Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Germany.
PLoS One. 2013 Apr 24;8(4):e61635. doi: 10.1371/journal.pone.0061635. Print 2013.
Macrocyclic lactones (MLs) represent the major drug class for control of parasitic infections in humans and animals. However, recently reports of treatment failures became more frequent. In addition to human and ruminant parasitic nematodes this also is the case for the horse-nematode Parascaris equorum. Nevertheless, to date the molecular basis of ML resistance is still not understood. Unspecific resistance mechanisms involving transporters such as P-glycoproteins (Pgps) are expected to contribute to ML resistance in nematodes. Here, complete sequences of two P. equorum Pgps were cloned and identified as orthologs of Caenorhabditis elegans Ppg-11 and an unnamed Caenorhabditis briggsae Pgp designated as Pgp-16 using phylogenetic analysis. Quantitative real-time PCR was used to compare expression between tissues. Significantly higher PeqPgp-11 expression was found in the gut for both genders, whereas for PeqPgp-16 the body wall was identified as predominant expression site. Furthermore, Pgps were analyzed regarding their participation in resistance development. Using SeqDoC analyses, Pgp-sequences of P. equorum populations with different ML susceptibility were compared. This approach revealed three single nucleotide polymorphisms (SNPs) causing missense mutations in the PeqPgp-11 sequence which correlated with decreased ML susceptibility. However, no resistance associated differences in mRNA expression levels were detected between embryonated eggs of these populations. In contrast, comparison of two pre-adult groups with different ivermectin (IVM) susceptibility revealed the presence of the three SNPs and in addition statistically significant PeqPgp-11 overexpression in the group of worms with reduced susceptibility. These results indicate that Pgp-11 might be involved in IVM resistance in P. equorum as it shows increased expression in an IVM exposed life-cycle stage of an IVM resistant population as well as occurrence of putatively resistance associated SNPs in populations with reduced IVM susceptibility. These SNPs are promising diagnostic candidates for detection of ML resistance with potential also for other parasitic nematode species.
大环内酯类（MLs）是人类和动物寄生虫感染控制的主要药物类别。然而，最近报告的治疗失败案例越来越多。除了人类和反刍动物寄生线虫外，马线虫 Parascaris equorum 也是如此。尽管如此，迄今为止，ML 耐药的分子基础仍不清楚。涉及转运蛋白（如 P-糖蛋白（Pgps））等非特异性耐药机制预计会导致线虫对 ML 的耐药性。在这里，使用系统发育分析，克隆并鉴定了两种 P. equorum Pgps 的完整序列，它们是秀丽隐杆线虫 Ppg-11 和未命名的秀丽新小杆线虫 Pgp-16 的同源物。使用定量实时 PCR 比较组织之间的表达。发现两性的肠道中 PeqPgp-11 的表达明显更高，而对于 PeqPgp-16，体壁被鉴定为主要表达部位。此外，还分析了 Pgps 参与耐药性发展的情况。使用 SeqDoC 分析，比较了 ML 敏感性不同的 P. equorum 种群的 Pgp 序列。这种方法揭示了导致 PeqPgp-11 序列错义突变的三个单核苷酸多态性（SNP），这些突变与 ML 敏感性降低相关。然而，在这些种群的胚胎卵之间未检测到与 mRNA 表达水平相关的耐药性差异。相比之下，比较两个具有不同伊维菌素（IVM）敏感性的前成虫组，发现了三个 SNP 的存在，并且在敏感性降低的组中 PeqPgp-11 过表达统计学上也显著。这些结果表明 Pgp-11 可能参与 P. equorum 的 IVM 耐药性，因为它在 IVM 暴露的生命周期阶段的 IVM 耐药种群中表达增加，并且在 ML 敏感性降低的种群中出现了假定的耐药相关 SNP。这些 SNP 是检测 ML 耐药性的有前途的诊断候选物，对其他寄生线虫物种也具有潜在用途。
