Inhibition of pyruvate-ferredoxin oxidoreductase gene expression in Giardia lamblia by a virus-mediated hammerhead ribozyme

Giardia lamblia is a primitive eukaryotic microorganism that derives its me tabolic energy primarily from anaerobic glycolysis. In trophozoites, pyruva te-ferredoxin oxidoreductase (PFOR) converts pyruvate to acetyl-CoA with th e transfer of a pair of electrons to ferredoxin, which can then reduce metr onidazole and activate it into a potent antigiardiasis agent. It is unclear , however, whether this anaerobic disposal of electrons is essential for th e energy metabolism in Giardia. In the present study, cDNAs encoding hammer head ribozyme flanked with various lengths of antisense PFOR RNA were clone d 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 trophozoit es by electroporation and stablized in the transfected cells via serial pas sages under puromycin selection. PFOR mRNA and enzyme activity in the trans fected cells were decreased by 46-60% with the ribozyme PRzS flanked with 2 0 nt PFOR antisense RNA on each arm and by 69-80% with the ribozyme PRzL fl anked with 600 and 1500 nt PFOR antisense RNA. PRzS without the inserted ri bozyme or ribozyme flanked with alcohol dehydrogenase E antisense RNA showe d no effect on PFOR mRNA and activity. The ribozyme-transfected cells demon strated significantly enhanced resistance to metronidazole and grew equally well under anaerobic and aerobic conditions. In contrast, the wild-type ce lls grew slightly better anaerobically than the transfectants but did not g row at all in aerobic conditions. Thus, the reduced PFOR expression enables Giardia to grow under molecular oxygen and the presence of PFOR enhances t he anaerobic growth of Giardia with an increased susceptibility towards met ronidazole. In addition, this study demonstrated for the first time the fea sibility of using a viral RNA vector to express a ribozyme targeted at a sp ecific mRNA in G. lamblia to reduce the expression of a specific gene.