1887

Abstract

Isolates of from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography–tandem mass spectrometry (μLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels. Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain. The proteins were identified by μLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.

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2000-10-01
2020-04-02
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References

  1. Bell A., Bains M., Hancock R. E.. 1991; Pseudomonas aeruginosa outer membrane protein OprH: expression from the cloned gene and function in EDTA and gentamicin resistance. J Bacteriol173:6657–6664
    [Google Scholar]
  2. Bini L., Sanchez-Campillo M., Santucci A..9 other authors 1996; Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting. Electrophoresis17:185–190[CrossRef]
    [Google Scholar]
  3. Brown S. M., Howell M. L., Vasil M. L., Anderson A. J., Hassett D. J.. 1995; Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J Bacteriol177:6536–6544
    [Google Scholar]
  4. Chu L., May T. B., Chakrabarty A. M., Misra T. K.. 1991; Nucleotide sequence and expression of the algE gene involved in alginate biosynthesis by Pseudomonas aeruginosa. Gene107:1–10[CrossRef]
    [Google Scholar]
  5. Cordwell S. J., Nouwens A. S., Verrills N. M., McPherson J. C., Hains P. G., Van Dyk D. D., Walsh B. D.. 1999; The microbial proteome database – an automated laboratory catalogue for monitoring protein expression in bacteria. . Electrophoresis20:3580–3588[CrossRef]
    [Google Scholar]
  6. Dean C. R., Franklund C. V., Retief J. D., Coyne J. M. J., Hatano K., Evans D. J., Pier G. B., Goldberg J. B.. 1999; Characterization of the serogroup O11 O-antigen locus of Pseudomonas aeruginosa PA103. J Bacteriol181:4275–4284
    [Google Scholar]
  7. Elkins J. G., Hassett D. J., Stewart P. S., Schweizer H. P., McDermott T. R.. 1999; Protective role of catalase in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide. Appl Environ Microbiol65:4594–4600
    [Google Scholar]
  8. Ernst R. K., Yi E. C., Guo L., Lim K. B., Burns J. L., Hackett M., Miller S. I.. 1999; Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science286:1561–1565[CrossRef]
    [Google Scholar]
  9. Folders J., Tommassen J., Van Loon L. C., Bitter W.. 2000; Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa. J Bacteriol182:1257–1263[CrossRef]
    [Google Scholar]
  10. Goldberg J. B., Ohman D. E.. 1987; Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate. J Bacteriol169:1593–1602
    [Google Scholar]
  11. Govan J. R. W., Deretic V.. 1996; Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev60:539–574
    [Google Scholar]
  12. Grabert E., Wingender J., Winkler U. K.. 1990; An outer membrane protein characteristic of mucoid strains of Pseudomonas aeruginosa. FEMS Microbiol Lett68:83–88[CrossRef]
    [Google Scholar]
  13. Hancock R. E. W., Nikaido H.. 1978; Outer membranes of Gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J Bacteriol136:381–390
    [Google Scholar]
  14. Hancock R. E. W., Mutharia L. M., Chan L., Darveau R. P., Speert D. P., Pier G. B.. 1983; Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains. Infect Immun42:170–177
    [Google Scholar]
  15. Hancock R., Siehnel R., Martin N.. 1990; Outer membrane proteins of Pseudomonas. Mol Microbiol4:1069–1075[CrossRef]
    [Google Scholar]
  16. Hassett D. J., Woodruff W. A., Wozniak D. J., Vasil M. L., Cohen M. S., Ohman D. E.. 1993; Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria. J Bacteriol175:7658–7665
    [Google Scholar]
  17. Hassett D. J., Schweizer H. P., Ohman D. E.. 1995; Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism. J Bacteriol177:6330–6337
    [Google Scholar]
  18. Holloway B. W., Römling U., Tümmler B.. 1994; Genomic mapping of Pseudomonas aeruginosa PAO. . Microbiology140:2907–2929[CrossRef]
    [Google Scholar]
  19. Kelly N. M., MacDonald M. H., Martin N., Nicas T., Hancock R. E. W.. 1990; Comparion of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa. . J Clin Microbiol28:2017–2021
    [Google Scholar]
  20. Kersulyte D., Struelens M. J., Deplano A., Berg D. E.. 1995; Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients. J Clin Microbiol33:2216–2219
    [Google Scholar]
  21. Kropinski A. M., Chan L. C., Milazzo F. H.. 1979; The extraction and analysis of lipopolysaccharides from Pseudomonas aeruginosa strain PAO, and three rough mutants. Can J Microbiol25:390–398[CrossRef]
