Abstract
Recombinant Treponema pallidum surface antigen 4D isolated from Escherichia coli formed a protease-resistant ordered ring structure composed of 19,000-dalton subunits. On gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the higher oligomers of recombinant 4D migrated with molecular masses that were nearly multiples of the 190,000-dalton basic ordered ring. Reduction at room temperature with 2-mercaptoethanol converted the 190,000-dalton ordered ring and the higher oligomers to a 160,000-dalton form and the dissociated monomer. A 190,000-dalton form of 4D was identified in sodium dodecyl sulfate-solubilized T. pallidum after reduction at room temperature. Disulfide bonds stabilized both native and recombinant 4D oligomers against dissociation by heating in detergent without a reducing agent. Electron microscopy of recombinant 4D revealed that the characteristic ordered ring structure was maintained after reduction. Reduction of 4D under conditions that preserved the ordered ring structure did not affect the resistance of the molecule to digestion with proteinase K. The properties of 4D suggest that it may fulfill an important structural role in the T. pallidum outer membrane.
Full text
PDF






Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bavoil P., Ohlin A., Schachter J. Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infect Immun. 1984 May;44(2):479–485. doi: 10.1128/iai.44.2.479-485.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blattler D. P., Reithel F. J. Molecular weight determinations and the influence of gel density, protein charges and protein shape in polyacrylamide gel electrophoresis. J Chromatogr. 1970 Feb 4;46(3):286–292. doi: 10.1016/s0021-9673(00)84000-5. [DOI] [PubMed] [Google Scholar]
- Butler C. A., Street E. D., Hatch T. P., Hoffman P. S. Disulfide-bonded outer membrane proteins in the genus Legionella. Infect Immun. 1985 Apr;48(1):14–18. doi: 10.1128/iai.48.1.14-18.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- CRESTFIELD A. M., MOORE S., STEIN W. H. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem. 1963 Feb;238:622–627. [PubMed] [Google Scholar]
- Chang J. J., Leonard K., Arad T., Pitt T., Zhang Y. X., Zhang L. H. Structural studies of the outer envelope of Chlamydia trachomatis by electron microscopy. J Mol Biol. 1982 Nov 15;161(4):579–590. doi: 10.1016/0022-2836(82)90409-0. [DOI] [PubMed] [Google Scholar]
- Fehniger T. E., Radolf J. D., Lovett M. A. Properties of an ordered ring structure formed by recombinant Treponema pallidum surface antigen 4D. J Bacteriol. 1986 Mar;165(3):732–739. doi: 10.1128/jb.165.3.732-739.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fehniger T. E., Walfield A. M., Cunningham T. M., Radolf J. D., Miller J. N., Lovett M. A. Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen. Infect Immun. 1984 Nov;46(2):598–607. doi: 10.1128/iai.46.2.598-607.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gabay J. E., Blake M., Niles W. D., Horwitz M. A. Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin. J Bacteriol. 1985 Apr;162(1):85–91. doi: 10.1128/jb.162.1.85-91.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hackstadt T., Todd W. J., Caldwell H. D. Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae? J Bacteriol. 1985 Jan;161(1):25–31. doi: 10.1128/jb.161.1.25-31.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hancock R. E., Carey A. M. Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins. J Bacteriol. 1979 Dec;140(3):902–910. doi: 10.1128/jb.140.3.902-910.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanff P. A., Norris S. J., Lovett M. A., Miller J. N. Purification of Treponema pallidum, Nichols strain, by Percoll density gradient centrifugation. Sex Transm Dis. 1984 Oct-Dec;11(4):275–286. doi: 10.1097/00007435-198410000-00003. [DOI] [PubMed] [Google Scholar]
