Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance.
Ferredoxin, Fd, is often deficient in metronidazole-resistant strains of Trichomonas vaginalis and is thought to be necessary for drug activation. To directly test whether Fd is essential for metronidazole susceptibility, gene replacement technology has been developed for T. vaginalis. The selectable marker gene neomycin phosphotransferase (NEO) flanked by approximately 2.6 and approximately 2.0 kBp of the Fd 5' and 3' flanking regions (pKO-FD-NEO) was introduced into cells on linear DNA and selected for NEO gene expression. Stable transformants were shown to contain the NEO gene in the Fd locus and to have completely lost the Fd gene. Northern and immunoblot analyses confirm the loss of Fd mRNA and protein in pKO-FD-NEO cells. Analyses of the activity of hydrogenosomal proteins in Fd KO cells show a fourfold increase in hydrogenase activity and a 95% decrease in pyruvate/ferredoxin oxidoreductase (PFO) activity. In contrast, PFO and hydrogenase mRNA levels are unchanged. Surprisingly, Fd KO cells are not resistant to metronidazole under aerobic or anaerobic conditions. These cells are capable of producing molecular hydrogen, albeit at 50% the level of the parental strain, demonstrating that the Fd gene product eliminated in KO cells is neither necessary for hydrogen production nor metronidazole activation. Together these data indicate the presence of unidentified Fds or flavodoxins capable of drug activation or an unidentified mechanism that does not require either PFO or Fd for metronidazole activation.
Land
Kirkwood M
KM
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles School of Medicine, Los Angeles, California 90095-1489, USA.
Delgadillo-Correa
Maria G
MG
Tachezy
Jan
J
Vanacova
Stepanka
S
Hsieh
Christine L
CL
Sutak
Robert
R
Johnson
Patricia J
PJ
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004351:Drug Resistance / Q000187:drug effects*; D005288:Ferredoxins / Q000235:genetics*; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000235:genetics; D008795:Metronidazole / Q000494:pharmacology*; D008954:Models, Biological; D010957:Plasmids / Q000235:genetics; D014158:Transcription, Genetic; D014169:Transformation, Bacterial / Q000235:genetics; D014246:Trichomonas vaginalis / Q000187:drug effects / Q000235:genetics*
D017931:DNA Primers; D005288:Ferredoxins; D005819:Genetic Markers; D005839:Gentamicins; D008795:Metronidazole; C010680:antibiotic G 418; D019868:Kanamycin Kinase