Abstract
Antioxidants produced by the parasite Schistosoma mansoni are believed to be involved in the maintenance of cellular redox balance, thus contributing to larval survival in their intermediate snail host, Biomphalaria glabrata. Here, we focused on specific antioxidant enzymes, including glutathione-S-transferases 26 and 28 (GST26 and 28), glutathione peroxidase (GPx), peroxiredoxin 1 and 2 (Prx1 and 2) and Cu/Zn superoxide dismutase (SOD), known to be involved in cellular redox reactions, in an attempt to evaluate their endogenous antioxidant function in the early-developing primary sporocyst stage of S. mansoni. Previously we demonstrated a specific and consistent RNA interference (RNAi)-mediated knockdown of GST26 and 28, Prx1 and 2, and GPx transcripts, and an unexpected elevation of SOD transcripts in sporocysts treated with gene-specific double-stranded (ds)RNA. In the present followup study, in vitro transforming sporocysts were exposed to dsRNAs for GST26 and 28, combined Prx1/2, GPx, SOD or green-fluorescent protein (GFP, control) for 7 days in culture, followed by assessment of the effects of specific dsRNA treatments on protein levels using semi-quantitative Western blot analysis (GST26, Prx1/2 only), and larval susceptibility to exogenous oxidative stress in in vitro killing assays. Significant decreases (80% and 50%) in immunoreactive GST26 and Prx1/2, respectively, were observed in sporocysts treated with specific dsRNA, compared to control larvae treated with GFP dsRNA. Sporocysts cultured with dsRNAs for GST26, GST28, Prx1/2 and GPx, but not SOD dsRNA, were significantly increased in their susceptibility to H2O2 oxidative stress (60–80% mortalities at 48 hr) compared to GFP dsRNA controls (∼18% mortality). H2O2-mediated killing was abrogated by bovine catalase, further supporting a protective role for endogenous sporocyst antioxidants. Finally, in vitro killing of S. mansoni sporocysts by hemocytes of susceptible NMRI B. glabrata snails was increased in larvae treated with Prx1/2, GST26 and GST28 dsRNA, compared to those treated with GFP or SOD dsRNAs. Results of these experiments strongly support the hypothesis that endogenous expression and regulation of larval antioxidant enzymes serve a direct role in protection against external oxidative stress, including immune-mediated cytotoxic reactions. Moreover, these findings illustrate the efficacy of a RNAi-type approach in investigating gene function in larval schistosomes.
Highlights
Miracidial penetration and entry into the molluscan intermediate host represent a critical transition period in which the previously free-living larval stage is confronted with a potentially hostile environment as it attempts to establish a viable infection [1,2]
Anti-oxidant enzymes produced by the parasite Schistosoma mansoni are believed to play a critical role in the maintenance of cellular redox balance, contributing to larval survival in their snail host, Biomphalaria glabrata
We have incorporated a RNA interference approach attempting to knock down specific anti-oxidant enzymes, including gluthatione-S-transferases 26 and 28 (GST26 and 28), gluthatione peroxidase (GPx), peroxiredoxins 1 and 2 (Prx1/2) and superoxide dismutase (SOD), and to evaluate their endogenous anti-oxidant function in the sporocyst stage of S. mansoni
Summary
Miracidial penetration and entry into the molluscan intermediate host represent a critical transition period in which the previously free-living larval stage is confronted with a potentially hostile environment as it attempts to establish a viable infection [1,2]. Miracidia of the human blood fluke Schistosoma mansoni shed their ciliated epidermal plates soon after entry into the host snail Biomphalaria spp., transforming to primary or mother sporocysts It is during this time of transition and early sporocyst development that larvae are especially vulnerable to oxidative stress generated from products of oxidized plasma hemoglobin [3], or reactive oxygen or nitrogen species (ROS and RNS, respectively) resulting from hemocyte-mediated immune responses [4,5,6,7]. Included among these enzymes are the following: (i) glutathione-S-transferases 26 and 28 (GST26 and GST28) that function to neutralize potential membrane damage by the linked catalysis of glutathione (GSH) reduction with detoxification reactions involving thiol-conjugation to xenobiotics [19], (ii) peroxiredoxin (Prx and Prx2) that are involved in maintaining redox balance, by reducing hydrogen
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