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The parasite Fasciola hepatica causes major global disease of livestock, with increasing reports of human infection. Vaccine candidates with varying protection rates have been identified by pre-genomic approaches. As many candidates are part of protein superfamilies, sub-proteomics offers new possibilities to systematically reveal the relative importance of individual family proteins to vaccine formulations within populations. The superfamily glutathione transferase (GST) from liver fluke has phase II detoxification and housekeeping roles, and has been shown to contain protective vaccine candidates. GST were purified from cytosolic fractions of adult flukes using glutathione- and S-hexylglutathione-agarose, separated by 2-DE, and identified by MS/MS, with the support of a liver fluke EST database. All previously described F. hepatica GST isoforms were identified in 2-DE. Amongst the isoforms mapped by 2-DE, a new GST, closely related to the Sigma class enzymes is described for the first time in the liver fluke. We also describe cDNA encoding putative Omega class GST in F. hepatica.

BackgroundLiver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised.Methodology/Principal FindingsIn this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection.Conclusions/SignificanceWe have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity.

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

The phase II detoxification system glutathione transferase (GST) is associated with the establishment of parasitic nematode infections within the gastrointestinal environment of the mammalian host. We report the functional analysis of a GST from an important worldwide parasitic nematode of small ruminants, Haemonchus contortus. This GST shows limited activity with a range of classical GST substrates but effectively binds hematin. The high-affinity binding site for hematin was not present in the GST showing the most identity, CE07055 from the free-living nematode Caenorhabditis elegans. This finding suggests that the high-affinity binding of hematin may represent a parasite adaptation to blood or tissue feeding from the host.The gastrointestinal blood-feeding nematode Haemonchus contortus represents a major economic burden to agricultural communities worldwide, causing infections resulting in anemia, weight loss, and ultimately death in small ruminants. There are presently no commercial vaccines available for H. contortus infections, and the most effective method of control is a combination of pasture management and the use of chemical agents (anthelmintics). Increasing reports of drug-resistant H. contortus indicate that this current control strategy is not sustainable (26), with chemicals no longer effectively controlling H. contortus infections in several parts of the world (33). Understanding the host-parasite relationship at the mucosal feeding surface is important in identifying new therapeutic approaches. The levels of the phase II detoxification system glutathione transferase (GST) have been shown to increase in parasitic helminths during chronic infection (3). Previous research has attempted to correlate this overexpression with the ability of GST to detoxify immune-initiated cytotoxic products of lipid peroxidation (8, 10) or has associated the overexpression of GST, including H. contortus GST, with drug resistance (18,19). In this paper, we analyze a new GST from the sheep strongylid H. contortus and show that this GST does not appear to have a broad immune defense or drug metabolism role but possibly has a more focused detoxification function within the nematode. MATERIALS AND METHODSIsolation, recombinant expression, and purification of H. contortus GST. mRNA was isolated from adult H. contortus nematodes (CAVR, a drug-resistant strain) with a Quickprep Micro mRNA purification kit (Pharmacia). H. contortus cDNA was obtained with a First Strand cDNA synthesis kit (Pharmacia). The H. contortus GST-encoding DNA was isolated by an established strategy with an upstream primer derived from the N-terminal sequence of the native H. contortus GST protein (27) and a downstream oligo(dT)-based anchor primer (1, 5). The PCR product (approximately 650 bp) was cloned into pUC18 (SureClone ligation kit; Pharmacia), and the insert was sequenced. The H. contortus GST was directionally cloned into pET23d and sequenced in both directions (Long-read LI-COR, GenBank accession number AF281663). The recombinant H. contortu...

Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis.

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 TCBZ-SO at physiologically relevant concentrations, which may indicate a role in TCBZ metabolism and resistance.

Male genital schistosomiasis (MGS) is a gender specific manifestation of urogenital schistosomiasis (UGS) first described in 1911 by Madden in Egypt. Today, while affecting millions of men and boys worldwide, MGS receives insufficient attention, especially in sub-Saharan Africa (SSA). To provide a systematic review with an epidemiological update of MGS, we inspected both online and hardcopy resources in our appraisal. A total of 147 articles were eventually identified, only 31 articles were exclusively focused on MGS with original or targeted research. From these, we discuss pertinent clinico-pathological features of MGS, highlight the possible connection and interplay with HIV, and assess current diagnostic techniques alongside consideration of their use and application in SSA. To appreciate the burden of MGS more fully, especially in endemic areas, there is a clear need for better surveillance and longitudinal population research to investigate the best point-of-care (POC) diagnostic and its performance through time. Furthermore, to optimise individual case management, exploration of alternative praziquantel dosing regimens is needed for MGS in men with or without HIV co-infection.