title: A new enabling proteomics methodology to investigate membrane associated proteins from parasitic nematodes: case study using ivermectin resistant and ivermectin susceptible isolates of Caenorhabditis elegans and Haemonchus contortus.
abstract: The mechanisms involved in anthelmintic resistance (AR) are complex but a greater understanding of AR management is essential for effective and sustainable control of parasitic helminth worms in livestock. Current tests to measure AR are time consuming and can be technically problematic, gold standard diagnostics are therefore urgently required to assist in combatting the threat from drug resistant parasites. For anthelmintics such as ivermectin (IVM), target proteins may be present in the cellular membrane. As proteins usually act in complexes and not in isolation, AR may develop and be measurable in the target associated proteins present in the parasite membrane. The model nematode Caenorhabditis elegans was used to develop a sub-proteomic assay to measure protein expression differences, between IVM resistant and IVM susceptible isolates in the presence and absence of drug challenge. Evaluation of detergents including CHAPS, ASB-14, C7BzO, Triton ×100 and TBP (tributyl phosphine) determined optimal conditions for the resolution of membrane proteins in Two Dimensional Gel Electrophoresis (2DE). These sub-proteomic methodologies were then translated and evaluated using IVM-susceptible and IVM-resistant Haemonchus contortus; a pathogenic blood feeding parasitic nematode which is of global importance in livestock health, welfare and productivity. We have demonstrated the successful resolution of membrane associated proteins from both C. elegans and H. contortus isolates, using a combination of CHAPS and the zwitterionic amphiphilic surfactant ASB-14 to further support the detection of markers for AR.
authors.affiliation: Institute of Biological, Environmental and Rural Sciences (IBERS), Edward Llwyd Building, Aberystwyth University, SY23 3DA, UK. Electronic address: elh18@aber.ac.uk.
authors.forename: Elizabeth H
authors.initials: EH
authors.lastname: Hart
authors.affiliation: Institute of Biological, Environmental and Rural Sciences (IBERS), Edward Llwyd Building, Aberystwyth University, SY23 3DA, UK.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Brophy
authors.affiliation: Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Prescott
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, Scotland EH26 OPZ, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Bartley
authors.affiliation: Institute of Biological, Environmental and Rural Sciences (IBERS), Edward Llwyd Building, Aberystwyth University, SY23 3DA, UK.
authors.forename: Basil T
authors.initials: BT
authors.lastname: Wolf
authors.affiliation: Institute of Biological, Environmental and Rural Sciences (IBERS), Edward Llwyd Building, Aberystwyth University, SY23 3DA, UK.
authors.forename: Joanne V
authors.initials: JV
authors.lastname: Hamilton
mesh_terms: D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology; D017173:Caenorhabditis elegans / Q000737:chemistry* / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation; D006189:Haemonchus / Q000737:chemistry* / Q000235:genetics; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics; D007559:Ivermectin / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
chemical_list: D000871:Anthelmintics; D015801:Helminth Proteins; D008565:Membrane Proteins; D007559:Ivermectin
keywords: 2DE optimisation; Caenorhabditis elegans; Haemonchus contortus; Membrane associated proteins; Sub-proteome analysis