Comparative Proteomics of Excretory-Secretory Proteins Released by the Liver Fluke Fasciola hepatica in Sheep Host Bile and during in Vitro Culture ex Host

Abstract

Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates. Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates. The parasitic fluke Fasciola hepatica infects humans and ruminant livestock worldwide. An estimated 2.4 million people are infected with Fasciola species, and a further 180 million are at risk (1AnonymousControl of Foodborne Trematode Infections. World Health Organization, Geneva1995Google Scholar). In addition, F. hepatica causes an estimated loss of $3 billion worldwide per annum through livestock mortality, especially in sheep, and by decreased productivity via reduction of milk and meat yields in cattle (2Boray J.C. Chemotherapy of infections with fasciolidae.in: Boray J.C. Immunology, Pathobiology and Control of Fasciolosis. MSD AGVET, Rahway, NJ1994: 83-97Google Scholar). Presently the rate of fluke infection in parts of northwest Europe has been estimated as greater than 30%. In the absence of commercial vaccines, the benzimidazole derivative triclabendazole (TCBZ; 1The abbreviations used are: TCBZ, triclabendazole; ES, excretory-secretory; FABP, fatty acid-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMF, peptide mass fingerprint; TIC, trypsin inhibitor complex; Tpx, thioredoxin peroxidase; TTBS, Tris-buffered saline with 1% Tween 20; 2DE, two-dimensional gel electrophoresis; e, expectancy; BLAST, Basic Local Alignment Search Tool. 1The abbreviations used are: TCBZ, triclabendazole; ES, excretory-secretory; FABP, fatty acid-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMF, peptide mass fingerprint; TIC, trypsin inhibitor complex; Tpx, thioredoxin peroxidase; TTBS, Tris-buffered saline with 1% Tween 20; 2DE, two-dimensional gel electrophoresis; e, expectancy; BLAST, Basic Local Alignment Search Tool. Fasinex®) is the drug most extensively used against Fasciola. Unlike other fasciolicides, TCBZ shows activity against both juvenile flukes, which are responsible for the damage to the liver of acute fasciolosis, and the mature flukes, which cause the debilitation of chronic fasciolosis. Recently resistance to TCBZ has been reported in several countries suggesting that chemotherapeutic control of this infection may soon be compromised (3Moll L. Gaasenbeek C.P.H. Vellema P. Borgsteede F.H.M. Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in The Netherlands.Vet. Parasitol. 2000; 91: 153-158Crossref PubMed Scopus (184) Google Scholar, 4Mitchell G.B.B. Maris L. Bonniwell M.A. Triclabendazole-resistant liver fluke in Scottish sheep.Vet. Rec. 1998; 143 (399): 399PubMed Google Scholar, 5Thomas I. Coles G.C. Duffus K. Triclabendazole-resistant Fasciola hepatica in southwest Wales.Vet. Rec. 2000; 146 (200): 200PubMed Google Scholar). The most common diagnostic field method used to detect Fasciola infection requires counting fluke eggs in fecal samples. However, egg counts are time-consuming and expensive and have limited ability to diagnose early acute stages of infection. New immunological diagnostic methods with commercially protected antigen identification are available in the form of a rapid on-site field test, DriDot™ (Biotechnology Ireland) based on latex agglutination and a companion ELISA for laboratory veterinary applications. Further diagnostics-based research is required to freely validate current tests and to provide alternative molecular approaches for liver fluke populations. In well characterized cattle models, the immune response to F. hepatica infection is proinflammatory lasting for about 4–6 weeks postinfection. This immune response appears to be switched off at around the time adults enter the bile duct. The presence of flukes modify Th1 helper T cell responses, inhibiting the induction of the protective immune system, which makes the host more susceptible to further infections. It is likely that liver fluke secretions modify host mac ro phage-based signaling events to switch the proinflammatory immune response “off” before the infection has been appropriately controlled. Juvenile F. hepatica develop and mature within the intrahepatic bile duct and gall bladder of infected hosts, continuing to release their excretory-secretory (ES) survival products that can ultimately lead to fibrosis and calcification of host tissues. In vitro biochemical studies have predicted that ES products of F. hepatica have roles in feeding behavior, detoxification of bile components, and the evasion of the immune system. In addition, in vitro proteomics (6Jefferies J.R. Campbell A.M. Van Rossum A.J. Barrett J. Brophy P.M. Proteomic analysis of Fasciola hepatica excretory-secretory products.Proteomics. 2001; 1: 1128-1132Crossref PubMed Scopus (0) Google Scholar) support the release of several major from liver For detoxification with immune evasion roles were to be by in vitro liver flukes P.M. Barrett J. transferase in PubMed Scopus Google Scholar). of cathepsin L proteases are also in vitro by adult F. hepatica (6Jefferies J.R. Campbell A.M. Van Rossum A.J. Barrett J. Brophy P.M. Proteomic analysis of Fasciola hepatica excretory-secretory products.Proteomics. 