Kopp Steven R, Coleman Glen T, Traub Rebecca J, McCarthy James S, Kotze Andrew C
School of Veterinary Science, University of Queensland, Australia.
Int J Parasitol. 2009 Mar;39(4):435-41. doi: 10.1016/j.ijpara.2008.08.005. Epub 2008 Sep 13.
The molecular mechanism of resistance to nicotinic agonist anthelmintics such as pyrantel and levamisole in nematodes of medical and veterinary significance is poorly understood. The identification of pyrantel-resistant isolates of the canine hookworm, Ancylostoma caninum, provides an opportunity to explore, at a molecular level, the mechanism of cholinergic resistance in a species that is a model for the human hookworms. Here we describe the cloning of three A. caninum genes orthologous to components of the pyrantel-sensitive nicotinic acetylcholine receptor in Caenorhabditis elegans (UNC-29, -38, -63). Analysis of mRNA levels by quantitative PCR was also performed on these genes, plus an additional three nicotinic acetylcholine receptor subunit genes thought not to be constituents of the pyrantel-sensitive receptor, for which a partial sequence was obtained. Gene sequences and mRNA levels were compared between two isolates of A. caninum showing either high- or low-level resistance to pyrantel (as shown previously by in vivo efficacy and in vitro comparative studies). While no polymorphisms of likely significance between the two A. caninum isolates were observed, quantitative analysis of transcription revealed significantly lower levels for the three putative pyrantel receptor subunits (AAR-29, -38 and -63) in the highly pyrantel-resistant isolate compared with the isolate with low-level resistance. In contrast, transcription of the three subunits thought not to constitute the pyrantel receptor (AAR-8, -15 and -19) was either not significantly different between the two isolates, or slightly higher in the highly-resistant isolate. This data suggests that reduced transcription of the mRNA coding for nicotinic acetylcholine receptor subunits that form the pyrantel-sensitive receptors may be a component of the pyrantel resistance mechanism in A. caninum.
对于医学和兽医学上具有重要意义的线虫对诸如噻嘧啶和左旋咪唑等烟碱激动剂驱虫药产生抗性的分子机制，人们了解甚少。犬钩虫（犬弓首线虫）噻嘧啶抗性分离株的鉴定为在分子水平上探索一种作为人类钩虫模型的物种中的胆碱能抗性机制提供了契机。在此，我们描述了三个犬弓首线虫基因的克隆，这些基因与秀丽隐杆线虫中对噻嘧啶敏感的烟碱型乙酰胆碱受体的组分（UNC-29、-38、-63）直系同源。还对这些基因以及另外三个被认为不是噻嘧啶敏感受体组分的烟碱型乙酰胆碱受体亚基基因进行了定量PCR分析，我们获得了后者的部分序列。在对噻嘧啶表现出高抗性或低抗性的两个犬弓首线虫分离株（如先前通过体内药效和体外比较研究所示）之间比较了基因序列和mRNA水平。虽然在两个犬弓首线虫分离株之间未观察到可能具有重要意义的多态性，但转录定量分析显示，与低抗性分离株相比，高度噻嘧啶抗性分离株中三个假定的噻嘧啶受体亚基（AAR-29、-38和-63）的水平显著降低。相比之下，被认为不构成噻嘧啶受体的三个亚基（AAR-8、-15和-19）的转录在两个分离株之间要么没有显著差异，要么在高抗性分离株中略高。该数据表明，编码构成噻嘧啶敏感受体的烟碱型乙酰胆碱受体亚基的mRNA转录减少可能是犬弓首线虫噻嘧啶抗性机制的一个组成部分。
