Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Aug;173(16):4932–4940. doi: 10.1128/jb.173.16.4932-4940.1991

Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure.

G Tipples 1, G McClarty 1
PMCID: PMC208181  PMID: 1860812

Abstract

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates but not deoxyribonucleotide triphosphates. Ribonucleotide reductase is the only enzyme known to catalyze the direct conversion of a ribonucleotide to a deoxyribonucleotide. Hydroxyurea inhibits ribonucleotide reductase by inactivating the tyrosine free radical present in the small subunit of the enzyme. In this report, we show that Chlamydia trachomatis growth is inhibited by hydroxyurea in both wild-type mouse L cells and hydroxyurea-resistant mouse L cells. Hydroxyurea was used as a selective agent in culture to isolate, by a stepwise procedure, a series of C. trachomatis isolates with increasing levels of resistance to the cytotoxic effects of the drug. One of the drug-resistant C. trachomatis isolates (L2HR-10.0) was studied in more detail. L2HR-10.0 retained its drug resistance phenotype even after passage in the absence of hydroxyurea for 10 growth cycles. In addition, L2HR-10.0 was cross resistant to guanazole, another inhibitor of ribonucleotide reductase. Results obtained from hydroxyurea inhibition studies using various host cell-parasite combinations indicated that inhibition of host cell and C. trachomatis DNA synthesis by hydroxyurea can occur but need not occur simultaneously. Crude extract prepared from highly purified C. trachomatis reticulate bodies was capable of reducing CDP to dCDP. The CDP reductase activity was not inhibited by monoclonal antibodies to the large and small subunits of mammalian ribonucleotide reductase, suggesting that the activity is chlamydia specific. The CDP reductase activity was inhibited by hydroxyurea. Crude extract prepared from drug-resistant L2HR-10.0 reticulate bodies contained an elevation in ribonucleotide reductase activity. In total, our results indicate that C. trachomatis obtains the precursors for DNA synthesis as ribonucleotides with subsequent conversion to deoxyribonucleotides catalyzed by a chlamydia-specific ribonucleotide reductase.

