Comparative Proteomic Analysis of Triclabendazole Response in the Liver Fluke Fasciola hepatica Gustavo Chemale,†,‡ Samirah Perally,†,‡ E. James LaCourse,*,‡,§ Mark C. Prescott,| Laura M. Jones,† Deborah Ward,| Myles Meaney,⊥ Elizabeth Hoey,⊥ Gerard P. Brennan,⊥ Ian Fairweather,⊥ Alan Trudgett,⊥ and Peter M. Brophy† Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, United Kingdom SY23 3DA, Liverpool School of Tropical Medicine, United Kingdom L3 5QA, School of Biological Sciences, Queens University Belfast, United Kingdom, and School of Biological Sciences, The University of Liverpool, Liverpool, United Kingdom L69 7ZB Received February 24, 2010
Control of Fasciola hepatica infections of livestock in the absence of vaccines depends largely on the chemical triclabendazole (TCBZ) because it is effective against immature and adult parasites. Overdependence on a single drug and improper application is considered a significant factor in increasing global reports of fluke resistant to TCBZ. The mode(s) of action and biological target(s) of TCBZ are not confirmed, delaying detection and the monitoring of early TCBZ resistance. In this study, to further understand liver fluke response to TCBZ, the soluble proteomes of TCBZ-resistant and TCBZ-susceptible isolates of F. hepatica were compared with and without in vitro exposure to the metabolically active form of the parent drug triclabendazole sulphoxide (TCBZ-SO), via two-dimensional gel electrophoresis (2-DE). Gel image analysis revealed proteins displaying altered synthesis patterns and responses both between isolates and under TCBZ-SO exposure. These proteins were identified by mass spectrometry supported by a F. hepatica expressed sequence tag (EST) data set. The TCBZ responding proteins were grouped into three categories; structural proteins, energy metabolism proteins, and “stress” response proteins. This single proteomic investigation supported the reductionist experiments from many laboratories that collectively suggest TCBZ has a range of effects on liver fluke metabolism. Proteomics highlighted differences in the innate proteome profile of different fluke isolates that may influence future therapy and diagnostics design. Two of the TCBZ responding proteins, a glutathione transferase and a fatty acid binding protein, were cloned, produced as recombinants, and both found to bind TCBZSO at physiologically relevant concentrations, which may indicate a role in TCBZ metabolism and resistance. Keywords: Fasciola hepatica; triclabendazole • 2-DE • glutathione transferase • fatty acid binding protein
Introduction The trematode liver fluke, Fasciola hepatica, along with Fasciola gigantica are the causative agents of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Liver fluke causes economic losses of over US$ 3 billion worldwide per annum to livestock via mortality, reduction in host fecundity, susceptibility to other infections, decrease in meat, milk, and wool production, and condemnation of livers.1 The disease is increasing in livestock worldwide,2,3 with a number of potential contributing factors: climate change (warmer winters and wetter summers supporting larger inter* To whom correspondence should be addressed. E. James LaCourse. E-mail:
[email protected]. Tel: +44 (0) 151 705 3153. Fax: +44 (0) 151 705 3370. † Aberystwyth University. ‡ These authors contributed equally to this work. § Liverpool School of Tropical Medicine. | The University of Liverpool. ⊥ Queens University Belfast.
