1887

Abstract

Development of resistance against metronidazole and mechanisms responsible for this process were studied in a sexually transmitted pathogen of humans, . Monitoring of changes in metabolism and protein expression that accompanied increasing resistance of strains derived from a common drug-susceptible parent (TV 10-02) showed the multistep character of the process. The aerobic type of resistance known to occur in isolates from patients non-responsive to treatment appeared at the earliest stage, followed by development of the anaerobic type of resistance which was accompanied by gradual loss of hydrogenosomal proteins associated with drug-activating pathways [pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin]. Unexpectedly, the loss of PFOR did not result in acquisition of full anaerobic resistance, thus indicating an alternative source of electrons required for the drug activation. These data suggest involvement of the oxidative decarboxylation of malate in hydrogenosomes, catalysed by NAD-dependent malic enzyme and subsequent transfer of reduced equivalents to the drug via NADH:ferredoxin oxidoreductase and ferredoxin. Accordingly, all components of this pathway were eliminated before the resistance was fully developed. Resistant compensated the impaired function of hydrogenosomes by enhanced conversion of pyruvate to lactate in the cytosol. Further analysis of the two key enzymes involved in metronidazole activation by Northern blotting and assay for nascent mRNA showed that the insufficient expression of the PFOR protein results from decreased gene transcription, while down-regulation of malic enzyme is controlled at the mRNA level.

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2002-08-01
2024-03-29
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References

  1. Bergmeyer H. U. 1963 Methods of Enzymatic Analysis New York: Academic Press;
    [Google Scholar]
  2. Bowtell D. D. 1987; Rapid isolation of eukaryotic DNA. Anal Biochem 162:463–465 [CrossRef]
    [Google Scholar]
  3. Brown D. M., Upcroft J. A., Dodd H. N., Chen N., Upcroft P. 1999; Alternative 2-keto acid oxidoreductase activities in Trichomonas vaginalis . Mol Biochem Parasitol 98:203–214 [CrossRef]
    [Google Scholar]
  4. C̆erkasov J., C̆erkasovová A., Kulda J., Vilhelmová D. 1978; Respiration of hydrogenosomes of Tritrichomonas foetus . J Biol Chem 253:1207–1214
    [Google Scholar]
  5. C̆erkasovová A., C̆erkasov J., Kulda J. 1984; Metabolic differences between metronidazole resistant and susceptible strains of Tritrichomonas foetus . Mol Biochem Parasitol 11:105–118 [CrossRef]
    [Google Scholar]
  6. C̆erkasovová A., Novák J., C̆erkasov J., Kulda J., Tachezy J. 1987; Metabolic properties of Trichomonas vaginalis resistant to metronidazole under anaerobic conditions. Acta Univ Carol Biol 30:505–512
    [Google Scholar]
  7. Chapman A., Cammack R., Linstead D., Lloyd D. 1985; The generation of metronidazole radicals in hydrogenosomes isolated from Trichomonas vaginalis . J Gen Microbiol 131:2141–2144
    [Google Scholar]
  8. de Carneri I., Achilli G., Monti G., Trane F. 1969; Induction of in-vivo resistance of Trichomonas vaginalis to metronidazole. Lancet 2:1308–1309
    [Google Scholar]
  9. Diamond L. S. 1957; The establishment of various trichomonads of animals and man in axenic cultures. J Parasitol 43:488–490
    [Google Scholar]
  10. Doran D. J. 1959; Studies on trichomonads: III. inhibitors, acid production, and substrate utilization by 4 strains of Tritrichomonas foetus . J Protozool 6:177–182 [CrossRef]
    [Google Scholar]
  11. Drmota T., Proost P., Van Ranst M., Weyda F., Kulda J., Tachezy J. 1996; Iron-ascorbate cleavable malic enzyme from hydrogenosomes of Trichomonas vaginalis : purification and characterization. Mol Biochem Parasitol 83:221–234 [CrossRef]
    [Google Scholar]
  12. Edwards D. I. 1993; Nitroimidazole drugs – action and resistance mechanisms. II. Mechanisms of resistance. J Antimicrob Chemother 31:201–210 [CrossRef]
