Hazel A. Wright

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

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.

Growth and energetics in the stickleback-Schistocephalus host-parasite system: a review of experimental infection studies

Summary Three-spined sticklebacks in natural lacustrine populations are often infected with plerocercoids of the indirectly transmitted pseudophyllidean cestode Schistocephalus solidus. Field studies typically show infections to be associated with reduced host condition, gonadogenesis and energy reserves, though infection phenotypes can vary considerably both between and within host populations. Experimental infection studies allow the impact of infections on hosts to be studied under a variety of rearing conditions, and so can be used to determine the environmental component of infection phenotypes. Here, we review recent laboratory studies undertaken by our group, examining the growth and condition of experimentally infected fish reared under conditions that differed in terms of absolute ration, temporal pattern of feeding and level of competition between fish. We compare infection phenotypes generated in our experimental studies with those of fish sampled in field based studies. Experimental studies in which infected fish were reared under competition for limited food resources, or were fed a restricted diet, generated infection phenotypes that most closely resembled those found in the majority of natural populations. When access to food was unrestricted, however, infected fish were able to sustain high growth rates and lay down energy reserves. If experimental studies are to be used to understand the impact of infection under natural conditions, husbandry protocols that closely match field conditions must be designed. We suggest that a full understanding of the impact of parasites on their hosts can only be gained by integrating controlled laboratory experiments with detailed field studies. The stickleback‐Schistocephalus system is ideally suited to examining these questions, and we provide several suggestions for future research.

Towards Delineating Functions within the Fasciola Secreted Cathepsin L Protease Family by Integrating In Vivo Based Sub-Proteomics and Phylogenetics

Background Fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES) products are also the most promising vaccine candidates, the cathepsin L (Cat L) protease family. Methodology/Principal Findings The sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples. Conclusions/Significance We have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase.

Proteomic analysis of embryonic Fasciola hepatica: Characterization and antigenic potential of a developmentally regulated heat shock protein

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35alpha based on predictions of molecular weight. Production of recombinant Fh-HSP35alpha reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35alpha as an egg-based diagnostic marker for liver fluke infection.

Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40

Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.

A Proteomics Approach To Quantify Protein Levels Following RNA Interference: Case Study with Glutathione Transferase Superfamily from the Model Metazoan Caenorhabditis elegans

Loss-of-function phenotypic analysis via interference RNA (RNAi) technology is a revolutionary approach to assigning gene function. While transcript-based methodologies commonly validate RNAi gene suppression investigations, protein-based validation is less developed. This report illustrates the potential for two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-DE) and gel analysis to quantify protein levels following RNAi. This case study involves three glutathione transferase (GST) genes targeted by RNAi from the model organism Caenorhabditis elegans.

The current state of sustainability in bioscience laboratories: a statistical examination of a UK tertiary institute

Purpose – This study aims to identify the current barriers to sustainability in the bioscience laboratory setting and to determine which mechanisms are likely to increase sustainable behaviours in this specialised environment.Design/methodology/approach – The study gathers qualitative data from a sample of laboratory researchers presently conducting experimentation in the biological sciences. A questionnaire, regarding sustainability in the laboratory, was developed and distributed to all bioscience researchers at Aberystwyth University.Findings – Although the majority of respondents had favourable attitudes to sustainability, almost three‐quarters (71 per cent) stated that they were not conducting their research in the most sustainable way possible. The factors most likely to hinder sustainable behaviour were lack of support, lack of information and time constraints. However, monetary costs and benefits, closely followed by “other” costs and benefits, were most likely to encourage sustainable behaviour i...

Sustainability in bioscience fieldwork: Practical information from a UK agricultural research institute

Purpose – Owing to the specialist nature of biological experimentation, scientific research staff have been largely neglected from the pro‐environmental initiatives which have inundated other areas of higher education. This dearth of studies is surprising given that scientific research is recognised as a substantial contributor to the environmental impact of tertiary institutes. The present study seeks to utilise the current sustainability literature to identify barriers to sustainability in scientific fieldwork and determines which methods or procedures might increase pro‐environmental behaviours in this technical environment. The resultant information serves to provide a comparison with previously identified barriers to sustainability in the laboratory environment and identifies which environmental initiatives might be successful in both the field and laboratory.Design/methodology/approach – This study gathers qualitative data from a sample of scientific researchers presently conducting field experiment...

Bovine viral diarrhoea initiative in Wales

THE awareness of bovine viral diarrhoea (BVD) in the cattle herd has never been higher among the veterinary profession, with a number of initiatives occurring at a national and regional level throughout the UK. Many practices throughout the UK have assisted clients in eradicating the disease on farms and in areas where they …