4940 Journal of Proteome Research 2010, 9, 4940–4951 Published on Web 07/19/2010
mediate mud snail host populations); fragmented disease management (only treating sheep not cattle and limiting veterinary interaction); encouragement of wet-lands; livestock movement; and/or failure/resistance of chemical control treatments in the absence of commercial vaccines. Fasciolosis is also a re-emerging human disease with estimates of between 2.4 and 17 million people infected worldwide.4 Climate changes, altered land use, socio-economic factors and livestock movements have been considered in providing the opportunity for increased spread and introduction of pathogenic isolates to humans. The World Health Organisation’s recent shift in policy for this emerging disease has paved the way for fasciolosis to be added to the preventative chemotherapy concept.5 This move is in response to the re-emergence of human fasciolosis and is supported by Novartis Pharma AG to implement largescale drug distributions where fasciolosis is a public health concern.6 Control programs for F. hepatica, in the absence of commercial vaccines, rely on chemical treatments.7 Most flukicidal 10.1021/pr1000785
2010 American Chemical Society
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Triclabendazole Response in Fasciola hepatica drugs target adult worms responsible for chronic fasciolosis and not acute pathogenic infections caused by immature fluke migrating through liver.8 Triclabendazole (TCBZ; Fasinex and Egaten, Novartis Animal Health), a novel benzimidazolederivative, offers high activity against both young pathogenic juvenile fluke and the adults present in chronic infections.9 TCBZ has been the front-line defense against liver fluke disease for over 20 years, both in domestic livestock and humans. However, TCBZ-resistant liver fluke are spreading throughout Europe and Australia, compromising control efforts.8-10 Following end of patent protection, generic forms of TCBZ will likely lead to wider application, potential misuse and exacerbation of the spread of resistance. The mode of action of TCBZ at the molecular level has yet to be resolved. Laboratories report a variety of effects of TCBZ on liver fluke including disruption of egg formation,11 protein synthesis inhibition12 and stimulation of glucose-derived formation of acetate and priopionate.13 Many studies focused on microtubule-disrupting action, inferring that, like other benzimidazole-derivative anthelmintics (BZs) in nematodes, TCBZ may also exert its effect directly or indirectly via tubulin.7,9 The inhibition of cell division in the vitelline, spermatogenic and oogonial cells11,14,15 the inhibition of transport of secretory bodies in the tegument16 and the inhibition of tubulin staining16 all support this idea. However, TCBZ-resistant isolates of F. hepatica remain susceptible to the benzimidazole, albendazole (ABZ), with TCBZ resistance appearing unrelated to single mutations in the β-tubulin gene from liver fluke.18-19 Moreover, TCBZ is not effective against other helminths. TCBZ must at least have different tubulin binding site(s) from other BZs if not different primary and/or biological system targets.20,21 TCBZ-resistant fluke have also been shown to accumulate less TCBZ and its metabolites than susceptible fluke,22 with drug entry into the parasite occurring mainly through passive lipophilic diffusion across the tegument rather than orally.23 Uptake of the related BZ compound albendazole (ABZ) does not differ between resistant/susceptible fluke, indicating that perhaps the efflux of drug is only one of several potential contributory factors to resistance.24 Possible transporters for drug efflux include the P-glycoproteins (Pgp), of which a homologue has been identified in F. hepatica, although only found to be expressed in immature fluke.25 Additionally, treatment of fluke with ivermectin, a substrate of Pgp, returns accumulation of TCBZ in resistant F. hepatica to that of susceptible isolates.28 Furthermore, it has been reported that the co-treatment of resistant fluke with TCBZ and another Pgp inhibitor, R(+)-verapamil, demonstrated severe disruption to the tegument surface, in contrast to the minimal disruption found in the absence of R(+)-verapamil, thus supporting a role for efflux pumps in TCBZ resistance.8 The increased efflux of TCBZ is unlikely to be solely responsible for resistance phenotype. Biotransformation and metabolism of TCBZ is also hypothesized to play a major role in detoxification. TCBZ is, mainly oxidized by host liver to form the major active metabolite, triclabendazole sulphoxide (TCBZSO), and a lesser proportion of other metabolities (triclabendazole Sulphone (TCBZ-SO2) and hydroxylated metabolites (OHTCBZ, OH-TCBZ-SO and OH-TCBZ-SO2).26 Fluke have also been shown to metabolize TCBZ, with TCBZ-resistant fluke oxidizing TCBZ more efficiently than susceptible isolates, converting the proposed major active form TCBZ-SO, to a potentially less active form, TCBZ-SO2.22,27 Although the enzymes mediating this biotransformation have yet to be identi-
fied, the use of cytochrome P450 and flavin mono-oxygenase substrates and inhibitors in studies with fluke support the potential for existence and involvement of these enzymes in the metabolism of and resistance to TCBZ.22,28 It may be that the increased production of oxidized and hydroxylated metabolites in resistant fluke allows more efficient detoxification via phase II and phase III conjugation, inactivation and efflux mechanisms. Passive protein-binding of TCBZ has also been proposed as a mode of resistance restricting drug availability at the specific intracellular target site of action.24 The multiple effects on the biochemistry and physiology on F. hepatica suggests that TCBZ interacts both directly and indirectly with many different biological systems.8 Unraveling such potentially complex interactions requires a nonreductionist approach. Array technologies offer such an opportunity to study fasciolicidal activity on a wider scale and, in the case of proteomics technologies supported by an EST data set29 for protein identification, at the functionally relevant phenotypedirecting protein level. We report differences between soluble proteome profiles from well-characterized TCBZ-susceptible (Cullompton isolate) and TCBZ-resistant (Sligo isolate) F. hepatica when exposed or not exposed to the active form TCBZ-SO.