    [Google Scholar]
  22. McAtee C. P., Lim M. Y., Fung K., Velligan M., Fry K., Chow T., Berg D. E.. 1998; Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by two-dimensional gel electrophoresis, sequence analysis, and serum profiling. Clin Diagn Lab Immunol5:537–542
    [Google Scholar]
  23. Mahenthiralingam E., Campbell M. E., Foster J., Lam J. S., Speert D. P.. 1996; Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol34:1129–1135
    [Google Scholar]
  24. Maher W. E., Kobe M., Fass R. J.. 1993; Restriction endonuclease analysis of clinical Pseudomonas aeruginosa strains: useful epidemiologic data from a simple and rapid method. J Clin Microbiol31:1426–1429
    [Google Scholar]
  25. Mandal A., Naaby-Hansen S., Wolkowicz M. J..9 other authors 1999; FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa. Biol Reprod61:1184–1197[CrossRef]
    [Google Scholar]
  26. Martin N. L., Rawling E. G., Wong R. S. Y., Rosok M., Hancock R. E. W.. 1993; Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF. FEMS Microbiol Lett113:261–266[CrossRef]
    [Google Scholar]
  27. Mathee K., Ciofu O., Sternberg C..9 other authors 1999; Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology145:1349–1357[CrossRef]
    [Google Scholar]
  28. Mehta A., Kinter M. T., Sherman N. E., Driscoll D. M.. 2000; Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA. Mol Cell Biol20:1846–1854[CrossRef]
    [Google Scholar]
  29. Ohman D. E., Chakrabarty A. M.. 1982; Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin. Infect Immun37:662–669
    [Google Scholar]
  30. Pier G. B.. 1998; Pseudomonas aeruginosa: a key problem in cystic fibrosis. Am Soc Microbiol News64:339–347
    [Google Scholar]
  31. Pollack M.. 2000; Pseudomonas aeruginosa. In Principles and Practice of Infectious Diseases pp.2310–2335Edited by Mandell G. L., Bennett J. E., Dolin R.. Philadelphia: Churchill Livingstone;
    [Google Scholar]
  32. Preston M. J., Fleiszig S. M. J., Zaidi T. S., Goldberg J. B., Shortridge V. D., Vasil M. L., Pier G. B.. 1995; Rapid and sensitive method for evaluating Pseudomonas aeruginosa virulence factors during corneal infections in mice. Infect Immun63:3497–3501
    [Google Scholar]
  33. Quadroni M., James P., Dainese-Hatt P., Kertesz M. A.. 1999; Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation-induced response in Pseudomonas aeruginosa PAO1. Eur J Biochem266:986–996[CrossRef]
    [Google Scholar]
  34. Rawling E. G., Brinkman F. S. L., Hancock R. E. W.. 1998; Roles of the carboxy-terminal half of Pseudomonas aeruginosa major outer membrane protein OprF in cell shape, growth in low-osmolarity medium, and peptidoglycan association. J Bacteriol180:3556–3562
    [Google Scholar]
  35. Rehm B. H. A., Grabert E., Hein J., Winkler U. K.. 1994; Antibody response of rabbits and cystic fibosis patients to an alginate-specific outer membrane protein of a mucoid strain of Pseudomonas aeruginosa. . Microb Pathog16:43–51[CrossRef]
    [Google Scholar]
  36. Römling U., Grothues D., Koopman U., Jahnke B., Greipel J., Tümmler B.. 1992; Pulsed-field gel electrophoresis analysis of a Pseudomonas aeruginosa pathovar. Electrophoresis13:646–648[CrossRef]
    [Google Scholar]
  37. Schmidt K. D., Tümmler B., Römling U.. 1996; Comparative genome mapping of Pseudomonas aeruginosa PAO with P. aeruginosa C, which belongs to a major clone in cystic fibrosis patients and aquatic habitats. J Bacteriol178:85–93
    [Google Scholar]
  38. Speert D., Campbell M., Puterman M. L..11 other authors 1994; A multicenter comparison of methods for typing strains of Pseudomonas aeruginosa predominantly from patients with cystic fibrosis. J Infect Dis169:134–142[CrossRef]
    [Google Scholar]
  39. Sprott G. C., Koval S. F., Schnaitman C. A.. 1994; Cell fractionation. In Methods for General and Molecular Bacteriology pp.73–103Edited by Gerhardt P., Murray R. G. E., Wood W. A., Krieg N. R.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  40. Tomb J., White O., Kerlavage A. R..39 other authors 1997; The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature388:539–548[CrossRef]
    [Google Scholar]
  41. Vandamme P., Mahenthiralingam E., Holmes B., Coenye T., Hoste B., De Vos P., Henry D., Speert D. P.. 2000; Identification and population structure of Burkholderia stabilis sp. nov. (formerly Burkholderia cepacia genomovar IV). J Clin Microbiol38:1042–1047
    [Google Scholar]
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