UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences

Abstract

UDP-glycosyltransferases (UGT), catalysing conjugation of UDP-activated sugar donors to small lipophilic chemicals, are widespread in living organisms from bacteria to fungi, plant, or animals. The progress of genome sequencing has enabled an assessment of the UGT multigene family in Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite of small ruminants. Here we report 32 putative UGT genes divided into 15 UGT families. Phylogenetic analysis in comparison with UGTs from Caenorhabditis elegans, a free-living model nematode, revealed several single member homologues, a lack of the dramatic gene expansion seen in C. elegans, but also several families (UGT365, UGT366, UGT368) expanded in H. contortus only. The assessment of constitutive UGT mRNA expression in H. contortus adults identified significant differences between females and males. In addition, we compared the expression of selected UGTs in the drug-sensitive ISE strain to two benzimidazole-resistant strains, IRE and WR, with different genetic backgrounds. Constitutive expression of UGT368B2 was significantly higher in both resistant strains than in the sensitive strain. As resistant strains were able to deactivate benzimidazole anthelmintics via glycosylation more effectively then the sensitive strain, UGT368B2 enhanced constitutive expression might contribute to drug resistance in H. contortus.

Keywords: Detoxification; Haemonchus contortus; Resistance; UDP-glycosyltransferase.

Graphical abstract

Fig. 1

Comparison of selected Hc_UGTs with human UGT2B7. Multiple alignment was performed by using Clustal Omega. Signal peptide in N-terminus predicted by Signal P4.1 is shown as black bar, signature motif and transmembrane domain in C-terminus half in grey and cytoplasmic tail in white above the alignment. Important catalytic residues H and D are indicated by asterisks (*) above the alignment. DBR refers to donor binding region, and several important residues interacting with sugar donor are indicated by symbols under the alignment.

Fig. 2

Relative abundancies of UGT genes from females and males of sensitive ISE strain. For each UGT the mean of relative expression is displayed as ΔCq, calculated as efficiency (UGT)^−Cq(UGT)/efficiency (Hc_gpd)^{−Cq(Hc_gpd)}, * indicates significant difference between female and male at P < 0.05, N = 3.

Fig. 3

Comparison of constitutive expression of UGTs in females and males of adult Haemonchus contortus. Preliminary RNA-seq expression, heat map (A) indicating transcript abundances of UGTs expressed as log₂ transformed normalized values (RPKM) across H. contortus female (F-RPKM) or male (M-RPKM) samples (N = 1). Relative expression (Female/Male, log₂) of Hc_UGTs mRNA from quantitative real-time PCR assay (B), (N = 3). n.d. not detected. Yellow border lines and the X-axis below the heat map correspond to the ratio of Female/Male log₂ relative transcript abundancies for direct comparison with the qPCR results. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Chromozome localization of each UGT gene. The arrow direction indicates location on forward (→) or reverse (←) strand.

Fig. 5

Phylogenetic tree of Haemonchus contortus (marked by black dots) and Caenorhabditis elegans UDP-glycosyltransferases (UGTs). A consensus phylogenetic tree was constructed using the Maximum Likelihood method based on the Le Gascuel 2008 model. The bootstrap consensus tree was calculated (500 replicates). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. UGT from C. elegans with distinct role in resistance is marked by * (Fontaine and Choe, 2018).

Fig. 6

Comparison of constitutive expression in different Haemonchus contortus strains analysed by qPCR. Relative expression of selected Hc_UGTs mRNA from females (A) and males (B). * indicates significant difference between ISE and WR or ISE and IRE at P < 0.05, N = 3.