McKown, Richard Dwayne (2004-12). Localization and partial immunological characterization of Fasciola hepatica Thioredoxin. Doctoral Dissertation.
This study reports the localization and partial characterization of thioredoxin from the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised in culture and infected with F. hepatica so that Western blotting and immunohistochemical techniques could be utilized to determine the presence of thioredoxin in different stages of the parasite?s development. The results of these experiments showed that thioredoxin was present in the tegument, gut epithelium, excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and gut of the redia and cercaria intermediate stages. In situ hybridization was used to determine the localization and possible differential mRNA expression of two different F. hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in situ hybridization results showed that both isotypes are expressed in the tegument and gut epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference in the amount of expression between the two isotypes. Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL? Protein Fusion and Expression System was used to test its affects on the production of super oxide anion by murine peritoneal macrophages, bovine monocyte-derived macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse peritoneal macrophages and bovine monocyte-derived macrophages. The results of the cellular assays were not definitive due to the fact that the maltose binding protein (MBP) moiety of the recombinant thioredoxin, when tested alone, increased production of nitric oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could not be effectively separated from the thioredoxin portion of the recombinant, allowing the thioredoxin affects to be tested independently, no true conclusions regarding its affects on the host immune cells tested could be drawn. This is the first report of the localization of thioredoxin in both the adult F. hepatica as well as in specific intermediate stages of the parasite. These studies demonstrate the possible affects that a protein tag can have on experimental results and demonstrate how such data may be interpreted when a non-cleaved recombinant protein is used in cellular or other assays when compared to native or cleaved recombinant proteins.