International Journal for Parasitology 41 (2011) 1347–1359 Contents lists available at SciVerse ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara Exploring the Fasciola hepatica tegument proteome ⇑ R. Alan Wilson a, , Janelle M. Wright b, William de Castro-Borges a,c, Sophie J. Parker-Manuel a, Adam A. Dowle d, Peter D. Ashton a, Neil D. Young e, Robin B. Gasser e, Terry W. Spithill b,f a Centre for Immunology and Infection, Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom b School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW 2650, Australia c Departamento de Ciências Biológicas, Universidade Federal de Ouro Preto, CEP – 35400-000 Ouro Preto, MG, Brazil d Centre of Excellence in Mass Spectrometry, Technology Facility, Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom e Department of Veterinary Science, The University of Melbourne, Parkville, Victoria 3052, Australia f Department of Agricultural Sciences and Centre for AgriBioscience, La Trobe University, Bundoora, Victoria 3083, Australia article info abstract Article history: The surface tegument of the liver fluke Fasciola hepatica is a syncytial cytoplasmic layer bounded exter- Received 15 June 2011 nally by a plasma membrane and covered by a glycocalyx, which constitutes the interface between the Received in revised form 29 August 2011 parasite and its ruminant host. The tegument’s interaction with the immune system during the fluke’s Accepted 30 August 2011 protracted migration from the gut lumen through the peritoneal cavity and liver parenchyma to the Available online 5 October 2011 lumen of the bile duct, plays a key role in the fluke’s establishment or elimination. However, little is known about proteins of the tegument surface or its secretions. We applied techniques developed for Keywords: the blood fluke, Schistosoma mansoni, to enrich a tegument surface membrane preparation and analyse Fasciola hepatica its composition by tandem mass spectrometry using new transcript databases for F. hepatica.We Liver fluke Tegument proteome increased the membrane and secretory pathway components of the final preparation to 30%, whilst Excretory–secretory proteins eliminating contaminating proteases. We identified a series of proteins or transcripts shared with the Tandem mass spectrometry schistosome tegument including annexins, a tetraspanin, carbonic anhydrase and an orthologue of a host Morphology protein (CD59) that inhibits complement fixation. Unique to F. hepatica, we also found proteins with lec- Vomitus tin, cubulin and von Willebrand factor domains plus 10 proteins with leader sequences or transmem- brane helices. Many of these surface proteins are potential vaccine candidates. We were hampered in collecting tegument secretions by the propensity of liver flukes, unlike blood flukes, to vomit their gut contents. We analysed both the ‘vomitus’ and a second supernatant released from haematin-depleted flukes. We identified many proteases, some novel, as well as a second protein with a von Willebrand fac- tor domain. This study demonstrates that components of the tegumental surface of F. hepatica can be defined using proteomic approaches, but also indicates the need to prevent vomiting if tegument secre- tions are to be characterised. Crown Copyright Ó 2011 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. 1. Introduction 180 million at risk. High but localised prevalence (72–100%) has been recorded in Bolivia (>1 million cases), Peru, Africa and the Fasciolosis is a major zoonotic disease caused by the liver flukes Middle East (Mas-Coma et al., 2005; McManus and Dalton, 2006) Fasciola hepatica and Fasciola gigantica, which are flatworm para- with up to 830,000 people infected in Egypt alone (Mas-Coma sites transmitted following ingestion of herbage contaminated et al., 2005). Triclabendazole is the drug of choice for treatment with the infective stage (metacercariae). The infection is a signifi- of fasciolosis but resistance, first observed in Australia in 1995, is cant constraint on ruminant productivity in Europe, Africa, Asia, now widespread in Europe (Fairweather, 2009). In endemic areas the Americas and Australasia, with prevalences in some regions such as Bolivia and Egypt, resistance threatens to make the disease of 80–100%, >600 million animals at risk, and annual economic untreatable and new methods of control are urgently needed. losses of >US$3 billion (Piedrafita et al., 2004, 2007). It is recogni- The production of an effective vaccine may be a sustainable sed by the World Health Organization (WHO) as a major control strategy. However, although there has been a considerable food-borne problem, with up to 17 million people infected