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Volume 139, Issue 5

Expression of a plasmid gene of Chlamydia trachomatis encoding a novel 28 kDa antigen Free

Abstract

Summary: Plasmid pCT is present in essentially all isolates of and may encode factors important for survival in the natural environment. However, no pCT-associated phenotype has been described so far. With the purpose of investigating the possibility of a role of pCT in pathogenicity, we examined the expression of an ORF (ORF3), potentially encoding a 28 kDa polypeptide (pgp3). Analysis of RNA extracted from chlamydia-infected Vero cells detected ORF3-specific transcripts, from 20 h post-infection onwards, mainly as discrete RNA species of 1390 nucleotides comprising the downstream ORF4 sequence. ORF3 DNA was cloned and expressed in as a 39 kDa fusion protein (MS2/pgp3). Antibodies raised against purified MS2/pgp3, specifically recognized a 28 kDa protein on Western blots of protein from purified chlamydial elementary bodies (EBs). The same antibodies detected chlamydial inclusions in methanol-fixed infected cells by immunofluorescence. Western blot analysis of EBs extracted with 2% Sarkosyl, showed that a large proportion of the 28 kDa antigen is associated with the detergent-insoluble (‘membrane’) fraction. Antibodies recognizing pgp3 epitopes were detected in sera from patients with chlamydial infections, but not in sero-negative control sera. The findings support the hypothesis that pCT may provide a function related to chlamydial cell physiology.

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© Society for General Microbiology 1993
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1993-05-01
2024-05-01
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References

