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@article{840516,
author = {Land, K.M. and DellgadilloandCorrea, M.G. and Tachezy, Jan and Vaňáčová, Štěpánka and Hsieh, C.L. and Sutak, R. and Johnson, Patricia J.},
article_number = {1},
keywords = {Trichomonads; ferredoxin; metronidazole; drug resistance; gene knock-out},
language = {eng},
issn = {0950-382X},
journal = {Molecular Microbiology},
title = {Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance},
volume = {51},
year = {2004}
}
TY - JOUR
ID - 840516
AU - Land, K.M. - Dellgadillo-Correa, M.G. - Tachezy, Jan - Vaňáčová, Štěpánka - Hsieh, C.L. - Sutak, R. - Johnson, Patricia J.
PY - 2004
TI - Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance
JF - Molecular Microbiology
VL - 51
IS - 1
SP - 115-122
EP - 115-122
SN - 0950382X
KW - Trichomonads
KW - ferredoxin
KW - metronidazole
KW - drug resistance
KW - gene knock-out
N2 - 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.
ER -
LAND, K.M., M.G. DELLGADILLO-CORREA, Jan TACHEZY, Štěpánka VAŇÁČOVÁ, C.L. HSIEH, R. SUTAK and Patricia J. JOHNSON. Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance. \textit{Molecular Microbiology}. 2004, vol.~51, No~1, p.~115-122. ISSN~0950-382X.
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