    [Google Scholar]
  13. Ellis J. E., Cole D., Lloyd D. 1992; Influence of oxygen on the fermentative metabolism of metronidazole-sensitive and resistant strains of Trichomonas vaginalis . Mol Biochem Parasitol 56:79–88 [CrossRef]
    [Google Scholar]
  14. Johnson P. J., d’Oliveira C. E., Gorrell T. E., Müller M. 1990; Molecular analysis of the hydrogenosomal ferredoxin of the anaerobic protist Trichomonas vaginalis . Proc Natl Acad Sci USA 87:6097–6101 [CrossRef]
    [Google Scholar]
  15. Kabı́c̆ková H., Kulda J., C̆erkasovová A., Nẽmcová H. 1987; Metronidazole resistant Tritrichomonas foetus : activities of hydrogenosomal enzymes in course of development of anaerobic resistance. Acta Univ Carol Biol 30:513–519
    [Google Scholar]
  16. Kulda J. 1999; Trichomonads, hydrogenosomes and drug resistance. Int J Parasitol 29:199–212 [CrossRef]
    [Google Scholar]
  17. Kulda J., Vojtĕchovská M., Tachezy J., Demes̆ P., Kunzová E. 1982; Metronidazole resistance of Trichomonas vaginalis as a cause of treatment failure in trichomoniasis. A case report. Br J Vener Dis 58:394–399
    [Google Scholar]
  18. Kulda J., C̆erkasov J., Demes̆ P., C̆erkasovová A. 1984; Tritrichomonas foetus : stable anaerobic resistance to metronidazole in vitro . Exp Parasitol 57:93–103 [CrossRef]
    [Google Scholar]
  19. Kulda J., Kabı́c̆ková H., Tachezy J., C̆erkasovová A., C̆erkasov J. 1989; Metronidazole resistant trichomonads: mechanisms of in vitro developed anaerobic resistance. In Biochemistry and Molecular Biology of ‘Anaerobic’ Protozoa pp 137–160 Edited by Lloyd D., Coombs G. H., Paget T. A. P. Chur, Switzerland: Harwood Academic Publishers;
    [Google Scholar]
  20. Kulda J., Tachezy J., C̆erkasovová A. 1993; In vitro induced anaerobic resistance to metronidazole in Trichomonas vaginalis . J Eukaryot Microbiol 40:262–269 [CrossRef]
    [Google Scholar]
  21. Lahti C. J., d’Oliveira C. E., Johnson P. J. 1992; Beta-succinyl-coenzyme A synthetase from Trichomonas vaginalis is a soluble hydrogenosomal protein with an amino-terminal sequence that resembles mitochondrial presequences. J Bacteriol 174:6822–6830
    [Google Scholar]
  22. Land K. M., Clemens D. L., Johnson P. J. 2001; Loss of multiple hydrogenosomal proteins associated with organelle metabolism and high-level drug resistance in trichomonads. Exp Parasitol 97:102–110 [CrossRef]
    [Google Scholar]
  23. Lindmark D. G., Müller M. 1973; Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus , and its role in pyruvate metabolism. J Biol Chem 248:7724–7728
    [Google Scholar]
  24. Liston D. R., Johnson P. J. 1999; Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Mol Cell Biol 19:2380–2388
    [Google Scholar]
  25. Liston D. R., Carrero J. C., Johnson P. J. 1999; Upstream regulatory sequences required for expression of the Trichomonas vaginalis alpha-succinyl CoA synthetase gene. Mol Biochem Parasitol 104:323–329 [CrossRef]
    [Google Scholar]
  26. Liu S. M., Brown D. M., O’Donoghue P., Upcroft P., Upcroft J. A. 2000; Ferredoxin involvement in metronidazole resistance of Giardia duodenalis . Mol Biochem Parasitol 108:137–140 [CrossRef]
    [Google Scholar]
  27. Lloyd D., Kristensen B. 1985; Metronidazole inhibition of hydrogen production in vivo in drug-sensitive and -resistant strains of Trichomonas vaginalis . J Gen Microbiol 131:849–853
    [Google Scholar]
  28. Lossick J. G., Müller M., Gorrell T. E. 1986; In vitro drug susceptibility and doses of metronidazole required for cure in cases of refractory vaginal trichomoniasis. J Infect Dis 153:948–955 [CrossRef]
    [Google Scholar]
  29. Meingassner J. G., Thurner J. 1979; Strain of Trichomonas vaginalis resistant to metronidazole and other 5-nitroimidazoles. Antimicrob Agents Chemother 15:254–257 [CrossRef]
    [Google Scholar]
  30. Meingassner J. G., Mieth H., Czok R., Lindmark D. G., Müller M. 1978; Assay conditions and the demonstration of nitroimidazole resistance in Tritrichomonas foetus . Antimicrob Agents Chemother 13:1–3 [CrossRef]