and effort to develop such a vaccine, current experimental approaches suffer from two limitations: efficacy is variable between animals ⇑ Corresponding author. Tel.: +44 1904 328600; fax: +44 1904 328505. and the level achieved to date (38–72%) falls short of the >80% E-mail address: [email protected] (R.A. Wilson). protection generally agreed as necessary for a commercially viable 0020-7519/$36.00 Crown Copyright Ó 2011 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. doi:10.1016/j.ijpara.2011.08.003 1348 R.A. Wilson et al. / International Journal for Parasitology 41 (2011) 1347–1359 product for cattle (Hillyer, 2005; McManus and Dalton, 2006). Two Lastly, a detergent extract of F. hepatica tegument proteins with candidate vaccines (leucine amino peptidase and SAP2) have undefined composition was shown to suppress the maturation shown an efficacy of >80% in single animal trials but require vali- and function of murine bone marrow-derived dendritic cells (Ham- dation in cattle (Piacenza et al., 1999; Espino and Hillyer, 2004; ilton et al., 2009). Clearly, further definition of the proteins on the Acosta et al., 2008). The development of anti-Fasciola vaccines tegument surface of the fluke is needed to inform vaccine has been hindered by lack of insights into natural acquired im- development. mune mechanisms expressed by ruminant hosts against fluke In contrast, the tegument proteins of the related flukes Schisto- infection and knowledge of immune correlates of protection. A bet- soma mansoni (van Balkom et al., 2005; Braschi et al., 2006; Braschi ter understanding of the targets of acquired immunity in cattle is and Wilson, 2006), Schistosoma japonicum (Mulvenna et al., 2010a) required if we are to devise a commercial vaccine for large and Opisthorchis viverrini (Mulvenna et al., 2010b) have recently ruminants. been subjected to proteomic analysis. The studies have ranged There is good evidence from several studies that livestock can from the simple compositional analysis of material sloughed off acquire resistance to Fasciola. Vaccination using either irradiated the fluke surface by freeze/thaw/vortexing (van Balkom et al., metacercariae, drug abbreviated infection, parasite extracts or de- 2005) to more sophisticated protocols that enrich for tegument fined antigens can induce 48–89% reductions in fluke burdens in membrane proteins using differential extraction (Braschi et al., ruminants (Dalton et al., 1996; Morrison et al., 1996; Piacenza 2006). Proteins accessible to impermeant probes on live schisto- et al., 1999; Hoyle et al., 2003; Piedrafita et al., 2004; Hillyer, somes have been tagged with biotin to facilitate their recovery 2005; McManus and Dalton, 2006; Golden et al., 2010). These re- and identification (Braschi and Wilson, 2006; Mulvenna et al., sults demonstrate that Fasciola antigens can elicit high levels of 2010a). Finally, live schistosomes have also been subjected to a immunity in cattle and sheep, suggesting that the goal of >80% pro- complementary approach of enzymatic shaving that releases ex- tection is achievable. The key step now is to identify the parasite posed proteins into the medium for recovery and identification stage and antigens driving the acquired protective response. In cat- (Castro-Borges et al., 2011). Such studies have provided a wealth tle or sheep where protection has been demonstrated following of information about the surface organisation of the blood dwelling vaccination or due to natural resistance, clinical serology has re- schistosomes and it is notable that two surface-exposed tegument vealed that killing of parasites occurs within approximately proteins of S. mansoni have shown efficacy as vaccines in the 6 weeks of infection but only after some damage to the liver paren- mouse model of schistosomiasis (Tran et al., 2006; Cardoso et al., chyma (Dalton et al., 1996; Roberts et al., 1997; Piacenza et al., 2008). Here, as the first step towards a molecular definition of 1999; Hoyle et al., 2003; Piedrafita et al., 2004). These observations the tegument surface, the parasite-host interface, the better to suggest that the newly excysted juvenile (NEJ) or immature para- understand the interaction of F. hepatica with the immune system, site migrating in the liver, not the adult fluke, is the target of the we report the analysis of tegument proteins isolated from the adult acquired immune response but the nature of this effector response fluke. acting in vivo is not clear. Moreover, a vaccine targeting the NEJ, which suppressed or eliminated invasion of the liver parenchyma, 2. Materials and methods would minimise liver pathology and reduce production losses in livestock.