1887

Abstract

This study utilized suppressive subtractive hybridization between the clinical isolate J2315 and the closely related environmental isolate ATCC 25416 to isolate DNA fragments specific to J2315. Analysis of the resulting pools of -specific DNAs identified several fragments that may be part of putative virulence factors. Further analysis of a single fragment indicated that it was internal to a gene of which the predicted product had characteristics of repeat in toxin (RTX)-like proteins and high similarity to proteins in other human or plant pathogens. In conjunction with this finding, phenotypic traits associated with known RTX proteins were assessed. A haemagglutinating activity of J2315 was identified that was absent in ATCC 25416. The expression of this activity appeared to be growth phase-dependent. Analysis of the gene presence and haemagglutinating activity across the species of the complex showed that both were common to the ET12 lineage of , but were absent in the other species examined. Haemagglutinating activity was limited to isolates with the RTX-like gene. Expression studies utilizing quantitative PCR demonstrated an association between onset of haemagglutinating activity and increased expression of the gene, which suggests that the putative RTX determinant encodes a haemagglutinating activity.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.46138-0
2006-01-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/jmm/55/1/11.html?itemId=/content/journal/jmm/10.1099/jmm.0.46138-0&mimeType=html&fmt=ahah

References

  1. Adderson E. E., Takahashi S., Wang Y., Armstrong J., Miller D. V., Bohnsack J. F. 2003; Subtractive hybridization identifies a novel predicted protein mediating epithelial cell invasion by virulent serotype III group B Streptococcus agalactiae . Infect Immun 71:6857–6863 [CrossRef]
    [Google Scholar]
  2. Agron P. G., Macht M., Radnedge L., Skowronski E. W., Miller W., Andersen G. L. 2002; Use of subtractive hybridization for comprehensive surveys of prokaryotic genome differences. FEMS Microbiol Lett 211:175–182 [CrossRef]
    [Google Scholar]
  3. Akopyants N. S., Fradkov A., Diatchenko L., Hill J. E., Siebert P. D., Lukyanov S. A., Sverdlov E. D., Berg D. E. 1998; PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori . Proc Natl Acad Sci U S A 95:13108–13113 [CrossRef]
    [Google Scholar]
  4. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410 [CrossRef]
    [Google Scholar]
  5. Ames T. R., Markham R. J., Opuda-Asibo J., Leininger J. R., Maheswaran S. K. 1985; Pulmonary response to intratracheal challenge with Pasteurella haemolytica and Pasteurella multocida . Can J Comp Med 49:395–400
    [Google Scholar]
  6. Bateman A., Birney E., Cerruti L. & 7 other authors; 2002; The Pfam protein families database. Nucleic Acids Res 30:276–280 [CrossRef]
    [Google Scholar]
  7. Bernier S. P., Silo-Suh L., Woods D. E., Ohman D. E., Sokol P. A. 2003; Comparative analysis of plant and animal models for characterization of Burkholderia cepacia virulence. Infect Immun 71:5306–5313 [CrossRef]
    [Google Scholar]
  8. Berriman M., Rutherford K. 2003; Viewing and annotating sequence data with Artemis. Brief Bioinform 4:124–132 [CrossRef]
    [Google Scholar]
  9. Betsou F., Sebo P., Guiso N. 1995; The C-terminal domain is essential for protective activity of the Bordetella pertussis adenylate cyclase-hemolysin. Infect Immun 63:3309–3315
    [Google Scholar]
  10. Chidambaram M., Sharma B., Petras S. F., Reese C. P., Froshauer S., Weinstock G. M. 1995; Isolation of Pasteurella haemolytica leukotoxin mutants. Infect Immun 63:1027–1032
    [Google Scholar]
