Proteomic analysis of triclabendazole response in Fasciola hepatica

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The objective is to determine changes in the expression proteomes of triclabendazole (TCBZ) resistant and susceptible isolates of the liver fluke, Fasciola hepatica, as a consequence of exposure to TCBZ. The project will use defined and well characterised fluke isolates, two are susceptible to TCBZ and two are resistant, allowing reference to published work in Belfast, and avoiding individual isolate differences, as opposed to differences associated with TCBZ metabolism and excretion. Material for proteomic analysis and studies on drug metabolism will be provided by time-course experiments (50micrograms/ml of triclabendazole sulphoxide (TCBZ-SO) for 3 h, 6h and 9h in vitro at 37ºC) utilising three week old juveniles and adults. Major TCBZ-SO responding spots detected from two Dimensional Eletrophoresis (2 DE) image analysis will be identified by Mass Spectrometry, and antibodies raised to recombinant proteins for localisation by immunochemistry. Proteomics will be undertaken according to techniques developed in our laboratory for F. hepatica (Jefferies et al 2000, 2001 ¿ in reference list) using high-throughput 2DE equipment with automated staining and spot excision. Proteins will be identified incorporating the new F. hepatica EST database by two MS strategies, either MALDI-TOF-MS: peptide mass fingerprinting (PMF)/post-source decay (PSD) or LC-ESI MS/MS for the sequencing of selected peptides. For metabolic studies fluke homogenates will be prepared and analysed by HPLC using a Shimadzu SPD-6A UV detector (Shimadzu, Kyoto, Japan), LC-6A pumps with 50ml loop injection, an SCL-6B system controller and SIL-6B auto-injector (Shimadzu). Separation will be performed on a Bio-Rad C18 Hi-Pore 250mm x 4.6mm reversed phase column (pore size 300 Angstrom; particle size 5 micron) with a Waters guard pack. TCBZ-SO and other TCBZ metabolites will be identified and quantified using internal standards (for this purpose we have 7 pure TCBZ metabolites and related compounds which have been supplied by the drug manufacturer, Novartis). For the first time, the relative roles of soluble phase I and phase II drug metabolising enzymes (including Glutathione transferase, GST) and membrane drug detoxification proteins such as P-glycoprotein in TCBZ-resistance in the liver fluke will be resolved. F. hepatica will be incubated with TCBZ-SO in vitro as described above. During the incubations, flukes will also be exposed to inhibitors and reversal agents of detoxification proteins (Sauvage et al. in press). Fluke homogenates will be prepared and analysed by HPLC to assess the relative contribution of detoxification pathways. The levels of TCBZ-SO and other TCBZ metabolites will be determined by the internal standards. These experiments will be complementary to the proteomics studies and will provide information relating to the interpretation of the proteomes. Joint with BB/C00082X/1