- Hatch T. P., Allan I., Pearce J. H. Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of Chlamydia spp. J Bacteriol. 1984 Jan;157(1):13–20. doi: 10.1128/jb.157.1.13-20.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hougen K. H. The ultrastructure of cultivable treponemes. Acta Pathol Microbiol Scand B. 1975 Apr;83(2):91–99. doi: 10.1111/j.1699-0463.1975.tb00076.x. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lugtenberg B., Van Alphen L. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim Biophys Acta. 1983 Mar 21;737(1):51–115. doi: 10.1016/0304-4157(83)90014-x. [DOI] [PubMed] [Google Scholar]
- Morrison M. Lactoperoxidase-catalyzed iodination as a tool for investigation of proteins. Methods Enzymol. 1980;70(A):214–220. doi: 10.1016/s0076-6879(80)70051-4. [DOI] [PubMed] [Google Scholar]
- Newhall W. J., Jones R. B. Disulfide-linked oligomers of the major outer membrane protein of chlamydiae. J Bacteriol. 1983 May;154(2):998–1001. doi: 10.1128/jb.154.2.998-1001.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Newhall W. J., Wilde C. E., 3rd, Sawyer W. D., Haak R. A. High-molecular-weight antigenic protein complex in the outer membrane of Neisseria gonorrhoeae. Infect Immun. 1980 Feb;27(2):475–482. doi: 10.1128/iai.27.2.475-482.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Norgard M. V., Miller J. N. Cloning and expression of Treponema pallidum (Nichols) antigen genes in Escherichia coli. Infect Immun. 1983 Nov;42(2):435–445. doi: 10.1128/iai.42.2.435-445.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Radolf J. D., Fehniger T. E., Silverblatt F. J., Miller J. N., Lovett M. A. The surface of virulent Treponema pallidum: resistance to antibody binding in the absence of complement and surface association of recombinant antigen 4D. Infect Immun. 1986 May;52(2):579–585. doi: 10.1128/iai.52.2.579-585.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Radolf J. D., Lernhardt E. B., Fehniger T. E., Lovett M. A. Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purified recombinant Treponema pallidum antigen 4D. J Infect Dis. 1986 Jun;153(6):1023–1027. doi: 10.1093/infdis/153.6.1023. [DOI] [PubMed] [Google Scholar]
- Rosenbusch J. P. Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J Biol Chem. 1974 Dec 25;249(24):8019–8029. [PubMed] [Google Scholar]
- Sleytr U. B. Regular arrays of macromolecules on bacterial cell walls: structure, chemistry, assembly, and function. Int Rev Cytol. 1978;53:1–62. doi: 10.1016/s0074-7696(08)62240-8. [DOI] [PubMed] [Google Scholar]
- Stamm L. V., Folds J. D., Bassford P. J., Jr Expression of Treponema pallidum antigens in Escherichia coli K-12. Infect Immun. 1982 Jun;36(3):1238–1241. doi: 10.1128/iai.36.3.1238-1241.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sykes J. A., Miller J. N. Ultrastructural studies of treponemes: location of axial filaments and some dimensions of Treponema pallidum (Nichols strain), Treponema denticola, and Treponema reiteri. Infect Immun. 1973 Jan;7(1):100–110. doi: 10.1128/iai.7.1.100-110.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
- Valentine R. C., Shapiro B. M., Stadtman E. R. Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry. 1968 Jun;7(6):2143–2152. doi: 10.1021/bi00846a017. [DOI] [PubMed] [Google Scholar]
- Walfield A. M., Hanff P. A., Lovett M. A. Expression of Treponema pallidum antigens in Escherichia coli. Science. 1982 Apr 30;216(4545):522–523. doi: 10.1126/science.7041257. [DOI] [PubMed] [Google Scholar]
- van Embden J. D., van der Donk H. J., van Eijk R. V., van der Heide H. G., de Jong J. A., van Olderen M. F., Osterhaus A. B., Schouls L. M. Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12. Infect Immun. 1983 Oct;42(1):187–196. doi: 10.1128/iai.42.1.187-196.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]