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Protein expression analysis ofChlamydia pneumoniae persistence by combined surface-enhanced laser desorption ionization time-of-flight mass spectrometry and two-dimensional polyacrylamide gel electrophoresis

Clinical Proteomics volume 2, pages 205–215 (2006)Cite this article

Abstract

The aim of this study was to examine the protein expression profiles of persistentChlamydia pneumoniae by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Although 2D PAGE is still the method of choice for separating and detecting components of complex protein mixtures, it has several distinct disadvantages; i.e., being labor-intensive and having a bias toward proteins within the dynamic range of the gel condition. Hence, SELDI-TOF-MS technology was used to complement 2D PAGE.C. pneumoniae-infected HEp2 cells were treated with or without IFN-γ, and protein expression profiles were determined at 48 h postinfection (hpi). Unfractionated monolayers were also used for protein profiling by SELDI-TOF, using two different chip surface types: weak cation exchanger and hydrophobic surface. Under IFN-γ-induced persistence,C. pneumoniae expresses an altered protein expression profile. Twenty chlamydial proteins showed differential regulatory patterns by SELDI-TOF-MS, two of which, HSP-70 cofactor, and a hypothetical protein, were identified by 2D PAGE and mass spectrometry. Two additional proteins, phosphatidylserine decarboxylase and 30S ribosomal protein S17, were exclusively identified by SELDI TOF-MS analysis, as these were not present in sufficient quantity for detection by 2D PAGE. We propose that a combination of 2D-PAGE and SELDI-TOF-MS may complement the disadvantages of each technique alone and may provide a rapid and precise screening technique.

References

  1. Ramirez, J. A., Ahkee, S., Summersgill, J. T., et al. (1996) Isolation ofChlamydia pneumoniae from the coronary artery of a patient with coronary atherosclerosis.Ann. Intern. Med. 125, 979–982.

    PubMed  CAS  Google Scholar 

  2. Saikku, P., Leinonen, M., Mattila, K., et al. (1988) Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction.Lancet 8618, 983–986.

    Article  Google Scholar 

  3. Kuo, C.-C., Gown, A. M., Benditt, E. P., and Grayston, J. T. (1992) Detection ofChlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain.Artheroscler. Thromb. 13 1501–1504.

    Google Scholar 

  4. Kuo, C.-C., Shor, A., Campbell, L. A., Fukushi, H., Patton, D. L., and Gryaston, J. T. (1993) Demonstration ofChlamydia pneumoniae in atherosclerotic lesions of coronary arteries.J. Infect. Dis. 167, 841–849.

    PubMed  CAS  Google Scholar 

  5. Grayston, J. T., Kuo, C.-C., Coulson, A. S., Campbell, L. A., Lawrence, R. D., and Ming-Jong, L. (1995)Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery.Circulation 93, 3397–3400.

    Google Scholar 

  6. Raulston, J. E. (1995) Chlamydial envelope components and pathogen-host cell interactions.Mol. Microbiol. 15, 607–616.

    Article  PubMed  CAS  Google Scholar 

  7. McClarty, G. (1994). Chlamydiae and the biochemistry of intracellular parasitism.Trends Microbiol. 2, 157–164.

    Article  PubMed  CAS  Google Scholar 

  8. Moulder, J. W. (1991) Interaction of chlamydiae and host cells in vitro.Microbiol. Rev. 55, 143–190.

    PubMed  CAS  Google Scholar 

  9. Hatch, T. (1999) Developmental biology, inChlamydia: Intracellular Biology, Pathogenesis, and Immunity, (Stephens, R. S., eds.), American Society for Microbiology, Washington, DC, pp. 29–67.

    Google Scholar 

  10. Wolf, K., Fischer, E., and Hackstadt, T. (2000) Ultrastructural analysis of developmental events inChlamydia pneumoniae-infected cells.Infect. Immun. 68, 2379–2385.

    Article  PubMed  CAS  Google Scholar 

  11. Beatty, W. L., Morrison, R. P., and Byrne, G. I. (1994) Persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis.Microbiol. Rev. 58, 686–699.

    PubMed  CAS  Google Scholar 

  12. Hogan, R. J., Mathews, S. A., Mukhopadhyay, S., Summersgill, J. T., and Timms, P. (2004) Chlamydial persistence: beyong the biphasic paradigm.Infect. Immun. 72, 1843–1855.

    Article  PubMed  CAS  Google Scholar 

  13. Stephens, R. S., Kalman, S., Lammel, C., et al. (1998) Genome sequence of an obligate intracellular pathogen of humans:Chlamydia trachomatis.Science 282, 754–759.

