Schistosomiasis is estimated to afflict over 200 million people worldwide. Until a practical vaccine is available, chemotherapy will continue to be important both for treating infected individuals and in programs aimed at controlling infection transmission within populations. Because only three drugs are available for clinical use, the appearance of drug resistance is of continuing concern. Elucidation of the biochemical basis of schistosome-drug interactions is important for understanding the molecular mechanisms of both drug efficacy and drug resistance. Adult Schistosoma mansoni express at least six biochemically distinct forms of glutathione S-transferases (EC 2.5.1.18) that have been shown to play a role in cellular protection by catalyzing the conjugation of glutathione with a structurally diverse group of xenobiotics and endogenously produced electrophiles. Although a few cDNAs encoding schistosome glutathione S-transferases have been isolated and overexpressed in bacteria to produce candidate vaccine antigens, nothing is known about how this important family of enzymes is regulated within the parasite. We have discovered that exposure of adult S. mansoni to three prototypic inducing agents (2(3)-t-butyl-4-hydroxyanisole, 3-methylcholanthrene, and phenobarbital) elevates the steady-state level of mRNA that hybridizes to one of three S. mansoni glutathione S-transferase class-specific cDNA probes. This observation suggests that expression of those genes is subject to regulatory mecbanisms that allow the parasite to respond to chemical stimuli, including perhaps antischistosomal drugs, encountered in its environment. This proposal is designed to test that hypothesis. Specific experimental objectives include: 1) Survey the stage- and gender-selective expression of glutathione S-transferase genes by Northern hybridization analysis and high pressure liquid chromatography; 2) Determine whether 2,3,7,8-tetrachlordibenzo-p-dioxin and oltipraz induce S. mansoni glutathione S-transferase gene expression using Northern hybridization analysis to quantitate specific mRNA levels; 3) Using nuclear """"""""run-on"""""""" assays and measurements of mRNA turnover, test the hypothesis that induction of schistosome glutathione S-transferase gene expression by prototypic inducing agents is transcriptionally regulated; and 4) Identify putative regulatory sequences (xenobiotic responsive elements) in the 5'-flanking region of a S. mansoni glutathione S-transferase isoenzyme subunit gene that are required for transcriptional activation. The latter experiments will entail construction and expression of glutathione S-transferase-firefly luciferase fusion genes in eukaryotic cell lines.
