Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.
Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.
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DOI：
10.1371/journal.ppat.1006996
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发表时间：
2018-05
期刊：
影响因子：
6.7
通讯作者：
Neveu C
中科院分区：
文献类型：
--
作者：
Blanchard A;Guégnard F;Charvet CL;Crisford A;Courtot E;Sauvé C;Harmache A;Duguet T;O'Connor V;Castagnone-Sereno P;Reaves B;Wolstenholme AJ;Beech RN;Holden-Dye L;Neveu C
胆碱能激动剂如左旋咪唑和吡喃酮被广泛用作驱虫剂来治疗寄生虫感染。这些药物通过激活线虫体壁肌肉中表达的乙酰胆碱受体（achr）引起痉挛性麻痹。在模型线虫秀丽隐杆线虫中，遗传筛选鉴定出5个编码水平甲氨醇敏感achr （L-AChR）亚基的基因：unc-38、unc-63、unc-29、level -1和level -8。这些亚基在非洲爪蟾卵母细胞中异种表达时形成功能性的L-AChR。在这里，我们发现大多数对左旋咪唑敏感的寄生物种缺乏与秀丽隐杆线虫的同源基因level -8。这就提出了关于构成胆碱能驱虫药靶标的天然受体的性质的重要问题。研究人员发现，在非洲爪蟾卵母细胞中表达的线虫L-AChR中，与之亲缘关系密切的ACR-8亚基在功能上替代了LEV-8。ACR-8在寄生线虫对胆碱能驱虫药敏感性中的重要性通过“模型跳跃”的方法得到了加强，我们证明了来自嗜血的弯曲血线虫的ACR-8能够完全恢复左旋咪唑的敏感性，当在线虫的体壁肌肉中表达时，ACR-8对吡喃嘧啶具有高敏感性。acr-8对体内药物敏感性的关键作用已被成功证明为RNAi基因沉默Hco-acr-8，降低了H. contortus幼虫对左旋咪唑的敏感性。有趣的是，吡喃嘧啶的敏感性保持不变，从而为这些重要的驱虫剂在寄生物种与秀丽隐杆线虫中的不同作用模式提供了新的证据。更广泛地说，这突出了秀丽隐杆线虫作为一种预测模型来破译寄生线虫物种的胆碱能激动剂靶点的局限性，并为发现下一代驱虫剂化合物提供了关键的分子见解。寄生虫对全球健康和经济都有影响。它们会感染动物，包括牲畜、人类和包括所有主要粮食作物在内的植物。它们在人类和兽药中的控制依赖于驱虫药物，但现在这受到了耐药蠕虫的挑战，特别是在牲畜中。重要的是，对于像左旋咪唑和其他胆碱能激动剂这样的驱虫药，耐药性似乎不太常见，这强调了研究它们在寄生线虫中的分子靶点的必要性。左旋咪唑受体最初是在自由生活的模式线虫C.秀丽隐杆线虫中发现的，但现在很明显，这并不是许多寄生物种的一个很好的预测指标。我们特别发现，在秀丽隐杆线虫中参与左旋咪唑敏感性的LEV-8亚基在许多对左旋咪唑敏感的寄生物种中并不存在。本研究利用异源表达系统和基因沉默在体内提供功能证明，ACR-8亚基在线虫的左旋咪唑敏感性中起关键作用，而ACR-8不是秀丽隐杆线虫左旋咪唑受体的必需组分。这对了解胆碱能驱虫药的分子靶点和解决日益严峻的耐药性挑战具有重要意义。
Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a ‘model hopping’ approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds. Parasitic nematodes have global health and economic impacts. They infect animals, including livestock, humans, and plants including all major food crops. Their control in human and veterinary medicine is reliant on anthelmintic drugs but this is now challenged by resistant worms especially in livestock. Importantly, for anthelmintics such as levamisole and other cholinergic agonists, resistance appears to be less frequent stressing the need to investigate their molecular target in parasitic nematodes. The levamisole receptor was first identified in the free-living model nematode C. elegans but it is now becoming apparent that this is not a good predictor for many parasitic species. In particular we have found that the LEV-8 subunit which is involved in levamisole sensitivity in C. elegans, is not present in many levamisole-sensitive parasitic species. Here we used heterologous expression systems and gene silencing to provide the functional in vivo demonstration that the ACR-8 subunit, which is not an essential component of the levamisole receptor in C. elegans, has a critical role in the levamisole sensitivity of parasitic nematodes. This has important significance for understanding the molecular targets of cholinergic anthelmintics and addresses the increasing challenge of drug resistance.
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影响因子：
25
作者：
Boulin, Thomas;Rapti, Georgia;Bessereau, Jean-Louis
通讯作者：
Bessereau, Jean-Louis
影响因子：
3.8
作者：
Duguet, Thomas B.;Charvet, Claude L.;Beech, Robin N.
通讯作者：
Beech, Robin N.
DOI：
10.1002/ps.2780160612
发表时间：
1985-12-01
期刊：
PESTICIDE SCIENCE
影响因子：
--
作者：
HARROW, ID;GRATION, KAF
通讯作者：
GRATION, KAF
影响因子：
6.7
作者：
Courtot E;Charvet CL;Beech RN;Harmache A;Wolstenholme AJ;Holden-Dye L;O'Connor V;Peineau N;Woods DJ;Neveu C
通讯作者：
Neveu C
DOI：
10.1073/pnas.0806933105
发表时间：
2008-11-25
影响因子：
11.1
作者：
Boulin, Thomas;Gielen, Marc;Bessereau, Jean-Louis
通讯作者：
Bessereau, Jean-Louis