JamesR.Jefferies AlisonM.Campbell ArjanJ.vanRossum JohnBarrett PeterM.Brophy Institute of Biological Sciences, University of Wales, Aberystwyth, Wales Proteomicanalysisof Fasciola hepatica excretory-secretoryproducts This paper describes a global investigation of the components of Fasciola hepatica excretory-secretory (ES) products by a proteomic approach. Despite the absence of a F. hepatica genome sequencing project we have shown that it was possible to iden- tify 29 of the 60 prominent proteins found using two-dimensional gel electrophoresis. As well as cathepsin L proteases, a number of enzymes implicated in parasite protec- tion from the host immune system were also found to be present in relatively large abundance. These included superoxide dismutase, thioredoxin peroxidase, gluta- thione S-transferases and fatty acid binding proteins, all of which may play a part in the detoxification of reactive oxygen intermediates. Interestingly, ovine superoxide dis- mutase was the only protein from the host identified on the gel. We suggest that the relative abundance and protective nature of the components of the ES products of this organism play an important role in its survival within the host. The precise identification, to individual NCBI database entries, of a number of glutathione S-transferases and cathepsin Ls from F.hepatica, by peptide mass fingerprinting, was hampered by multi- database submissions of the two protein superfamilies from this organism. Keywords: Excretory-secretory produce / Fasciola hepatica / Peptide mass fingerprinting / Glutathione-S-transferase / Fatty acid binding protein PRO 0091 1 Introduction The excretory-secretory (ES) products of parasitic organ- isms are generally thought to play an important role in host-parasite interactions. These ES products are impor- tant not only in host tissue invasion and digestion, but also in the survival of the parasite in a hostile host environ- ment, either by evasion [1], subversion of [2], or defence against [3], the immune system. The components of the ES products arise from a number of parasite tissues. Fas- ciola hepatica, in common with other flukes has a blind ending gut, so faeces, as well as a number of secreted components are shed, via the mouth, through which food is also ingested. Other components may be excreted through the median pore situated at the posterior of the body, whilst the tegument may also be considered to be a secretory organ as its surface is also actively shed. Proteins from the ES products of F. hepatica have been shown to have a number of effects on the host immune system. F. hepatica ES products are capable of immuno- suppresive and modulatory effects in rat and sheep lym- phocytes, respectively [4, 7]. ES products have been shown to suppress the metabolic burst of sheep neutro- phils [5], whilst a similar phenomenon was demonstrated using Fasciola gigantica ES products [6]. A number of cathepsin proteases have also been implicated in immune evasion by their ability to cleave host antibody [1]. Parasites have evolved methods of detoxifying reactive oxygen intermediates produced by eosinophils, neutrophils and other cell types, via defence enzymes such as super- oxide dismutase (SOD) and glutathione-S-transferases (GSTs). ES products have previously been studied individu- ally and in a number of different laboratories, giving a frag- mented picture of their complex biochemical interaction with host components. We now take a proteomic approach, to look for the first time, at a global view of the components of the ES products from this parasite, to obtain information on the proportion and relative abundance of the proteins present. This work will also help to develop a proteomic based methodology to provide a complete overview of host-parasite interactions at the interface between the two organisms. Individual components of the ES products have already been investigated as potential vaccination targets [8], but with limited success in respect to protection. With a better understanding of how this parasite interacts with its host, it will be possible to significantly increase the pros- pects of identifying effective vaccination or drug targets. 2 Materialsandmethods Livers from naturally infected sheep were collected on the day of slaughter from a local slaughterhouse, and the para- sites removed and washed six times in PBS (pH 7.3) to Correspondence: Dr. J. R. Jefferies, Institut of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, Wales SY23 3DA, UK E-mail: jrj@aber.ac.uk Fax: +44-1970-622350 Abbreviations:ES, excretory-secretory; FABP , fatty acid binding protein; GST , glutathione-S-transferase; PMF , peptide mass fin- gerprint; SOD, superoxide dismutase 1128 Proteomics 2001, 1, 1128–1132  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 1615-9853/01/0909–1128 $17.50+.50/0