2001; 1: 1128-1132Crossref PubMed Scopus (0) Google Scholar, L. L. The cathepsin of the Fasciola J. PubMed Scopus Google Scholar). These proteases can host P. Fasciola hepatica cathepsin and potential in the of first liver fluke J. Parasitol. PubMed Scopus Google Scholar) and proteins P. F. by Fasciola hepatica and Parasitol. PubMed Scopus Google Scholar) for and are also at in of host A.M. A.J. Fasciola a cathepsin host J. Parasitol. PubMed Scopus Google immune evasion In vitro studies can only to the by F. hepatica into host is that the ES of F. hepatica be by from natural host and the in a proteomics can proteins from a of host and proteins this the potential to validate in vitro ex host studies in vivo and provide the for time In vivo proteomics during infection also the of identifying targets and therapy for and and our understanding of the the immune system. of sheep bile have to be and a study of bile a of to identification J. and of in J. PubMed Scopus Google Scholar, J. P. analysis of PubMed Scopus Google Scholar). from sheep has only been characterized at a with to the major bile of proteins and of bile from sheep and Google Scholar). in bile, including sheep, is within a of in they and they J. Google Scholar). proteins in the bile, as are from the from the of the and bile duct and have been in several of bile J. PubMed Scopus Google Scholar). In the gall bile is to with the with the of of bile J. PubMed Scopus Google Scholar) of other in they and they J. Google Scholar). has been to be an of However, is not to be as within the bile of of and have been in host bile the host has been infected with F. hepatica than that in serum I. and bile and during Fasciola hepatica infection in sheep.Vet. Parasitol. PubMed Scopus Google Scholar). In this study proteomics approaches were used for time course analysis of release from F. hepatica during in vitro culture and to released from the into the sheep bile for the first time, the ES produced by F. hepatica into a host in vivo identified and with the in vitro ES from ex host and from and infected sheep were from a confirmed by to of with In addition, fluke infection also confirmed by the presence of adult F. hepatica within the liver and bile and by within bile samples. from gall and liver gall were via a and fluke from per infected to of in infected bile samples. The bile were at to of and other and with to further in bile of bile J. PubMed Scopus Google Scholar). The bile were further via for at at the with were in and F. hepatica from infected sheep on the of were for a of in at to of the and to host (6Jefferies J.R. Campbell A.M. Van Rossum A.J. Barrett J. Brophy P.M. Proteomic analysis of Fasciola hepatica excretory-secretory products.Proteomics. 2001; 1: 1128-1132Crossref PubMed Scopus (0) Google Scholar). ES products were as (6Jefferies J.R. Campbell A.M. Van Rossum A.J. Barrett J. Brophy P.M. Proteomic analysis of Fasciola hepatica excretory-secretory products.Proteomics. 2001; 1: 1128-1132Crossref PubMed Scopus (0) Google Scholar) with liver flukes per flukes were for and at to only culture flukes, and all were at the time The ES products were via at for at The ES further at for of of ES products from time course with The were into and the time as ES products from F. a of the flukes were in 1% in and in an as the The liver flukes were to at for The released products from liver flukes, were as the ES products from flukes as of of both bile and ES and with an for bile and for were used to at for in the first were for and the for in of and J. J.R. 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Campbell A.M. Van Rossum A.J. Barrett J. Brophy P.M. Proteomic analysis of Fasciola hepatica excretory-secretory products.Proteomics. 2001; 1: 1128-1132Crossref PubMed Scopus (0) Google Scholar). However, the proteins by (6Jefferies J.R. Campbell A.M. Van Rossum A.J. Barrett J. Brophy P.M. 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The of in Fasciola PubMed Scopus Google Scholar) have that are to L proteases were the only F. hepatica proteins identified in both bile and in vitro In addition, our in vitro studies that cathepsin L proteases are the major adult ES proteins to at and enolase were also released from F. hepatica for at during in vitro ES These proteins have all been detected in vitro ES products from F. hepatica (6Jefferies J.R. Campbell A.M. Van Rossum A.J. Barrett J. Brophy P.M. 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Google Scholar, in vitro of and in bile of PubMed Google for activity of suggesting that the detoxification in on the of the The potential of F. hepatica may to an by that are first to the immune system. has been on the of other species, and in 2001; PubMed Scopus Google Scholar) with immune P. and biochemical and of an and by PubMed Scopus Google and has been predicted on the of the P. of an from the that PubMed Scopus Google Scholar) and in in vitro culture studies in other M.A. J. from in vitro mac ro PubMed Scopus Google Scholar) and J. analysis of the proteins of the and immune PubMed Scopus Google Scholar). Several proteins showed in the in vitro at including FABP, actin, and glyceraldehyde-3-phosphate dehydrogenase proteins are in vitro especially the of in vivo and absence of immune that in in adult F. hepatica in ES in in vitro studies on F. hepatica (6Jefferies J.R. Campbell A.M. Van Rossum A.J. Barrett J. Brophy P.M. 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