the BZ susceptible C. elegans N2 genetic background using the CRISPR-Cas9 system. These genome-edited strains were exposed to both albendazole and fenbendazole to quantitatively measure animal responses to BZs. We used a range of concentrations for each BZ compound to define response curves and found that all five of the alleles conferred resistance to BZ compounds equal to a loss of the entire beta-tubulin gene. These results prove that the parasite beta-tubulin alleles cause resistance. The E198V allele is found at low frequencies along with the E198L allele in natural parasite populations, suggesting that it could affect fitness. We performed competitive fitness assays and demonstrated that the E198V allele reduces animal health, supporting the hypothesis that this allele might be less fit in field populations. Overall, we present a powerful platform to quantitatively assess anthelmintic resistance and effects of specific resistance alleles on organismal fitness in the presence or absence of the drug.