    Article  PubMed  CAS  Google Scholar 

  14. Kalman, S., Mitchell, W., Marathe, R., et al. (1999) Comparative genomes ofChlamydia pneumoniae andC. trachomatis.Nat. Genet. 21, 385–389.

    Article  PubMed  CAS  Google Scholar 

  15. Read, T. D., Myers, G. S., Brunham, R. C., et al. (2003). Genome sequence ofChlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae.Nucleic Acids Res. 31, 2134–2147.

    Article  PubMed  CAS  Google Scholar 

  16. Belland, R. J., Zhong, G., Crane, D. D., et al. (2003). Genomic transcriptional profiling of the developmental cycle ofChlamydia trachomatis.Proc. Natl. Acad. Sci. USA 100, 8478–8483.

    Article  PubMed  CAS  Google Scholar 

  17. Gerard, H. C., Freise, J., Wang, Z., et al. (2002)Chlamydia trachomatis genes whose products are related to energy metabolism are expressed differentially in active vs. persistent infection.Microbes Infect 4, 13–22.

    Article  PubMed  CAS  Google Scholar 

  18. Molestina, R. E., Klein, J. B., Miller, R. D., Pierce, W. H., Ramirez, J. A., and Summersgill, J. T. (2002) Proteomic analysis of differentially expressedChlamydia pneumonia genes during persistent infection of HEp-2 cells.Infect. Immun. 70, 2976–2981.

    Article  PubMed  CAS  Google Scholar 

  19. Mukhopadhyay, S., Miller, R. D., and Summersgill, J. T. (2004a). Analysis of altered protein expression patterns ofChlamydia pneumoniae by an integrated Proteome-Works system.J. Proteome Res. 3, 878–883.

    Article  PubMed  CAS  Google Scholar 

  20. Nicholson, T. L., Olinger, L., Chong, K., Schoolnik, G., and Stephens, R. S. (2003) Global stage-specific gene regulation during the developmental cycle ofChlamydia trachomatis.J. Bacteriol. 185, 3179–3189.

    Article  PubMed  CAS  Google Scholar 

  21. Byrne, G. I., Ouellette, S. P., Wang, Z., et al. (2001)Chlamydia pneumoniae expresses genes required for DNA replication but not cytokines during persistent infection of HEp2 cells.Infect. Immun. 69, 5423–5429.

    Article  PubMed  CAS  Google Scholar 

  22. Mathews, S. A., George, C., Flegg, C. Stenzel, D., and Timms, P. (2001) Differential expression ofompA, ompB, pyk, nlpD and Cpn0585 genes between normal and interferon-γ treated cultures ofChlamydia pneumoniae.Microb. Pathog 30, 337–345.

    Article  PubMed  CAS  Google Scholar 

  23. Hogan, R. J., Mathews, S. A., Kutlin, A., Hammerschlag, M. R., and Timms, P. (2003) Differential expression of genes, encoding membrane proteins between acute and continuousChlamydia pneumoniae infections.Microb. Pathog. 34, 11–16.

    Article  PubMed  CAS  Google Scholar 

  24. Vandahl, B. B., Birkeland, S., Demol, H., et al. (2001) Proteome analysis of theChlamydia pneumoniae elementary body.Electrophoresis 22, 1204–1223.

    Article  PubMed  CAS  Google Scholar 

  25. Rapkiewicz, A. V., Espina, V., Petricoin, E. F., 3rd, and Liotta, L. A. (2004) Biomarkers of ovarian tumors.Eur. J. Cancer 40, 2604–2612.

    Article  PubMed  CAS  Google Scholar 

  26. Liu, A. Y., Zhang, H., Sorensen, C. M., and Diamond, D. L. (2005) Analysis of prostate cancer by proteomics using tissue specimens.J. Urol. 173, 73–78.

    Article  PubMed  CAS  Google Scholar 

  27. Gravett, M. G., Novy, M. J., Rosenfeld, R. G., et al (2004) Diagnosis of intra-amniotic infection by proteomic profiling and identification of novel biomarkers.JAMA 292, 462–469.

    Article  PubMed  CAS  Google Scholar 

  28. Mazzulli, T., Low, D. E., and Poutanen, S. M. (2005) Proteomics and severe acute respiratory syndrome (SARS): emerging technology meets emerging pathogen.Clin. Chem. 51, 6–7.

