Comparison of constitutive and thiabendazole-induced expression of five cytochrome P450 genes in fourth-stage larvae of Haemonchus contortus isolates with different drug susceptibility identifies one gene with high constitutive expression in a multi-resistant isolate

Abstract

Benzimidazoles (BZs) remain amongst the most widely used anthelmintic drug classes against gastro-intestinal nematode infections, although their efficacy is increasingly compromised by resistance. The primary underlying mechanisms for BZ resistance are single-nucleotide polymorphisms (SNPs) in the isotype 1 β-tubulin gene causing the substitutions F167Y, E198A or F200Y. However, resistance is believed to be multi-genic and previous studies have shown that isolates carrying 90–100% F200Y can vary considerably in their resistance level in the egg hatch assay (EHA). Cytochrome P450 monooxygenases (CYPs) are associated with drug resistance in mammals and arthropods and have been considered as mediators of anthelmintic resistance. In Caenorhabditis elegans, several members of the CYP34/35 and CYP31 families are BZ and/or xenobiotic inducible and thiabendazole (TBZ) is metabolised by CYP35D1. Here, expression of all 5 CYPs closely related to the C. elegans CYP34/35 and CYP31 families was investigated in fourth-stage larvae of two susceptible and three BZ-resistant Haemonchus contortus isolates following in vitro exposure to TBZ for 3 and 6 h using real-time RT-PCR. The resistance status of all isolates was determined using EHAs and quantification of resistance-associated β-tubulin SNPs using pyrosequencing. While none of the CYPs was TBZ inducible, constitutive expression of CYP34/35 family member HCOI100383400 was significantly 2.4–3.7-fold higher in the multi-drug resistant WR isolate with the strongest BZ resistance phenotype compared to susceptible and intermediate-level BZ-resistant isolates. Although this increase is only moderate, HCOI100383400 might still be involved in high-level BZ resistance by further decreasing susceptibility in isolates already carrying 100% of a β-tubulin SNP causing BZ resistance. Lower transcript levels were observed for all CYPs in the intermediately resistant IRE isolate in comparison to the susceptible HcH isolate, which, except for CYP HCOI01579500, were statistically non-significant. This suggests that none of the investigated CYPs may contribute to protection against TBZ in this particular isolate.

A Whole Genome Re-Sequencing Based GWA Analysis Reveals Candidate Genes Associated with Ivermectin Resistance in Haemonchus contortus

“…However, this view has been changed with the discovery of CYPs-encoding genes in C. elegans, where more than 80 CYP proteins are present [85,86], and these have been shown to be xenobiotics inducible [85,[87][88][89][90]. The CYPs family have also been characterized recently in H. contortus [91,92]. ABC transporter and pgp fall under the phase III of xenobiotic detoxification process.…”

Antifungal benzimidazoles disrupt vasculature by targeting one of nine β-tubulins

“…Benzimidazole suppressor screens in both Saccharomyces cerevisiae and Caenorhabditis elegans have independently identified resistance mutations occuring in β-tubulin genes, giving some insight into the binding site 29,30 . The case for a β-tubulin binding site is strengthened by numerous animal and agricultural studies also demonstrating resistance mutations arising repeatedly and independently across multiple parasitic nematode and fungal species infecting farm livestock and crops 22,27,[31][32][33][34][35][36][37][38][39][40][41][42][43] .…”

“…Three commonly observed mutations in fungal and nematode β-tubulins (F200Y, E198A, and F167Y) confer resistance to TBZ ( Fig. 2A, B), suggesting that its binding site is in the vicinity of these residues 22,27,[31][32][33][34][35][36][37][38][39][40][41][42][43] (File S1). Based on the previously observed benzimidazole suppressor 6 mutations, we used 3D structural modeling to evaluate TBZ's potential binding sites in a fungal β-tubulin.…”

“…Given the plethora of fungal and nematode studies on benzimidazole pesticide resistance in agriculture 22,[33][34][35][36]38,[40][41][42][43][44][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71] (Fig. 4A), we reasoned that TBZ's molecular mechanism may extend to other commercially used benzimidazole compounds.…”

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

“…The phase I enzymes of the cytochrome P450-family (CYP) have been shown to be involved in drug resistance in insects (27), but the involvement of phase I and II enzymes in anthelmintic resistance of parasitic nematodes has not been thoroughly investigated, particularly not in ascarids. However, constitutively higher expression of a CYP34/35 family member (28) as well as the phase II enzyme uridine 5'-diphospho-glucuronosyltransferase-glucosyltransferase (UGT) (29) have been found in BZ resistant strains of H. contortus. Several studies have also shown evidence for upregulation of genes encoding drug efflux pumps, such as ATP-binding cassette (ABC) transporters, in macrocyclic lactone resistant strongyle nematodes (30)(31)(32).…”