  11. Choi J. Y., Sifri C. D., Goumnerov B. C., Rahme L. G., Ausubel F. M., Calderwood S. B. 2002; Identification of virulence genes in a pathogenic strain of Pseudomonas aeruginosa by representational difference analysis. J Bacteriol 184:952–961 [CrossRef]
    [Google Scholar]
  12. Cieri M. V., Mayer-Hamblett N., Griffith A., Burns J. L. 2002; Correlation between an in vitro invasion assay and a murine model of Burkholderia cepacia lung infection. Infect Immun 70:1081–1086 [CrossRef]
    [Google Scholar]
  13. Coenye T., Vandamme P., Govan J. R. W., LiPuma J. J. 2001; Taxonomy and identification of the Burkholderia cepacia complex. J Clin Microbiol 39:3427–3436 [CrossRef]
    [Google Scholar]
  14. Coenye T., Vandamme P., LiPuma J. J., Govan J. R. W., Mahenthiralingam E. 2003; Updated version of the Burkholderia cepacia complex experimental strain panel. J Clin Microbiol 41:2797–2798 [CrossRef]
    [Google Scholar]
  15. Czuprynski C. J., Noel E. J., Ortiz-Carranza O., Srikumaran S. 1991; Activation of bovine neutrophils by partially purified Pasteurella haemolytica leukotoxin. Infect Immun 59:3126–3133
    [Google Scholar]
  16. Daborn P. J., Waterfield N., Blight M. A., Ffrench-Constant R. H. 2001; Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection. J Bacteriol 183:5834–5839 [CrossRef]
    [Google Scholar]
  17. Fehlner-Gardiner C. C., Hopkins T. M.-H., Valvano M. A. 2002; Identification of a general secretory pathway in a human isolate of Burkholderia vietnamiensis (formerly B. cepacia complex genomovar V) that is required for the secretion of hemolysin and phospholipase C activities. Microb Pathog 32:249–254 [CrossRef]
    [Google Scholar]
  18. Fullner K. J., Lencer W. I., Mekalanos J. J. 2001; Vibrio cholerae -induced cellular responses of polarized T84 intestinal epithelial cells are dependent on production of cholera toxin and the RTX toxin. Infect Immun 69:6310–6317 [CrossRef]
    [Google Scholar]
  19. Fullner K. J., Boucher J. C., Hanes M. A., Haines G. K. III, Meehan B. M., Walchle C., Sansonetti P. J., Mekalanos J. J. 2002; The contribution of accessory toxins of Vibrio cholerae O1 El Tor to the proinflammatory response in a murine pulmonary cholera model. J Exp Med 195:1455–1462 [CrossRef]
    [Google Scholar]
  20. Govan J. R. W., Brown P. H., Maddison J., Doherty C. J., Nelson J. W., Dodd M., Greening A. P., Webb A. K. 1993; Evidence for transmission of Pseudomonas cepacia by social contact in cystic fibrosis. Lancet 342:15–19 [CrossRef]
    [Google Scholar]
  21. Highlander S. K., Fedorova N. D., Dusek D. M., Panciera R., Alvarez L. E., Rinehart C. 2000; Inactivation of Pasteurella ( Mannheimia ) haemolytica leukotoxin causes partial attenuation of virulence in a calf challenge model. Infect Immun 68:3916–3922 [CrossRef]
    [Google Scholar]
  22. Keig P. M., Ingham E., Vandamme P. A. R., Kerr K. G. 2002; Differential invasion of respiratory epithelial cells by members of the Burkholderia cepacia complex. Clin Microbiol Infect 8:47–49 [CrossRef]
    [Google Scholar]
  23. Koronakis V. 2003; TolC – the bacterial exit duct for proteins and drugs. FEBS Lett 555:66–71 [CrossRef]
    [Google Scholar]
  24. Köthe M., Antl M., Huber B., Stoecker K., Ebrecht D., Steinmetz I., Eberl L. 2003; Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system. Cell Microbiol 5:343–351 [CrossRef]
    [Google Scholar]
  25. Lessie T. G., Hendrickson W., Manning B. D., Devereux R. 1996; Genomic complexity and plasticity of Burkholderia cepacia . FEMS Microbiol Lett 144:117–128 [CrossRef]
    [Google Scholar]