Full text

PDF
4932

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bose S. K., Liebhaber H. Deoxyribonucleic acid synthesis, cell cycle progression, and division of Chlamydia-infected HeLa 229 cells. Infect Immun. 1979 Jun;24(3):953–957. doi: 10.1128/iai.24.3.953-957.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caldwell H. D., Kromhout J., Schachter J. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect Immun. 1981 Mar;31(3):1161–1176. doi: 10.1128/iai.31.3.1161-1176.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ehrenberg A., Reichard P. Electron spin resonance of the iron-containing protein B2 from ribonucleotide reductase. J Biol Chem. 1972 Jun 10;247(11):3485–3488. [PubMed] [Google Scholar]
  4. Eliasson R., Fontecave M., Jörnvall H., Krook M., Pontis E., Reichard P. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor. Proc Natl Acad Sci U S A. 1990 May;87(9):3314–3318. doi: 10.1073/pnas.87.9.3314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Engström Y. Monoclonal antibodies against mammalian ribonucleotide reductase. Acta Chem Scand B. 1982;36(5):343–344. doi: 10.3891/acta.chem.scand.36b-0343. [DOI] [PubMed] [Google Scholar]
  6. Engström Y., Rozell B. Immunocytochemical evidence for the cytoplasmic localization and differential expression during the cell cycle of the M1 and M2 subunits of mammalian ribonucleotide reductase. EMBO J. 1988 Jun;7(6):1615–1620. doi: 10.1002/j.1460-2075.1988.tb02987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fraiz J., Jones R. B. Chlamydial infections. Annu Rev Med. 1988;39:357–370. doi: 10.1146/annurev.me.39.020188.002041. [DOI] [PubMed] [Google Scholar]
  8. Frutos R., Pages M., Bellis M., Roizes G., Bergoin M. Pulsed-field gel electrophoresis determination of the genome size of obligate intracellular bacteria belonging to the genera Chlamydia, Rickettsiella, and Porochlamydia. J Bacteriol. 1989 Aug;171(8):4511–4513. doi: 10.1128/jb.171.8.4511-4513.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hatch T. P. Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis. J Bacteriol. 1975 May;122(2):393–400. doi: 10.1128/jb.122.2.393-400.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hatch T. P. Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells. J Bacteriol. 1976 Feb;125(2):706–712. doi: 10.1128/jb.125.2.706-712.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hurta R. A., Wright J. A. Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells. Biochem Biophys Res Commun. 1990 Feb 28;167(1):258–264. doi: 10.1016/0006-291x(90)91759-l. [DOI] [PubMed] [Google Scholar]
  12. Jones R. B., Ridgway G. L., Boulding S., Hunley K. L. In vitro activity of rifamycins alone and in combination with other antibiotics against Chlamydia trachomatis. Rev Infect Dis. 1983 Jul-Aug;5 (Suppl 3):S556–S561. doi: 10.1093/clinids/5.supplement_3.s556. [DOI] [PubMed] [Google Scholar]
  13. Keshishyan H., Hanna L., Jawetz E. Emergence of rifampin-resistance in Chlamydia trachomatis. Nature. 1973 Jul 20;244(5412):173–174. doi: 10.1038/244173a0. [DOI] [PubMed] [Google Scholar]
  14. Larsen I. K., Sjöberg B. M., Thelander L. Characterization of the active site of ribonucleotide reductase of Escherichia coli, bacteriophage T4 and mammalian cells by inhibition studies with hydroxyurea analogues. Eur J Biochem. 1982 Jun 15;125(1):75–81. doi: 10.1111/j.1432-1033.1982.tb06653.x. [DOI] [PubMed] [Google Scholar]
  15. Lewis W. H., Kuzik B. A., Wright J. A. Assay of ribonucleotide reduction in nucleotide-permeable hamster cells. J Cell Physiol. 1978 Mar;94(3):287–298. doi: 10.1002/jcp.1040940306. [DOI] [PubMed] [Google Scholar]
  16. Lin H. S. Inhibition of thymidine kinase activity and deoxyribonucleic acid synthesis in L cells infected with the meningopneumonitis agent. J Bacteriol. 1968 Dec;96(6):2054–2065. doi: 10.1128/jb.96.6.2054-2065.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McClarty G. A., Chan A. K., Engstrom Y., Wright J. A., Thelander L. Elevated expression of M1 and M2 components and drug-induced posttranscriptional modulation of ribonucleotide reductase in a hydroxyurea-resistant mouse cell line. Biochemistry. 1987 Dec 1;26(24):8004–8011. doi: 10.1021/bi00398a068. [DOI] [PubMed] [Google Scholar]
  18. McClarty G. A., Chan A. K., Wright J. A. Characterization of a mouse cell line selected for hydroxyurea resistance by a stepwise procedure: drug-dependent overproduction of ribonucleotide reductase activity. Somat Cell Mol Genet. 1986 Mar;12(2):121–131. doi: 10.1007/BF01560659. [DOI] [PubMed] [Google Scholar]