Experimental Procedures Fluke Isolates. Fluke isolates were maintained at Queens University Belfast. TCBZ-susceptible F. hepatica isolate (termed “Cullompton isolate”) was originally obtained from an abattoir in Cullompton, Devon, U.K. in 1998 and was shown to be susceptible to TCBZ.30,31 TCBZ-resistant F. hepatica isolate (termed “Sligo isolate”) was originally obtained from sheep livers in Sligo, Ireland in 1998 and shown to be resistant to TCBZ.30,32 In Vitro Drug Treatment. Eight-week-old fluke of Cullompton and Sligo isolates were removed from the bile ducts of rat hosts under sterile conditions, and washed in sterile NCTC 135 culture medium at 37 °C (Flow Laboratories, Oxfordshire, U.K.), containing antibiotics (penicillin, 50 IU/mL; streptomycin, 50 mg/mL. Sigma, U.K.). Whole fluke were transferred independently to fresh culture medium (1 fluke per 5 mL) containing either solvent control or TCBZ-SO at a clinically relevant concentration of 15 µg/mL or a higher concentration routinely used within the laboratory to ensure fluke exhibit the resistant phenotype, of 50 µg/mL26,33 and incubated for 6 or 12 h at 37 °C. Following incubation, fluke from the range of cultures were visually assessed regarding their relative movement. This relative assessment was to ensure tolerance or susceptibility to drug exposure remained evident at a phenotypic level in the isolates. A relative scoring system based on the extent of movement in comparison to that of controls was used and is displayed in Table 1. ++++ represented movement similar to controls while a decrease in the number of + means a relative decrease in movement. Survival at the end of culture time is indicated by “/S”. Finally, fluke were removed, washed in phosphate buffered saline, snap frozen in liquid nitrogen and stored at -80 °C. At least four individual F. hepatica were used in each experiment. Cytosol Preparations. F. hepatica extracts were obtained by homogenization of frozen fluke at 4 °C in a glass grinder in lysis buffer, containing 50 mM Tris-HCl, pH 7.4, 0.2% TritonX100, 5 mM DTT and a cocktail of protease inhibitors (Roche, Mini-Complete, EDTA-free). After homogenization, samples were sonicated with four pulses of 30 s on ice and centrifuged Journal of Proteome Research • Vol. 9, No. 10, 2010 4941
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Table 1. Evaluation of Activity and Survival of Cullompton (Susceptible) and Sligo (Resistant) Liver Fluke Isolates after TCBZ-SO Treatmenta treatment
Cullompton fluke (susceptible)
Sligo fluke (resistant)
culture time
6h
12h
6h
12h
Nontreated 15 µg/mL 50 µg/mL
++++/S +++/S +/S
++++/S ++/S ND
++++/S ++++/S ++/S
++++/S ++++/S ND
a Fluke were exposed to TCBZ-SO and their movement assessed to provide information about activity and tolerance to drug exposure. ++++ represents movement similar to controls while a decrease in the number of + means a decrease in movement. Survival at the end of culture time is indicated by “/S”.