  26. Lewenza S., Sokol P. A. 2001; Regulation of ornibactin biosynthesis and N -acyl-l-homoserine lactone production by CepR in Burkholderia cepacia . J Bacteriol 183:2212–2218 [CrossRef]
    [Google Scholar]
  27. Li M.-S., Farrant J. L., Langford P. R., Kroll J. S. 2003; Identification and characterization of genomic loci unique to the Brazilian purpuric fever clonal group of H. influenzae biogroup aegyptius: functionality explored using meningococcal homology. Mol Microbiol 47:1101–1111 [CrossRef]
    [Google Scholar]
  28. Lin W., Fullner K. J., Clayton R., Sexton J. A., Rogers M. B., Calia K. E., Calderwood S. B., Fraser C., Mekalanos J. J. 1999; Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc Natl Acad Sci U S A 96:1071–1076 [CrossRef]
    [Google Scholar]
  29. LiPuma J. J., Dulaney B. J., McMenamin J. D., Whitby P. W., Stull T. L., Coenye T., Vandamme P. 1999; Development of rRNA-based PCR assays for identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patients. J Clin Microbiol 37:3167–3170
    [Google Scholar]
  30. LiPuma J. J., Spilker T., Gill L. H., Campbell P. W. III, Liu L., Mahenthiralingam E. 2001; Disproportionate distribution of Burkholderia cepacia complex species and transmissibility markers in cystic fibrosis. Am J Respir Crit Care Med 164:92–96 [CrossRef]
    [Google Scholar]
  31. Livak K. J., Schmittgen T. D. 2001; Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ C T method. Methods 25:402–408 [CrossRef]
    [Google Scholar]
  32. Mahenthiralingam E., Coenye T., Chung J. W., Speert D. P., Govan J. R. W., Taylor P., Vandamme P. 2000; Diagnostically and experimentally useful panel of strains from the Burkholderia cepacia complex. J Clin Microbiol 38:910–913
    [Google Scholar]
  33. Mahenthiralingam E., Baldwin A., Vandamme P. 2002; Burkholderia cepacia complex infection in patients with cystic fibrosis. J Med Microbiol 51:533–538
    [Google Scholar]
  34. Maheswaran S. K., Weiss D. J., Kannan M. S., Townsend E. L., Reddy K. R., Whiteley L. O., Srikumaran S. 1992; Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals. Vet Immunol Immunopathol 33:51–68 [CrossRef]
    [Google Scholar]
  35. Martin D. W., Mohr C. D. 2000; Invasion and intracellular survival of Burkholderia cepacia . Infect Immun 68:24–29 [CrossRef]
    [Google Scholar]
  36. Miller D. A., Mahenthiralingam E. 2003; Sequencing of the Pseudomonas aeruginosa and Burkholderia cepacia genomes and their applications in relation to cystic fibrosis. J R Soc Med 96 (Suppl. 43):57–65
    [Google Scholar]
  37. Morrow B. J., Graham J. E., Curtiss R. III 1999; Genomic subtractive hybridization and selective capture of transcribed sequences identify a novel Salmonella typhimurium fimbrial operon and putative transcriptional regulator that are absent from the Salmonella typhi genome. Infect Immun 67:5106–5116
    [Google Scholar]
  38. Moxon R., Tang C. 2000; Challenge of investigating biologically relevant functions of virulence factors in bacterial pathogens. Philos Trans R Soc Lond B Biol Sci 355:643–656 [CrossRef]
    [Google Scholar]
  39. Oelschlaeger T. A., Dobrindt U., Hacker J. 2002; Virulence factors of uropathogens. Curr Opin Urol 12:33–38 [CrossRef]
    [Google Scholar]
  40. Pitt T. L., Kaufmann M. E., Patel P. S., Benge L. C., Gaskin S., Livermore D. M. 1996; Type characterisation and antibiotic susceptibility of Burkholderia ( Pseudomonas ) cepacia isolates from patients with cystic fibrosis in the United Kingdom and the Republic of Ireland. J Med Microbiol 44:203–210 [CrossRef]
    [Google Scholar]
  41. Rowe G. E., Welch R. A. 1994; Assays of hemolytic toxins. Methods Enzymol 235:657–667
    [Google Scholar]
