UDP-Glycosyltransferases and Albendazole Metabolism in the Juvenile Stages of Haemonchus contortus

Abstract

The nematode Haemonchus contortus, a gastrointestinal parasite of ruminants, can severely burden livestock production. Although anthelmintics are the mainstay in the treatment of haemonchosis, their efficacy diminishes due to drug-resistance development in H. contortus. An increased anthelmintics inactivation via biotransformation belongs to a significant drug-resistance mechanism in H. contortus. UDP-glycosyltransferases (UGTs) participate in the metabolic inactivation of anthelmintics and other xenobiotic substrates through their conjugation with activated sugar, which drives the elimination of the xenobiotics due to enhanced solubility. The UGTs family, in terms of the biotransformation of commonly used anthelmintics, has been well described in adults as a target stage. In contrast, the free-living juvenile stages of H. contortus have attracted less attention. The expression of UGTs considerably varies throughout the life cycle of the juvenile nematodes, suggesting their different roles. Furthermore, the constitutive expression in a susceptible strain with two resistant strains shows several resistance-related changes in UGTs expression, and the exposure of juvenile stages of H. contortus to albendazole (ABZ) and ABZ-sulfoxide (ABZSO; in sublethal concentrations) leads to the increased expression of several UGTs. The anthelmintic drug ABZ and its primary metabolite ABZSO biotransformation, tested in the juvenile stages, shows significant differences between susceptible and resistant strain. Moreover, higher amounts of glycosidated metabolites of ABZ are formed in the resistant strain. Our results show similarly, as in adults, the UGTs and glycosidations significant for resistance-related differences in ABZ biotransformation and warrant further investigation in their individual functions.

The induction and inhibition of UDP-glycosyltransferases in Haemonchus contortus and their role in the metabolism of albendazole

“…As generally recommended for a transcriptional response study, two exposure times (4 h and 12 h) were used. Not many UGTs were upregulated by ABZ nor ABZSO after 4-h exposure both in the males and females, with more transcripts upregulated in males after 12 h. Similarly, the 4-h exposure to ABZ did not lead to any induction of UGTs in the juvenile stages ( Kellerova et al, 2020a ), a result also found with ABZ in very high concentrations in H. contortus adults ( Stasiuk et al, 2019 ). On the other hand, the exposure of H. contortus adults to sub-lethal concentrations of ABZ and ABZSO (1 μM and lower) for 4 and 12 h ex vivo ( Kellerova et al, 2020b ), and for several weeks in vivo ( Dimunová et al, 2022 ) led to the transcriptional response of several UGTs ( Table 4 ).…”

“…enhanced survival ( Fontaine and Choe, 2018 ). The transcription level of UGTs in H. contortus fluctuates during ontogenetic development, with most of the UGTs showing a higher expression in juvenile stages, possibly due to the greater levels of environmental toxins to which the free-living stages are exposed ( Kellerova et al, 2020a ). The connection between UGTs and anthelmintics resistance in H. contortus has been suggested due to the enhanced production of glycosides ( Stuchlikova et al, 2018 ) and higher constitutive expression of UGT368B2 in resistant than in sensitive strains ( Matouskova et al, 2018 ).…”

“…RNA extraction, cDNA synthesis and quantitative qPCR was performed according to previous studies ( Kellerova et al, 2020a , 2020b ). In brief, the samples were homogenized in the FastPrep-24 5G Homogenizer (MP Biomedicals, France); using TriReagent® the total RNA was extracted according to the manufacturer's protocol.…”

“…qPCR was performed in the 384-Well PCR Thermal Cycler; QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, CA, USA) with SYBR Green I detection as described previously ( Kellerova et al, 2020b ). Two housekeeping genes, glyceraldehyde-3P-dehydrogenase (gpd) and nuclear-cap binding protein subunit 2-like (ncbp), were used as reference genes, with 31 UGT primer sets used based on a previous study ( Kellerova et al, 2020a ).…”

“…Interestingly, a higher number and greater amounts of these glucosides were found in the resistant strains compared to the susceptible strain, a finding which indicates the connection between the increased glycosylation of anthelmintics and anthelmintic resistance ( Vokral et al, 2012 , 2013 ; Stuchlikova et al, 2018 ; Stasiuk et al, 2019 ). In resistant strains of H. contortus , increased constitutive expression of several UGT genes was found ( Matouskova et al, 2018 ; Kellerova et al, 2020a ). In the free-living nematode Caenorhabditis elegans, the nonsense point mutation of ugt-22 , which catalyses deactivation of anthelmintic albendazole (ABZ), increased the efficacy of ABZ ( Fontaine and Choe, 2018 ).…”