Godoy Pablo, Che Hua, Beech Robin N, Prichard Roger K
Institute of Parasitology, Macdonald campus, McGill University, 21,111 Lakeshore Road, Sainte Anne-de-Bellevue, H9X3V9, QC, Canada.
Parasit Vectors. 2016 Jan 28;9:52. doi: 10.1186/s13071-016-1317-8.
The existence nematodes of veterinary importance such as Haemonchus contortus resistant to anthelmintic drugs, including the macrocyclic lactones, has become a major concern in animal health. Macrocyclic lactone resistance in H. contortus seems to be multigenic including the active efflux of these drugs by P-glycoproteins, members of the ABC transporter family, present in this parasite. The goals of the present work were to determine the activity of H. contortus P-glycoprotein 9.1 (Hco-PGP-9.1) and its interaction with the avermectins, ivermectin, abamectin, and also the milbemycin, moxidectin. Additionally, the localisation of Hco-PGP-9.1 was sought in adult worms.
Hco-Pgp-9.1 was cloned and expressed in mammalian cells and its expression profile was determined at the transcriptional and protein level by qRT-PCR and Western-blot, respectively. The nematode transport activity was assessed using the tracer dye Rhodamine 123. A ligand competition assay between different macrocyclic lactones and Rhodamine 123 was used to establish whether or not there was interaction between Hco-PGP-9.1 and the avermectins (abamectin and ivermectin) or moxidectin. In addition, immunostaining was carried out to localise Hco-PGP-9.1 expression in the transgenic cells and in adult female parasites.
Hco-PGP-9.1 was expressed in the cell membrane of the transfected host cells and was able to extrude Rhodamine 123. Ivermectin and abamectin, but not moxidectin, had a pronounced inhibitory effect on the ability of Hco-PGP-9.1 to transport Rhodamine 123. Antibodies raised against Hco-PGP-9.1 epitopes localised to the uterus of adult female H. contortus.
These results suggest a strong interaction of the avermectins with Hco-PGP-9.1. However, possibly due to its physico-chemical properties, moxidectin had markedly less effect on Hco-PGP-9.1. Because of the greater interaction of the avermectins than moxidectin with this transporter, it is more likely to contribute to avermectin resistance than to moxidectin resistance in H. contortus. Possible over expression of Hco-PGP-9.1 in the female reproductive system in resistant worms could reduce paralysis of the uterus by macrocyclic lactones, allowing continued egg release in drug challenged resistant worms.
诸如捻转血矛线虫等具有兽医重要性的线虫对包括大环内酯类在内的驱虫药产生抗性，已成为动物健康领域的一个主要问题。捻转血矛线虫对大环内酯类的抗性似乎是多基因的，包括由该寄生虫中存在的ABC转运蛋白家族成员P -糖蛋白对这些药物的主动外排。本研究的目的是确定捻转血矛线虫P -糖蛋白9.1（Hco - PGP - 9.1）的活性及其与阿维菌素、伊维菌素、阿巴美丁以及米尔贝霉素、莫西菌素的相互作用。此外，还研究了Hco - PGP - 9.1在成虫中的定位。
克隆Hco - Pgp - 9.1并在哺乳动物细胞中表达，分别通过qRT - PCR和蛋白质印迹在转录和蛋白质水平测定其表达谱。使用示踪染料罗丹明123评估线虫转运活性。利用不同大环内酯类与罗丹明123之间的配体竞争试验来确定Hco - PGP - 9.1与阿维菌素（阿巴美丁和伊维菌素）或莫西菌素之间是否存在相互作用。此外，进行免疫染色以定位Hco - PGP - 9.1在转基因细胞和成年雌性寄生虫中的表达。
Hco - PGP - 9.1在转染宿主细胞的细胞膜中表达，并能够排出罗丹明123。伊维菌素和阿巴美丁，而非莫西菌素，对Hco - PGP - 9.1转运罗丹明123的能力有显著抑制作用。针对Hco - PGP - 9.1表位产生的抗体定位于成年雌性捻转血矛线虫的子宫。
这些结果表明阿维菌素与Hco - PGP - 9.1之间存在强烈相互作用。然而，可能由于其物理化学性质，莫西菌素对Hco - PGP - 9.1的作用明显较小。由于阿维菌素比莫西菌素与该转运蛋白的相互作用更强，在捻转血矛线虫中，它比莫西菌素更有可能导致对阿维菌素的抗性。抗性蠕虫雌性生殖系统中Hco - PGP - 9.1可能的过度表达可减少大环内酯类对子宫的麻痹作用，使受药物攻击的抗性蠕虫能够持续产卵。