  42. Rutherford K., Parkhill J., Crook J., Horsnell T., Rice P., Rajandream M.-A., Barrell B. 2000; Artemis: sequence visualization and annotation. Bioinformatics 16:944–945 [CrossRef]
    [Google Scholar]
  43. Saini L. S., Galsworthy S. B., John M. A., Valvano M. A. 1999; Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation. Microbiology 145:3465–3475
    [Google Scholar]
  44. Sajjan U. S., Corey M., Karmali M. A., Forstner J. F. 1992; Binding of Pseudomonas cepacia to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis. J Clin Invest 89:648–656 [CrossRef]
    [Google Scholar]
  45. Sajjan U., Wu Y., Kent G., Forstner J. 2000; Preferential adherence of cable-piliated Burkholderia cepacia to respiratory epithelia of CF knockout mice and human cystic fibrosis lung explants. J Med Microbiol 49:875–885
    [Google Scholar]
  46. Sambrook J., Russell D. 2001 Molecular Cloning: a Laboratory Manual , 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  47. Seah J. N., Frey J., Kwang J. 2002; The N-terminal domain of RTX toxin ApxI of Actinobacillus pleuropneumoniae elicits protective immunity in mice. Infect Immun 70:6464–6467 [CrossRef]
    [Google Scholar]
  48. Sharma R. C., Schimke R. T. 1996; Preparation of electrocompetent E. coli using salt-free growth medium. Biotechniques 20:42–44
    [Google Scholar]
  49. Speert D. P. 2002; Advances in Burkholderia cepacia complex. Paediatr Respir Rev 3:230–235 [CrossRef]
    [Google Scholar]
  50. Speert D. P., Steen B., Halsey K., Kwan E. 1999; A murine model for infection with Burkholderia cepacia with sustained persistence in the spleen. Infect Immun 67:4027–4032
    [Google Scholar]
  51. Speert D. P., Henry D., Vandamme P., Corey M., Mahenthiralingam E. 2002; Epidemiology of Burkholderia cepacia complex in patients with cystic fibrosis, Canada. Emerg Infect Dis 8:181–187 [CrossRef]
    [Google Scholar]
  52. Srinivasan U., Foxman B., Marrs C. F. 2003; Identification of a gene encoding heat-resistant agglutinin in Escherichia coli as a putative virulence factor in urinary tract infection. J Clin Microbiol 41:285–289 [CrossRef]
    [Google Scholar]
  53. Tatum F. M., Briggs R. E., Sreevatsan S. S., Zehr E. S., Ling Hsuan S., Whiteley L. O., Ames T. R., Maheswaran S. K. 1998; Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence. Microb Pathog 24:37–46 [CrossRef]
    [Google Scholar]
  54. Tomich M., Mohr C. D. 2003; Adherence and autoaggregation phenotypes of a Burkholderia cenocepacia cable pilus mutant. FEMS Microbiol Lett 228:287–297 [CrossRef]
    [Google Scholar]
  55. Tomich M., Mohr C. D. 2004; Transcriptional and posttranscriptional control of cable pilus gene expression in Burkholderia cenocepacia . J Bacteriol 186:1009–1020 [CrossRef]
    [Google Scholar]
  56. Welch R. A. 2001; RTX toxin structure and function: a story of numerous anomalies and few analogies in toxin biology. Curr Top Microbiol Immunol 257:85–111
    [Google Scholar]
  57. Whitby P. W., Carter K. B., Hatter K. L., LiPuma J. J., Stull T. L. 2000; Identification of members of the Burkholderia cepacia complex by species-specific PCR. J Clin Microbiol 38:2962–2965
    [Google Scholar]
  58. Winstanley C. 2002; Spot the difference: applications of subtractive hybridisation to the study of bacterial pathogens. J Med Microbiol 51:459–467
    [Google Scholar]
  59. Yoo H. S., Rajagopal B. S., Maheswaran S. K., Ames T. R. 1995; Purified Pasteurella haemolytica leukotoxin induces expression of inflammatory cytokines from bovine alveolar macrophages. Microb Pathog 18:237–252 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.46138-0
Loading
/content/journal/jmm/10.1099/jmm.0.46138-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error