    Article  PubMed  CAS  Google Scholar 

  29. Barzaghi, D., Isbister, J. D., Lauer, K. P., and Born, T. L. (2004) Use of surface-enhanced laser desorption/ionization-time of flight to explore bacterial proteomes.Proteomics 4, 2624–2628.

    Article  PubMed  CAS  Google Scholar 

  30. Mukhopadhyay, S., Clark, A. P., Sullivan, E. D., Miller, R. D., and Summersgill, J. T. (2004b) Detailed protocol for purification ofChlamydia pneumoniae elementary bodies.J. Clin. Microbiol. 42, 3288–3290.

    Article  PubMed  Google Scholar 

  31. Hendrick, J. P. and Hartl, F. U. (1993) Molecular chaperone functions of heat-shock proteins.Annu. Rev. Biochem. 62, 349–384.

    Article  PubMed  CAS  Google Scholar 

  32. Gelinas, A. D., Toth, J., Bethoney, K. A., Langsetmo, K., Stafford, W. F., and Harrison, C. J. (2003) Thermodynamic linkage in the GrpE nucleotide exchange factor, a molecular thermosensor.Biochemistry 42, 9050–9059.

    Article  PubMed  CAS  Google Scholar 

  33. Ohta, T., Okuda, S., and Takahashi, H. (1977). Relationship between phospholipid compositions and transport activities of amino acids in Escherichia coli membrane vesicles.Biochem. Biophys. Acta 466, 44–56.

    Article  PubMed  CAS  Google Scholar 

  34. Hawrot, E. and Kennedy, E. P. (1975) Biogenesis of membrane lipids: mutants of Escherichia coli with temperature-sensitive phosphatidylserine decarboxylase.Proc. Natl. Acad. Sci. USA 72, 1112–1116.

    Article  PubMed  CAS  Google Scholar 

  35. Langley, K. E., Yaffe, M. P., and Kennedy, E. P. (1979) Biosynthesi of phospholipids inBacillus megaterium.J. Bacteriol. 140, 996–1007.

    PubMed  CAS  Google Scholar 

  36. Dodt, J., Kohler, S., and Baici, A. (1988) Interaction of site-specific hirudin variants with alpha-thrombin.FEBS Lett. 229, 87–90.

    Article  PubMed  CAS  Google Scholar 

  37. Nakashima, T., Higa, H. Matsubara, H., Benson, A. M., and Yasunobu, K. T. (1966) The amino acid sequence of bovine heart cytochrome c.J. Biol., Chem. 241, 1166–1177.

    CAS  Google Scholar 

  38. Bowen, W. J. (1949). The absorption spectra and extinction coefficients of myoglobin.J. Biol. Chem. 179, 235–245.

    CAS  Google Scholar 

  39. Deutsch, H. F. (1987) Carbonic anhydrases.Int. J. Biochem. 19, 101–113.

    Article  PubMed  CAS  Google Scholar 

  40. Chin, C. C., Brewer, J. M., and World, F. (1981) The amino acid sequence of yeast enolase.J. Biol. Chem. 256, 1377–1384.

    PubMed  CAS  Google Scholar 

  41. Hirayama, K., Akashi, S., Furuya, M., and Fukuhara, K. (1990) Rapid confirmation and revision of the primary structure of bovine serum albumin by ESIMS and Frit-FAB LC/MS.Biochem. Biophys. Res. Commun. 173, 639–646.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Division of Infectious Diseases, Department of Medicine, University of Louisville School of Medicine, Louisville, KY

    Sanghamitra Mukhopadhyay, Erin D. Sullivan & James T. Summersgill

  2. Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY

    Saeed A. Jortani, Roland Valdes & Christine Q. Simmons

  3. Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY

    Richard D. Miller & James T. Summersgill

  4. Biological Division Directorate, Naval Medical Research Center, Rockville, MD

    James T. Summersgill

Authors
  1. Sanghamitra Mukhopadhyay
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  2. Saeed A. Jortani
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  3. Roland Valdes
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  4. Christine Q. Simmons
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  5. Erin D. Sullivan
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  6. Richard D. Miller
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Correspondence to James T. Summersgill.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Mukhopadhyay, S., Jortani, S.A., Valdes, R. et al. Protein expression analysis ofChlamydia pneumoniae persistence by combined surface-enhanced laser desorption ionization time-of-flight mass spectrometry and two-dimensional polyacrylamide gel electrophoresis. Clin Proteom 2, 205–215 (2006). https://doi.org/10.1007/BF02752501

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Clinical Proteomics

ISSN: 1559-0275