  1. Bavoil P., Ohlin A., Schachter J. 1984; Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infection and Immunity 44:479–485
     [Google Scholar]
  2. Berk A. J. 1989; Characterization of RNA molecules by S1 nuclease analysis. Methods in Enzymology 180:334–347
     [Google Scholar]
  3. Birkelund S., Lundemose A. G., Christiansen G. 1990; The 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide is a DnaK-like protein. Infection and Immunity 58:2098–3104
     [Google Scholar]
  4. Boorstein W. R., Craig E. A. 1989; Primer extension analysis of RNA. Methods in Enzymology 180:347–369
     [Google Scholar]
  5. Caldwell H. D., Kromhout J., Schachter J. 1981; Purification andpartial characterization of the major outer membraneprotein of Chlamydia trachomatis. Infection and Immunity 31:1161–1176
     [Google Scholar]
  6. Cevenini R., Donati M., Sambri V., Rumpianesi F., Laplaca M. 1987; Reactivity of elementary and reticulate bodies of Chlamydia trachomatis LGV2 with monoclonal antibodies specific for the major outer membrane protein. FEMS Microbiology Letters 42:47–51
     [Google Scholar]
  7. Cevenini R., Sambri V., Pileri S., Ratti G., Laplaca M. 1991; Development of transplantable ascites tumours which continuously produce polyclonal antibodies in pristane primed BALB/c mice immunized with bacterial antigens and complete Freund’s adjuvant. Journal of Immunological Methods 140:111–118
     [Google Scholar]
  8. Chomczynski P., Sacchi N. 1987; Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162:156–159
     [Google Scholar]
  9. Comanducci M., Ricci S., Ratti G. 1988; The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells. Molecular Microbiology 2:531–538
     [Google Scholar]
  10. Comanducci M., Cevenini R., Armellini D., Moroni A., Ratti G. 1990a; Identification and characterization of a 28 kDa protein of C. trachomatis encoded by the 7·5 kb common plasmid.. In Chlamydial Infections Proceedings of the Seventh International Symposium on Human Chlamydial Infections pp. 121–124 Bowie W. R. others Edited by Cambridge, MA, USA: Cambridge University Press;
     [Google Scholar]
  11. Comanducci M., Ricci S., Cevenini R., Ratti G. 1990b; Diversity of the chlamydial common plasmid in biovars with different pathogenicity. Plasmid 23:149–154
     [Google Scholar]
  12. Cowing D. W., Bardwell J. C. A., Craig E. A., Woolford C., Hendrix R. W., Gross C. A. 1985; Consensus sequence for Escherichia coli heat shock gene promoters. Proceedings of the National Academy of Sciences of the United States of America 82:2679–2683
     [Google Scholar]
  13. Dean D., Palmer L., Chet Raj Pant, Courtright P., Falkow S., O’Hanley P. 1989; Use of a Chlamydia trachomatis DNA probe for the detection of ocular chlamydiae. Journal of Clinical Microbiology 27:1062–1067
     [Google Scholar]
  14. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysisprograms forthe VAX.. Nucleic Acids Research 12:387–395
     [Google Scholar]
  15. Eberhard W. G. 1989; Why do bacterial plasmids carry some genes and not others?. Plasmid 21:167–174
     [Google Scholar]
  16. Hackstadt T., Caldwell H. D. 1985; Effect of proteolitic cleavage of surface-exposed proteins on infectivity of Chlamydia trachomatis. Infection and Immunity 48:546–551
     [Google Scholar]
  17. Harlow E., Lane D. 1988 Antibodies: A Laboratory Manual Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press;
     [Google Scholar]
  18. Hatt C., Ward M. E., Clarke I. N. 1988; Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1. Evidence for involvement in DNA replication. Nucleic Acids Research 16:4053–4067
     [Google Scholar]
  19. Horn J. E., Hammer M. L., Falkow S., Quinn T. C. 1986; Detection of Chlamydia trachomatis in tissue culture and cervical scrapings by in situ DNA hybridization. Journal of Infectious Diseases 153:1155–1159
     [Google Scholar]
  20. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157:105–132
     [Google Scholar]
  21. Moulder J. W. 1985; Comparative biology of intracellular parasitism. Microbiological Reviews 49:298–337
     [Google Scholar]
  22. Moulder J. W., Hatch T. P., Kuo C. -C., Schachter J., Storz J. 1984; Genus I. Chlamydia Jones, Rate and Steams 1945 55AL. In Bergey’s Manual of Systematic Bacteriology l pp. 729–739 Krieg N. R., Holt J. G. Edited by Baltimore, MD: Williams & Wilkins;
     [Google Scholar]
  23. Nicosia A., Bartoloni A., Perugini M., Rappuoli R. 1987; Expression and immunological properties of the five subunits of pertussis toxin. Infection and Immunity 55:963–967
     [Google Scholar]
  24. Ostergaard L., Birkelund S., Christiansen G. 1990; Use of polymerase chain reaction for detection of Chlamydia trachomatis. Journal of Clinical Microbiology 28:1254–1260
     [Google Scholar]
  25. Palmer L., Falkow S. 1986; A common plasmid of Chlamydia trachomatis. Plasmid 16:52–62
     [Google Scholar]
  26. Peterson E. M., Markoff B. A., Schachter J., De La Maza L. M. 1990; The 7·5-kb plasmid present in Chlamydia trachomatis is not essential for the growth of this microorganism. Plasmid 23:144–148
     [Google Scholar]
  27. Ratti G., Moroni A., Cevenini R. 1991; Detection of Chlamydia trachomatis DNA in patients with non-gonococcal urethritis using the polymerase chain reaction. Journal of Clinical Pathology 44:564–568
     [Google Scholar]
  28. Remaut E., Tsao H., Fiers W. 1983; Improved plasmid vectors with a thermoinducible expression and temperature-regulated run-away replication. Gene 22:103–113
     [Google Scholar]
  29. Ricci S., Cevenini R., Cosco E., Comanducci M., Ratti G., Scarlato V. 1993; Transcription analysis of the Chlamydia trachomatis plasmid pCT identifies temporally regulated transcripts, anti-sense RNA and σ70-selected promoters. Molecular and General Genetics 237:318–326
     [Google Scholar]
  30. Saiki A. K., Gelfand D. H., Stoffell S., Scharf S. J., Higuchi R., Horne G. T., Mullis K. B., Erlich H. A. 1988; Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
     [Google Scholar]
  31. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn.. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press;
     [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74:5463–5467
     [Google Scholar]
  33. Scarlato V., Storlazzi A., Gargano S., Cascino A. 1989; Bacteriophage T4 late gene expression: overlapping promoters direct divergent transcription of the base plate gene cluster. Virology 171:475–483
     [Google Scholar]
  34. Sriprakash K. S., Macavoy E. S. 1987; Characterization and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis. Plasmid 18:205–214
     [Google Scholar]
  35. Strebel K., Beck E., Strohmaier K., Schaller H. 1986; Characterization of Foot-and-Mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. Journal of Virology 57:983–991
     [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academ y of Sciences of the United States of America 76:4650–6555
     [Google Scholar]
  37. Vieira J., Messing J. 1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
     [Google Scholar]
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