Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance
Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance
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DOI：
10.1111/j.1476-5381.2011.01420.x
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发表时间：
2011-11-01
影响因子：
7.3
通讯作者：
Neveu, C.
中科院分区：
文献类型：
--
作者：
Boulin, T.;Fauvin, A.;Neveu, C.
背景与目的胆碱能激动剂左旋咪唑被广泛用于治疗寄生虫感染。这种驱虫药物通过激活线虫肌肉细胞中表达的一类左旋咪唑敏感乙酰胆碱受体（l - achr）而使线虫瘫痪。然而，左旋咪唑的疗效已因耐药寄生虫的出现而受到损害，特别是在胃肠道线虫中，如弯曲血蜱。我们在此报道了H. contortus L-AChRs在异源表达系统中的首次功能重构和药理学表征。实验方法在秀丽隐杆线虫（Caenorhabditis elegans）中，5个AChR亚基和3个辅助蛋白基因在体内外合成l -AChR是必需的。我们克隆了这些基因的弯曲人同源基因，并在爪蟾卵母细胞中表达。我们通过结合不同的受体亚基，重组了两种具有不同药理作用的l - achr。Hco-ACR-8亚基在左旋咪唑的选择性敏感性中起关键作用。在秀丽隐杆线虫L-AChRs中观察到，H. tortus受体的表达需要辅助蛋白Hco-RIC-3、Hco-UNC-50和Hco-UNC-74。利用该实验系统，我们发现截断的Hco-UNC-63 L-AChR亚基在左旋咪唑耐药菌株中特异检测到，而在左旋咪唑敏感菌株中未检测到，当与全长对应的L-AChR共表达时，会阻碍L-AChR的正常功能。结论和意义：我们为任何寄生线虫左旋咪唑耐药的推测分子机制提供了第一个功能证据。该表达系统将提供一种在电生理水平上分析与耐药性相关的分子多态性的手段。
BACKGROUND AND PURPOSEThe cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole-sensitive acetylcholine receptors (L-AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug-resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L-AChRs in a heterologous expression system.EXPERIMENTAL APPROACHIn the free-living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L-AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L-AChRs with distinct pharmacologies by combining different receptor subunits.KEY RESULTSThe Hco-ACR-8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L-AChRs, expression of H. contortus receptors requires the ancillary proteins Hco-RIC-3, Hco-UNC-50 and Hco-UNC-74. Using this experimental system, we demonstrated that a truncated Hco-UNC-63 L-AChR subunit, which was specifically detected in a levamisole-resistant H. contortus isolate, but not in levamisole-sensitive strains, hampers the normal function of L-AChRs, when co-expressed with its full-length counterpart.CONCLUSIONS AND IMPLICATIONSWe provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level.