    [Google Scholar]
  31. Meri T., Jokiranta T. S., Suhonen L., Meri S. 2000; Resistance of Trichomonas vaginalis to metronidazole: report of the first three cases from Finland and optimization of in vitro susceptibility testing under various oxygen concentrations. J Clin Microbiol 38:763–767
    [Google Scholar]
  32. Mertens E., Van Schaftingen E., Müller M. 1992; Pyruvate kinase from Trichomonas vaginalis , an allosteric enzyme stimulated by ribose 5-phosphate and glycerate 3-phosphate. Mol Biochem Parasitol 54:13–20 [CrossRef]
    [Google Scholar]
  33. Moreno S. N., Mason R. P., Muniz R. P., Cruz F. S., Docampo R. 1983; Generation of free radicals from metronidazole and other nitroimidazoles by Tritrichomonas foetus . J Biol Chem 258:4051–4054
    [Google Scholar]
  34. Müller M. 1986; Reductive activation of nitroimidazoles in anaerobic microorganisms. Biochem Pharmacol 35:37–41 [CrossRef]
    [Google Scholar]
  35. Müller M. 1993; The hydrogenosome. J Gen Microbiol 139:2879–2889 [CrossRef]
    [Google Scholar]
  36. Müller M., Meingassner J. G., Miller W. A., Ledger W. J. 1980; Three metronidazole-resistant strains of Trichomonas vaginalis from the United States. Am J Obstet Gynecol 138:808–812
    [Google Scholar]
  37. Quon D. V., d’Oliveira C. E., Johnson P. J. 1992; Reduced transcription of the ferredoxin gene in metronidazole-resistant Trichomonas vaginalis . Proc Natl Acad Sci USA 89:4402–4406 [CrossRef]
    [Google Scholar]
  38. Quon D. V., Delgadillo M. G., Khachi A., Smale S. T., Johnson P. J. 1994; Similarity between a ubiquitous promoter element in an ancient eukaryote and mammalian initiator elements. Proc Natl Acad Sci USA 91:4579–4583 [CrossRef]
    [Google Scholar]
  39. Rasoloson D., Tomková E., Cammack R., Kulda J., Tachezy J. 2001; Metronidazole-resistant strains of Trichomonas vaginalis display increased susceptibility to oxygen. Parasitology 123:45–56
    [Google Scholar]
  40. Steinbüchel A., Müller M. 1986; Anaerobic pyruvate metabolism of Tritrichomonas foetus and Trichomonas vaginalis hydrogenosomes. Mol Biochem Parasitol 20:57–65 [CrossRef]
    [Google Scholar]
  41. Swain M., Ross N. W. 1995; A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis 16:948–951 [CrossRef]
    [Google Scholar]
  42. Tachezy J., Kulda J., Tomková E. 1993; Aerobic resistance of Trichomonas vaginalis to metronidazole induced in vitro . Parasitology 106:31–37 [CrossRef]
    [Google Scholar]
  43. Thong K. W., Coombs G. H. 1987; Comparative study of ferredoxin-linked and oxygen-metabolizing enzymes of trichomonads. Comp Biochem Physiol B 87:637–641
    [Google Scholar]
  44. Van̆ác̆ová S̆., Tachezy J., Ullu E., Tschudi C. 2001a; Unusual diversity in α-amanitin sensitivity of RNA polymerases in trichomonads. Mol Biochem Parasitol 115:239–247 [CrossRef]
    [Google Scholar]
  45. Van̆ác̆ová S̆., Rasoloson D., Rázga J., Hrdý I., Kulda J., Tachezy J. 2001b; Iron-induced changes in pyruvate metabolism of Tritrichomonas foetus and involvement of iron in expression of hydrogenosomal proteins. Microbiology 147:53–62
    [Google Scholar]
  46. Wang A. L., Wang C. C. 1985a; Isolation and characterization of DNA from Tritrichomonas foetus and Trichomonas vaginalis . Mol Biochem Parasitol 14:323–335 [CrossRef]
    [Google Scholar]
  47. Wang A. L., Wang C. C. 1985b; A linear double-stranded RNA in Trichomonas vaginalis . J Biol Chem 260:3697–3702
    [Google Scholar]
  48. Yarlett N., Yarlett N. C., Lloyd D. 1986a; Metronidazole-resistant clinical isolates of Trichomonas vaginalis have lowered oxygen affinities. Mol Biochem Parasitol 19:111–116 [CrossRef]
    [Google Scholar]
  49. Yarlett N., Yarlett N. C., Lloyd D. 1986b; Ferredoxin-dependent reduction of nitroimidazole derivatives in drug-resistant and susceptible strains of Trichomonas vaginalis . Biochem Pharmacol 35:1703–1708 [CrossRef]
    [Google Scholar]
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