Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance

LAND, K.M.; M.G. DELLGADILLO-CORREA; Jan TACHEZY; Štěpánka VAŇÁČOVÁ; C.L. HSIEH; R. SUTAK a Patricia J. JOHNSON. Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance. Molecular Microbiology. 2004, roč. 51, č. 1, s. 115-122. ISSN 0950-382X.

Základní údaje

Originální název

Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance

Název česky

Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance

Autoři

LAND, K.M.; M.G. DELLGADILLO-CORREA; Jan TACHEZY; Štěpánka VAŇÁČOVÁ; C.L. HSIEH; R. SUTAK a Patricia J. JOHNSON

Vydání

Molecular Microbiology, 2004, 0950-382X

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Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10600 1.6 Biological sciences

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Impakt faktor

Impact factor: 5.959

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14310/04:00036237

Organizační jednotka

Přírodovědecká fakulta

UT WoS

000186974100011

Klíčová slova česky

Trichomonads; ferredoxin; metronidazole; drug resistance; gene knock-out

Klíčová slova anglicky

Trichomonads; ferredoxin; metronidazole; drug resistance; gene knock-out

Štítky

drug resistance, ferredoxin, gene knock-out, metronidazole, Trichomonads

Příznaky

Mezinárodní význam, Recenzováno

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Anotace

V originále

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.

Česky

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.