at 100 000× g for 1 h at 4 °C and the supernatant was termed the cytosolic fraction. 2-DE. Cytosolic protein extracts were precipitated with an equal volume of ice-cold 20% TCA in acetone (w/v) and washed twice in ice-cold acetone before solubilization into isoelectric focusing buffer (IEF buffer) consisting of 7 M urea, 2 M thiourea, 4% w/v CHAPS, 66 mM DTT and 0.5% carrier ampholytes v/v (Biolyte 3-10, Bio-Rad). One mg of each replicate sample was passively in-gel rehydrated for 16 h and isoelectrically focused on 17 cm pH 3-10NL IPG strips (BioRad) to 60 000 Vh on a Protean IEF Cell (Bio-Rad). After focusing, strips were equilibrated for 15 min in reducing equilibration buffer (30% v/v glycerol, 6 M urea, 1% DTT) followed by 15 min in alkylating equilibration buffer (30% v/v glycerol, 6 M urea, 4% iodoacetamide). IPG strips were run upon sodium dodecyl sulfate polyacrylamde gel electrophoresis (SDS-PAGE) (12.5% acrylamide) using the Protean II xi 2-D Cell (BioRad). Gels were Coomassie blue stained (Phastgel Blue R, Amersham Biosciences) and scanned on a GS-800 calibrated densitometer (BioRad). Gel Image Analysis. Gel image quantitative differences between protein spots were analyzed via Progenesis PG220 software, version 2006 (Nonlinear Dynamics). Spots were manually detected on gels with background subtraction performed according to mode of nonspot, accepting the default margin parameter of 45 and normalization performed using total spot volume multiplied by 100. Gel images were then warped and spots manually matched to the reference gel. Averaged gels (based on mean normalized spot volumes) were created from four biological replicates. When creating an averaged gel the maximum number of gels in which spots may be absent was set to 0. Differences between normalized spot volumes with Student t test values of p < 0.05 were considered significant when average gels were compared. Protein Identification. Excision and In-Gel Tryptic Digest of Protein Spots. Protein spots were manually excised and destained in 50% (v/v) acetonitrile (ACN), 50 mM ammonium bicarbonate (Ambic) at 37 °C until clear of blue stain before dehydration in 100% ACN at 37 °C for 30 min. Proteins were digested by rehydrating gel plugs overnight with 10 ng/µL trypsin in 50 mM Ambic at 37 °C. Following digestion, 30 µL 60% (v/v) Ambic, 5% (v/v) formic acid was added to gel plugs before water bath sonication for 3 min. Gel plugs were centrifuged for 30 s at 16 000× g and supernatant removed to a clean tube. This “Ambic/formic acid/ sonication” step was repeated, supernatants pooled, and 4942
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vacuum-dried before resuspension of peptides in 20 µL 0.1% (v/v) formic acid for LC-MS/MS analysis. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Peptide mixtures from trypsin digested gel spots were separated using a LC Packings Ultimate nano-HPLC System. Sample injection was via an LC Packings Famos autosampler and the loading solvent was 0.1% formic acid. The precolumn used was a LC Packings C18 PepMap 100, 5 mm, 100 A and the nano HPLC column was a LC Packings PepMap C18, 3 mm, 100 A. The solvent system was: solvent A (2% ACN with 0.1% formic acid), and solvent B (80% ACN with 0.1% formic acid). The LC flow rate was 0.2 µL/min. The gradient employed was 5% solvent A to 100% solvent B in 1 h. The HPLC eluent was sprayed into the nano-ES source of a Waters Q-TOFµ MS via a New Objective Pico-Tip emitter. The MS was operated in the positive ion ES mode and multiply charged ions were detected using a data-directed MS/MS experiment. Collision induced dissociation (CID) MS/MS mass spectra were recorded over the mass range m/z 80-1400 Da with scan time 1 s. Mass Spectra Analysis. MassLynx v 3.5 (Waters, U.K.) ProteinLynx suite of tools was used to process raw fragmentation spectra. Each spectrum was combined and smoothed twice using the Savitzky-Golay method at (3 channels with background noise subtracted at polynomial order 15 and 10% below curve. Monoisotopic peaks were centered at 80% centroid setting. Sequest compatible (.dta) file peak mass lists for each spectrum were exported, and spectra common to each 2-DE spot were merged into a single MASCOT generic format (.mgf) file using the online Peak List Conversion Utility available at