P-glycoproteins of Haemonchus contortus: development of real-time PCR assays for gene expression studies

摘要

P-glycoproteins (P-gps) are proteins that function as efflux pumps, removing lipophilic xenobiotic compounds from cells. There is evidence that P-gps play a role in the resistance of parasitic nematodes to anthelmintic drugs such as benzimidazoles and macrocyclic lactones. As anthelmintic resistance becomes more common, it is important to identify candidate resistance genes with the aim of understanding the molecular basis of resistance, and of developing assays to detect these resistance-associated changes. We identified several sequences from the genome of the parasite Haemonchus contortus with convincing homology to the known P-gp coding genes of the model nematode Caenorhabditis elegans. Nine of these sequences were successfully amplified by polymerase chain reaction (PCR) and shown to be most similar to the C. elegans sequences for pgp-1, pgp-2, pgp-3, pgp-4, pgp-9, pgp-10, pgp-11, pgp-12 and pgp-14. These partial P-gp sequences from H. contortus were used to design and optimize a quantitative real-time PCR assay to investigate potential changes in the expression levels of P-gp transcripts associated with drug resistance. No significant changes in P-gp mRNA expression levels were found in a rapidly selected ivermectin-resistant parasite isolate compared to its drug-sensitive parent, but the assay has the potential to be used on other isolates in the future to further investigate resistance-associated changes in P-gp gene expression.