Glutathione transferases of Haemonchus contortus; Profiling and Responses to Ivermectin


S Summers6; P Millares3; H Rees3; M Prescott3; D Bartley5; J Hodgkinson2; A Morrison5; P Skuce5; E Lacourse4; P M Brophy1
1 Aberystwyth University - IBERS, UK;  2 Institute of Infection and Global Health, University of Liverpool, UK;  3 Institute of Integrative Biology, University of Liverpool, UK;  4 Liverpool School of Tropical Medicine / UoL, UK;  5 Moredun Research institute, UK;  6 University of Liverpool, UK


The most economically important gastrointestinal nematode parasite of sheep is Haemonchus contortus (the Barber’s Pole Worm). It has become increasingly difficult to control infections of H. contortus within the global sheep industry due to the rapid development of widespread anthelmintic resistance against multiple drug classes. There are concerns that the worldwide resistance observed against the macrocyclic lactone ivermectin (IVM) in particular may significantly threaten the feasibility of the sheep industry. The mechanisms for anthelmintic drug resistance are poorly understood and prior studies have focused on a range of potential mechanisms and drug targets. Amongst these potential mechanisms are the glutathione transferases (GSTs), a superfamily of phase II detoxification enzymes which may play a role in aiding nematode survival against anthelmintic drugs. However, the GST-specific proteome or ‘GST-ome’, and biochemical activity of H. contortus GSTs expressed under conditions of IVM exposure has yet to be revealed. In this study the cytosolic GSTs of three isolates of H. contortus, differing in susceptibility and resistance to IVM, were identified and compared via two-dimensional gel proteomics and enzymic activity assays following exposure to sub-lethal concentrations of IVM. Glutathione transferase enzyme activity was assayed using 1-chloro-2, 4-dinitrobenzene (CDNB) as a second substrate to reduced glutathione. Purification of GSTs was undertaken using S-hexylglutathione affinity chromatography, before the resolution and visualisation of purified GSTs via two-dimensional gel electrophoresis (2DE). Tandem mass spectrometry (MSMS) and protein database in-silico searches provided identification of GSTs present on 2DE. Comparative enzymic activities and GST identifications revealed class-specific profiles and responses across the three different isolates of H. contortus under sub-lethal IVM exposure. Adult stages of ivermectin resistant and susceptible H. contortus isolates displayed clear differences in protein expression, evident from protein profiles. These data correlate with previous findings in other anthelmintic resistant parasitic nematodes, including increased production of detoxification proteins in drug resistant isolates. The recombinant expression of GSTs identified in H. contortus isolates will aid in exploring the role GSTs may play in the detoxification of IVM. Understanding the mechanism of detoxification of IVM in H. contortus will aid in combatting the inevitable development of anthelmintic resistance in parasitic nematodes infecting both livestock and humans.

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