title: Metabolism of nitro drugs metronidazole and nitazoxanide in Giardia lamblia: characterization of a novel nitroreductase (GlNR2).
abstract: OBJECTIVE
  The protozoan parasite Giardia lamblia causes giardiasis, a persistent diarrhoea. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are used for the treatment of giardiasis. Nitroreductases may play a role in activating these drugs. G. lamblia contains two nitroreductases, GlNR1 and GlNR2. The aim of this work was to elucidate the role of GlNR2.
  
  
  METHODS
  Expression of GlNR2 was analysed by reverse transcription PCR. Recombinant GlNR2 was overexpressed in G. lamblia and drug susceptibility was analysed. Recombinant GlNR2 was subjected to functional assays. Escherichia coli expressing full-length or truncated GlNR1 and GlNR2 were grown in the presence of nitro compounds. Using E. coli reporter strains for nitric oxide and DNA damage responses, we analysed whether GlNR1 and GlNR2 elicited the respective responses in the presence, or absence, of the drugs.
  
  
  RESULTS
  G. lamblia trophozoites overexpressing GlNR2 were less susceptible to both nitro drugs as compared with control trophozoites. GlNR2 was a functional nitroreductase when expressed in E. coli. E. coli expressing GlNR1 was more susceptible to metronidazole under aerobic and semi-aerobic and to nitazoxanide under semi-aerobic growth conditions. E. coli expressing GlNR2 was not susceptible to either drug. In reporter strains, GlNR1, but not GlNR2, elicited nitric oxide and DNA repair responses, even in the absence of nitro drugs.
  
  
  CONCLUSIONS
  These findings suggest that GlNR2 is an active nitroreductase with a mode of action different from that of GlNR1. Thus, susceptibility to nitro drugs may depend not only on activation, but also on inactivation of the drugs by specific nitroreductases.
authors.affiliation: Institute of Parasitology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland.
authors.forename: Joachim
authors.initials: J
authors.lastname: Müller
authors.forename: Patricia
authors.initials: P
authors.lastname: Schildknecht
authors.forename: Norbert
authors.initials: N
authors.lastname: Müller
mesh_terms: D000981:Antiprotozoal Agents / Q000378:metabolism*; D003001:Cloning, Molecular; D004249:DNA Damage; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008795:Metronidazole / Q000378:metabolism*; D009569:Nitric Oxide / Q000633:toxicity; D009574:Nitro Compounds; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D021261:Parasitic Sensitivity Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013844:Thiazoles / Q000378:metabolism*
chemical_list: D000981:Antiprotozoal Agents; D009574:Nitro Compounds; D013844:Thiazoles; D008795:Metronidazole; D009569:Nitric Oxide; D009601:Nitroreductases; C041747:nitazoxanide
keywords: drug susceptibility; drug targets; mode of action