www.proteomecommons.org.34 Merged files were submitted to a MASCOT MS/MS ions search within a locally installed Mascot server (www.matrixscience.com) to search an “in-house” database constructed from 6260 (858 763 residues) Fasciola hepatica EST sequences downloaded and translated from the Sanger Institute (ftp://ftp.sanger.ac.uk/pub/pathogens/fasciola/ hepatica/ESTs/). Search parameters were as follows: enzyme set at trypsin with one missed cleavage allowed; fixed modification set for carbamidomethylation with variable modification considered for oxidation of methionine; monoisotopic masses with unrestricted protein masses were considered with peptide and fragment mass tolerances at (1.2 and (0.6 Da respectively for an ESI-QUAD-TOF instrument. Protein identifications resulting from MASCOT ions scores greater than 25 were considered as showing significant identity or extensive homology (p < 0.05) to the predicted identification displayed (www.matrixscience. com). Spectra that did not match any proteins, or scored nonsignificantly within F. hepatica EST database were researched through MASCOT against the NCBI nonredundant database (NCBI http://www.ncbi.nlm.nih.gov/ nr release 20091112 containing 10 032 801 sequences; 3 422 028 181 residues). MASCOT scores for individual ions greater than 23 indicate significant homology while scores above 51 indicate significant identity or extensive homology (p < 0.05). All MASCOT search parameters and settings were as described above except that taxonomy was restricted to “Metazoa” (1 804 634 sequences). Spectra containing large amounts of low abundance ion masses with mass peak lists too large to provide significant MASCOT scores, were interpreted via BioLynx automated peptide sequencing tools (MassLynx v 3.5). Raw spectra were
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Triclabendazole Response in Fasciola hepatica combined, smoothed, subtracted and centered as described above and deconvoluted using MaxEnt 3 software (MassLynx v 3.5). Precursor ion tolerance was set to 0.5 Da, fragment mass tolerance was set to 0.1 Da and intensity threshold was set at 1%. Amino acid modifications considered were carboxyamidomethylcysteine, cysteine acrylamide and methionine sulphoxide, and trypsin was specified as the enzyme used for protein digestion, two missed cleavages were permitted. Minimum mass standard deviation was set at 0.025 Da and the sequence display threshold (% Prob) set to 1. Highest ranked peptide sequences were accepted. Where significant low abundance mass peaks (noise) made automated sequencing problematic, fragmentation spectra were semiautomatically interpreted using the sequence tag function of peptide sequencing software (MassLynx v 3.5) with identical parameters as outlined above. Protein Identification and Annotation of F. hepatica EST Sequences. The F. hepatica ESTs used here are at present unannotated as to predicted protein identifications. F. hepatica proteins identified via LC-MS/MS, were assigned putative identification based upon similarity to proteins with existing annotation within GenBank nonredundant database (all nonredundant GenBank CDS translations+PDB+Swiss-Prot+PIR+ PRF excluding environmental samples from WGS projects (9 636 254 sequences; 3 294 494 089 total letters; Posted date Sep 2, 2009 5:42 p.m.; downloaded at ftp://ftp.ncbi.nih.gov/ blast/db/). Automated/manually interpreted peptide sequences from MS/MS spectra were searched via BLASTp35 within the BioEdit program (version 7.0.5.336 against an “in-house” database of Fasciola hepatica sequences (described earlier in Experimental Procedures). Where no significant matches were obtained, BLASTp was performed against GenBank (see below for database details). F. hepatica EST sequences identified by either; MASCOT MSMS ions searches, and/or BLASTp of LC-MS/MS automated/ manually interpreted peptides, were searched further via BLASTp (version 2.2.20 [Feb-08-2009]37,38 against GenBank (all nonredundant GenBank CDS translations+PDB+Swiss-Prot+ PIR+PRF excluding environmental samples from WGS projects (9 636 254 sequences; 3 294 494 089 total letters; Posted date Sep 2, 2009 5:42 p.m.; downloaded at ftp://ftp.ncbi.nih.gov/ blast/db/) according to default settings.37,38 Proteins showing similarity with significant E-values (
