Isolation and characterisation of four partial cDNA sequences encoding multidrug resistance-associated proteins (MRPs) in the salmon louse Lepeophtheirus salmonis (Kroyer, 1837)
Isolation and characterisation of four partial cDNA sequences encoding multidrug resistance-associated proteins (MRPs) in the salmon louse Lepeophtheirus salmonis (Kroyer, 1837)
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DOI：
10.1016/j.aquaculture.2013.12.015
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发表时间：
2014-03-20
期刊：
影响因子：
4.5
通讯作者：
Sturm, Armin
中科院分区：
文献类型：
--
作者：
Heumann, Jan;Carmichael, Stephen N.;Sturm, Armin
鲑虱Lepeophtheirus salmonis （Kroyer, 1837）是鲑盖鱼的一种体外寄生虫，严重影响北大西洋养殖鲑鱼的生产。目前养殖场对鲑鱼虱的控制部分依赖于兽药的使用；然而，由于鲑鱼除虫剂的数量有限，人们担心鲑鱼虱会产生抗药性。常见的抗鲑鱼虱药SLICE（默克动物保健公司）含有阿维菌素-阿维菌素-苯甲酸酯（EMB）。ABC （atp结合盒）转运体大基因超家族的成员已被确定为寄生线虫中潜在的阿维菌素抗性因子。在鲑鱼虱中，迄今为止只有三个ABC转运体被克隆和研究。我们在这里报道了四种新型沙门氏菌ABC转运蛋白的分离，并采用基于抑制剂的方法来评估沙门氏菌ABC转运蛋白在EMB毒理学中的作用。为了分离鲑鱼虱ABC转运蛋白，我们对公开的L鲑鱼表达序列标签（est）进行了同源性搜索，并将检索到的est组装成连续序列并进行了注释。通过这种方法分离的潜在ABC药物转运体包括四种多药耐药相关蛋白（MRPs）。此外，还获得了5种可能与耐药性无关的ABC蛋白。采用实时荧光定量PCR （RT-qPCR）对不同EMB易感性沙门氏菌菌株的MRPs mRNA水平进行分析。在没有EMB的情况下，所有研究的MRPs在药物敏感菌株S和中等EMB抗性菌株r中表现出相似的转录水平，而且，四种研究的MRPs的mRNA表达不受EMB暴露的影响。综上所述，RT-qPCR分析的结果并没有提供证据表明所研究的MRPs是影响EMB易感性的因素。进一步的实验使用基于抑制剂的方法来研究ABC转运蛋白在EMB毒性中的作用。在不动性生物测定中，两种菌株的鲑鱼虱暴露于EMB，单独或与环孢素A或维拉帕米联合提供，这是已知的p糖蛋白和MRPs抑制剂。环孢素A对两株菌株的EMB毒性增加程度相似，表明受该抑制剂影响的生化因子在两株菌株中表现出相似的水平。相反，维拉帕米仅在R菌株中增加EMB效应。这一结果可能表明，在中度抗emb的R菌株中，迄今为止未知的ABC转运蛋白表达增强。需要更多的研究来确定维拉帕米在鲑鱼虱中的靶标。(C) 2013 Elsevier B.V.版权所有
The salmon louse Lepeophtheirus salmonis (Kroyer, 1837) is an ectoparasite of salmo lid fish severely affecting cultured salmon production in the North Atlantic. Salmon louse control on farms currently relies in part upon the use of veterinary drugs; however, as only a limited number of salmon delousing agents are available, there are concerns about development of drug resistance in salmon lice. The common anti-salmon louse drug SLICE (Merck Animal Health) contains the avermectin emamectin benzoate (EMB). Members of the large gene superfamily of ABC (ATP-binding cassette) transporters have been identified as potential avermectin resistance factors in parasitic nematodes In salmon lice, only three ABC transporters have been cloned and studied to date. We report here upon the isolation of four novel L. salmonis ABC transporters, and employ an inhibitor-based approach to assess roles of L. salmonis ABC transporters in the toxicology of EMB. To isolate salmon louse ABC transporters, publicly available L salmonis expressed sequence tags (ESTs) were subjected to homology searches, and the retrieved ESTs assembled into contiguous sequences and annotated. Potential ABC drug transporters isolated by this approach included four multidrug resistance-associated proteins (MRPs). In addition, five ABC proteins likely having roles unrelated to drug resistance were obtained. Quantitative real time PCR (RT-qPCR) was used to analyse mRNA levels of the MRPs in L. salmonis strains differing in EMB susceptibility. In the absence of EMB, all studied MRPs showed similar transcript levels in the drug-susceptible strain S and the moderately EMB-resistant strain R. Moreover, mRNA expression of the four studied MRPs remained unaffected by exposure to EMB. Taken together, the results of RT-qPCR analyses did not provide evidence for roles of the studied MRPs as factors affecting EMB susceptibility. Further experiments used an inhibitor-based approach to investigate the roles of ABC transporters in EMB toxicity. In immotility bioassays, salmon lice of the two strains were exposed to EMB, provided alone or in combination with cyclosporin A or verapamil, which are known inhibitors of P-glycoprotein and MRPs. Cyclosporin A increased EMB toxicity to a similar degree with both strains, suggesting the biochemical factors affected by this inhibitor show similar levels in both strains. In contrast, verapamil increased EMB effects only in the R strain. This result could be indicative of an enhanced expression of hitherto unknown ABC transporters in the moderately EMB-resistant R strain. More research is required to identify the target of verapamil in salmon lice. (C) 2013 Elsevier B.V. All rights reserved.