Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones

Abstract

SUMMARY Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulateCon-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

Gene expression of ABC transporters in Cooperia oncophora after field and laboratory selection with macrocyclic lactones

“…In an IVM-resistant T. circumcincta isolate, increased gene expression of P-gp was most notable for Tci-pgp9 [13]. Increased expression of Con-pgp11 was recently reported in adult worms of a ML-resistant C. oncophora isolate [15]. The same P-gp isotype, Peqpgp11, is increased in P. equorum showing reduced susceptibility to ML [14].…”

“…Furthermore, 11 partial P-gp sequences have been identified in Teladorsagia circumcincta, an abomasal nematode of sheep [13], and two full P-gp sequences were recently cloned in the equine roundworm Parascaris equorum [14]. Furthermore, partial sequences of seven P-gp, five HAF and three MRP genes from C. oncophora have been described [15]. However, the degree of conservation of the biological role of ABC transporters between nematode species of veterinary interest and the nomenclature used are still unclear.…”

Acquired Tolerance to Ivermectin and Moxidectin after Drug Selection Pressure in the Nematode Caenorhabditis elegans

“…Interestingly, MOX interacts weakly with nematode Pgps (62)(63)(64)(65)(66), and this is in agreement with its higher level of toxicity in ML-resistant strains. Moreover, the cross-resistance phenotype between ML compounds in parasitic nematodes was associated with the overexpression of several Pgps (10,13,43,44). Given all the similarities between various different isolates with C. elegans, this model nematode clearly represents a relevant tool to study the mechanism of adaptation of parasitic nematodes to drugs.…”

“…Gene expression levels of ABC transporters or allele frequencies were modified after ML selection (8)(9)(10)(11)(12)(13), and they are involved in the tolerance of Caenorhabditis elegans (9,(14)(15)(16) and parasitic nematodes such as Haemonchus contortus or Cooperia oncophora (13,(17)(18)(19)(20) to MLs. In addition, mutation of the dyf-7 gene was associated with an IVM resistance phenotype in C. elegans and in H. contortus, leading to an abnormal dendritic morphology of amphid sensory neurons, as revealed by a dye-filling-defective phenotype (21).…”

Functional and Phylogenetic Characterization of Proteins Detected in Various Nematode Intestinal Compartments*

“…Interpro domain enrichment testing showed that "UDP-glucuronosyl/UDP-glucosyltransferase" domains were the most significantly enriched among IIM proteins (IPR002213; p ϭ 3.3 ϫ 10 Ϫ4 ; Table III); UDP-glycosyltransferases are found in all living organisms, and in C. elegans they are primarily responsible for the detoxification of foreign compounds (65,66). The IIM proteins were also enriched for many ABC transporter domain-containing proteins (Table III) that affect the absorption, distribution, and elimination of xenobiotics, including known anthelmintic drugs (67,68). Multiple examples of P-glycoprotein ABC transporters are located on the AIM of C. elegans (4).…”