  19. McClarty G., Tipples G. In situ studies on incorporation of nucleic acid precursors into Chlamydia trachomatis DNA. J Bacteriol. 1991 Aug;173(16):4922–4931. doi: 10.1128/jb.173.16.4922-4931.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morgan J. S., Creasey D. C., Wright J. A. Evidence that the antitumor agent hydroxyurea enters mammalian cells by a diffusion mechanism. Biochem Biophys Res Commun. 1986 Feb 13;134(3):1254–1259. doi: 10.1016/0006-291x(86)90385-2. [DOI] [PubMed] [Google Scholar]
  21. Moulder J. W. Interaction of chlamydiae and host cells in vitro. Microbiol Rev. 1991 Mar;55(1):143–190. doi: 10.1128/mr.55.1.143-190.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peeling R., Maclean I. W., Brunham R. C. In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein. Infect Immun. 1984 Nov;46(2):484–488. doi: 10.1128/iai.46.2.484-488.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Platz A., Karlsson M., Hahne S., Eriksson S., Sjöberg B. M. Alterations in intracellular deoxyribonucleotide levels of mutationally altered ribonucleotide reductases in Escherichia coli. J Bacteriol. 1985 Dec;164(3):1194–1199. doi: 10.1128/jb.164.3.1194-1199.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Platz A., Sjöberg B. M. Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region. J Bacteriol. 1980 Aug;143(2):561–568. doi: 10.1128/jb.143.2.561-568.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reichard P. Interactions between deoxyribonucleotide and DNA synthesis. Annu Rev Biochem. 1988;57:349–374. doi: 10.1146/annurev.bi.57.070188.002025. [DOI] [PubMed] [Google Scholar]
  26. Rosenkranz H. S., Gutter B., Becker Y. Studies on the developmental cycle of Chlamydia trachomatis: selective inhibition by hydroxyurea. J Bacteriol. 1973 Aug;115(2):682–690. doi: 10.1128/jb.115.2.682-690.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sardinia L. M., Segal E., Ganem D. Developmental regulation of the cysteine-rich outer-membrane proteins of murine Chlamydia trachomatis. J Gen Microbiol. 1988 Apr;134(4):997–1004. doi: 10.1099/00221287-134-4-997. [DOI] [PubMed] [Google Scholar]
  28. Schachter J., Caldwell H. D. Chlamydiae. Annu Rev Microbiol. 1980;34:285–309. doi: 10.1146/annurev.mi.34.100180.001441. [DOI] [PubMed] [Google Scholar]
  29. Schachter J. Rifampin in chlamydial infections. Rev Infect Dis. 1983 Jul-Aug;5 (Suppl 3):S562–S564. doi: 10.1093/clinids/5.supplement_3.s562. [DOI] [PubMed] [Google Scholar]
  30. Schachter J. The intracellular life of Chlamydia. Curr Top Microbiol Immunol. 1988;138:109–139. [PubMed] [Google Scholar]
  31. Slabaugh M. B., Mathews C. K. Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase. J Virol. 1986 Nov;60(2):506–514. doi: 10.1128/jvi.60.2.506-514.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spyrou G., Reichard P. Dynamics of the thymidine triphosphate pool during the cell cycle of synchronized 3T3 mouse fibroblasts. Mutat Res. 1988 Jul-Aug;200(1-2):37–43. doi: 10.1016/0027-5107(88)90069-3. [DOI] [PubMed] [Google Scholar]
  33. Steeper J. R., Steuart C. D. A rapid assay for CDP reductase activity in mammalian cell extracts. Anal Biochem. 1970 Mar;34:123–130. doi: 10.1016/0003-2697(70)90092-8. [DOI] [PubMed] [Google Scholar]
  34. Stubbe J. A. Protein radical involvement in biological catalysis? Annu Rev Biochem. 1989;58:257–285. doi: 10.1146/annurev.bi.58.070189.001353. [DOI] [PubMed] [Google Scholar]
  35. Stubbe J. Ribonucleotide reductases. Adv Enzymol Relat Areas Mol Biol. 1990;63:349–419. doi: 10.1002/9780470123096.ch6. [DOI] [PubMed] [Google Scholar]
  36. Thelander M., Thelander L. Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene. EMBO J. 1989 Sep;8(9):2475–2479. doi: 10.1002/j.1460-2075.1989.tb08383.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tribby I. I., Moulder J. W. Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis. J Bacteriol. 1966 Jun;91(6):2362–2367. doi: 10.1128/jb.91.6.2362-2367.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wright J. A., Chan A. K., Choy B. K., Hurta R. A., McClarty G. A., Tagger A. Y. Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis. Biochem Cell Biol. 1990 Dec;68(12):1364–1371. doi: 10.1139/o90-199. [DOI] [PubMed] [Google Scholar]
  39. Wright J. A., Lewis W. H. Evidence of a common site of action for the antitumor agents, hydroxyurea and guanazole. J Cell Physiol. 1974 Jun;83(3):437–439. doi: 10.1002/jcp.1040830314. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES