演者 Professor TIMOTHY P. YOSHINO
Department of Pathobiological Sciences, University of Wisconsin, School of Veterinary Medicine, Madison, WI 53706 USA
演題 ANTIOXIDANT RESPONSES IN LARVAL SCHISTOSOMES PROTECT AGAINST EXTERNAL OXIDATIVE STRESS
Abstract：In vitro-derived primary sporocysts of Schistosoma mansoni are highly sensitive to oxidative damage, especially that mediated by hydrogen peroxide (H2O2). Previous studies have shown that proteins released during in vitro transformation of the miracidial-to-sporocyst stage contain a variety of antioxidant (antiOx) enzymes that are hypothesized to serve a protective role against host reactive oxygen species (ROS). Moreover, exposure of sporocysts to sublethal H2O2 levels stimulates an upregulation in gene expression of at least one antiOx, namely peroxiredoxin (PRx). To explore the role of endogenous larval antiOx in protection against external oxidative stress, we employed an RNA interference (RNAi) approach to knockdown expression of the following S. mansoni genes: PRx, glutathione peroxidase (GPx), glutathione-S-transferases (GST26, GST28) and Cu/Zn superoxide dismutase (SOD). Consistent transcript knockdown, compared to a green fluorescent protein (GFP) double-stranded (ds) RNA control, was achieved for all enzymes except SOD, which unexpectedly was highly upregulated upon dsRNA treatment. In followup in vitro experiments, sporocysts treated with dsRNA for PRx, GPx, GST26 and GST28 were found to be significantly more sensitive to sublethal H2O2 treatments than the GFP control or SOD dsRNA-treated larvae as demonstrated by catalase-sensitive increases in larval mortality. Similarly, in vitro killing of S. mansoni sporocysts by hemocytes of susceptible NMRI Biomphalaria glabrata snails also was increased in PRx, GPx and GST26 dsRNA-treated larvae, compared to those treated with GFP or SOD dsRNAs. Results of these experiments strongly support the hypothesis that endogenous expression and regulation of larval antiOx enzymes serve a direct role in protection against external oxidative stress, including that produced through immune-mediated cytotoxic reactions. This study also illustrates how RNAi can be applied as an effective reverse-genetic approach in evaluating parasite gene function.
Yoshino, T. P., Davids, B.J., Castillo, M.G. and Johnston, L.A. (1998). Invertebrate cellular adhesion receptors: their structure and function as primordial recognition molecules in the immune system. Trends Comp Biochem. Physiol. 5:49-65.
Humphries, J. E. and Yoshino, T.P. (2001). Protein kinase C regulation of cell spreading in the molluscan Biomphalaria glabrata embryonic (Bge) cell line. BBA - Molecular Cell Research 1540:243-252.
Williams, D. L., A. A. Sayed, A. G. McArthur, A. Taft, J. J. Vermeire, and T. P. Yoshino. 2007. Profiling Schistosoma mansoni development using Serial Analysis of Gene Expression (SAGE). Exp. Parasitol., 117: 246-258 PMID: 17577588.
Vermeire, J. J. and T. P. Yoshino. 2007. Antioxidant gene expression and function in in vitro-developing Schistosoma mansoni mother sporocysts: possible role in self-protection. Parasitology, 134: 1369-1378.
Reinitz, D. A., T. P. Yoshino and R. A. Cole. 2007. A Ribeiroia spp. (Class:Trematoda) ﾐ Specific PCR-Based Diagnostic. J. Parasitol., 93: 1234-1238.
Humphries, J. E. and T. P. Yoshino. 2008. Regulation of hydrogen peroxide release in circulating hemocytes of the planorbid snail Biomphalaria glabrata. Dev. Comp. Immunol., 32: 554-562.
Yoshino, T. P., N. Dinguirard, and J. Kunert. 2008. Gene cloning and functional characterization of a tandem-repeat galectin from the freshwater snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni. Gene, 411: 46-58.
Wu, X.J., G. Sabat, J.F. Brown, M. Zhang, A. Taft, N. Peterson, A. Harms, and T.P. Yoshino. 2009. Proteomic analysis of Schistosoma mansoni proteins released during in vitro miracidium-to-sporocyst transformation. Mol. Biochem. Parasitol., 164:32-44.
Mourao, M., N. Dinguirard , G.R. Franco and T. P. Yoshino. Phenotypic screen of early-developing larval blood flukes, Schistosoma mansoni, using RNA interference. PLoS Negl Trop Dis, In revision.