title: Inhibition of pyruvate-ferredoxin oxidoreductase gene expression in Giardia lamblia by a virus-mediated hammerhead ribozyme.
abstract: Giardia lamblia is a primitive eukaryotic microorganism that derives its metabolic energy primarily from anaerobic glycolysis. In trophozoites, pyruvate-ferredoxin oxidoreductase (PFOR) converts pyruvate to acetyl-CoA with the transfer of a pair of electrons to ferredoxin, which can then reduce metronidazole and activate it into a potent antigiardiasis agent. It is unclear, however, whether this anaerobic disposal of electrons is essential for the energy metabolism in Giardia. In the present study, cDNAs encoding hammerhead ribozyme flanked with various lengths of antisense PFOR RNA were cloned into a viral vector pC631pac derived from the genome of giardiavirus (GLV). RNA transcripts of the plasmids showed high cleavage activities on PFOR mRNA in vitro. They were introduced into GLV-infected G. lamblia trophozoites by electroporation and stablized in the transfected cells via serial passages under puromycin selection. PFOR mRNA and enzyme activity in the transfected cells were decreased by 46-60% with the ribozyme PRzS flanked with 20 nt PFOR antisense RNA on each arm and by 69-80% with the ribozyme PRzL flanked with 600 and 1500 nt PFOR antisense RNA. PRzS without the inserted ribozyme or ribozyme flanked with alcohol dehydrogenase E antisense RNA showed no effect on PFOR mRNA and activity. The ribozyme-transfected cells demonstrated significantly enhanced resistance to metronidazole and grew equally well under anaerobic and aerobic conditions. In contrast, the wild-type cells grew slightly better anaerobically than the transfectants but did not grow at all in aerobic conditions. Thus, the reduced PFOR expression enables Giardia to grow under molecular oxygen and the presence of PFOR enhances the anaerobic growth of Giardia with an increased susceptibility towards metronidazole. In addition, this study demonstrated for the first time the feasibility of using a viral RNA vector to express a ribozyme targeted at a specific mRNA in G. lamblia to reduce the expression of a specific gene.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143-0446, USA.
authors.forename: M
authors.initials: M
authors.lastname: Dan
authors.forename: A L
authors.initials: AL
authors.lastname: Wang
authors.forename: C C
authors.initials: CC
authors.lastname: Wang
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D002460:Cell Line; D018076:DNA, Complementary; D016829:Giardia lamblia / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000821:virology*; D019198:Giardiavirus / Q000235:genetics*; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism; D008795:Metronidazole / Q000494:pharmacology; D010641:Phenotype; D050743:Pyruvate Synthase; D016372:RNA, Antisense; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
chemical_list: D000890:Anti-Infective Agents; D018076:DNA, Complementary; D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D008795:Metronidazole; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase