1887

Abstract

airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Various models have been developed to study pathobiology in the CF lung. In this study we produced a modified artificial-sputum medium (ASMDM) more closely resembling CF sputum than previous models, and extended previous work by using strain PAO1 arrays to examine the global transcription profiles of strain UCBPP-PA14 under early exponential-phase and stationary-phase growth. In early exponential phase, 38/39 nutrition-related genes were upregulated in line with data from previous models using UCBPP-PA14. Additionally, 23 type III secretion system (T3SS) genes, several anaerobic respiration genes and 24 quorum-sensing (QS)-related genes were upregulated in ASMDM, suggesting enhanced virulence factor expression and priming for anaerobic growth and biofilm formation. Under stationary phase growth in ASMDM, macroscopic clumps resembling microcolonies were evident in UCBPP-PA14 and CF strains, and over 40 potentially important genes were differentially expressed relative to stationary-phase growth in Luria broth. Most notably, QS-related and T3SS genes were downregulated in ASMDM, and iron-acquisition and assimilatory nitrate reductase genes were upregulated, simulating the iron-depleted, microaerophilic/anaerobic environment of CF sputum. ASMDM thus appears to be highly suitable for gene expression studies of in CF.

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2010-09-01
2019-12-06
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References

  1. Alvarez-Ortega, C. & Harwood, C. S. ( 2007; ). Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration. Mol Microbiol 65, 153–165.[CrossRef]
    [Google Scholar]
  2. Barken, K. B., Pamp, S. J., Yang, L., Gjermansen, M., Bertrand, J. J., Klausen, M., Givskov, M., Whitchurch, C. B., Engel, J. N. & Tolker-Nielsen, T. ( 2008; ). Roles of type IV pili, flagellum-mediated motility and extracellular DNA in the formation of mature multicellular structures in Pseudomonas aeruginosa biofilms. Environ Microbiol 10, 2331–2343.[CrossRef]
    [Google Scholar]
  3. Beatson, S. A., Whitchurch, C. B., Sargent, J. L., Levesque, R. C. & Mattick, J. S. ( 2002; ). Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 184, 3605–3613.[CrossRef]
    [Google Scholar]
  4. Benjamini, Y. & Hochberg, Y. ( 1995; ). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 57, 289–300.
    [Google Scholar]
  5. Berthelot, P., Attree, I., Plesiat, P., Chabert, J., de Bentzmann, S., Pozzetto, B. & Grattard, F. ( 2003; ). Genotypic and phenotypic analysis of type III secretion system in a cohort of Pseudomonas aeruginosa bacteremia isolates: evidence for a possible association between O serotypes and exo genes. J Infect Dis 188, 512–518.[CrossRef]
    [Google Scholar]
  6. Bjarnsholt, T., Jensen, P. O., Fiandaca, M. J., Pedersen, J., Hansen, C. R., Andersen, C. B., Pressler, T., Givskov, M. & Hoiby, N. ( 2009; ). Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr Pulmonol 44, 547–558.[CrossRef]
    [Google Scholar]
  7. Bolstad, B. M., Irizarry, R. A., Astrand, M. & Speed, T. P. ( 2003; ). A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193.[CrossRef]
    [Google Scholar]
  8. Brandt, T., Breitenstein, S., von der Hardt, H. & Tummler, B. ( 1995; ). DNA concentration and length in sputum of patients with cystic fibrosis during inhalation with recombinant human DNase. Thorax 50, 880–882.[CrossRef]
    [Google Scholar]
  9. Brint, J. M. & Ohman, D. E. ( 1995; ). Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR–RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR–LuxI family. J Bacteriol 177, 7155–7163.
    [Google Scholar]
  10. Davey, M. E., Caiazza, N. C. & O'Toole, G. A. ( 2003; ). Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 185, 1027–1036.[CrossRef]
    [Google Scholar]
  11. Dean, C. R., Neshat, S. & Poole, K. ( 1996; ). PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa. J Bacteriol 178, 5361–5369.
    [Google Scholar]
  12. De Kievit, T. R., Gillis, R., Marx, S., Brown, C. & Iglewski, B. H. ( 2001; ). Quorum-sensing genes in Pseudomonas aeruginosa biofilms: their role and expression patterns. Appl Environ Microbiol 67, 1865–1873.[CrossRef]
    [Google Scholar]
  13. Garcia-Medina, R., Dunne, W. M., Singh, P. K. & Brody, S. L. ( 2005; ). Pseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells. Infect Immun 73, 8298–8305.[CrossRef]
    [Google Scholar]
  14. Gautier, L., Cope, L., Bolstad, B. M. & Irizarry, R. A. ( 2004; ). affy – Analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 307–315.[CrossRef]
    [Google Scholar]
  15. Gentleman, R. C., Carey, V. J., Bates, D. M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y. & other authors ( 2004; ). Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5, R80 [CrossRef]
    [Google Scholar]
  16. Gupta, R., Gobble, T. R. & Schuster, M. ( 2009; ). GidA posttranscriptionally regulates rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 191, 5785–5792.[CrossRef]
    [Google Scholar]
  17. Hassett, D. J., Cuppoletti, J., Trapnell, B., Lymar, S. V., Rowe, J. J., Yoon, S. S., Hilliard, G. M., Parvatiyar, K., Kamani, M. C. & other authors ( 2002; ). Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets. Adv Drug Deliv Rev 54, 1425–1443.[CrossRef]
    [Google Scholar]
  18. Hassett, D. J., Sutton, M. D., Schurr, M. J., Herr, A. B., Caldwell, C. C. & Matu, J. O. ( 2009; ). Pseudomonas aeruginosa hypoxic or anaerobic biofilm infections within cystic fibrosis airways. Trends Microbiol 17, 130–138.[CrossRef]
    [Google Scholar]
  19. Henke, M. O., John, G., Germann, M., Lindemann, H. & Rubin, B. K. ( 2007; ). MUC5AC and MUC5B mucins increase in cystic fibrosis airway secretions during pulmonary exacerbation. Am J Respir Crit Care Med 175, 816–821.[CrossRef]
    [Google Scholar]
  20. Juhas, M., Wiehlmann, L., Salunkhe, P., Lauber, J., Buer, J. & Tummler, B. ( 2005; ). GeneChip expression analysis of the VqsR regulon of Pseudomonas aeruginosa TB. FEMS Microbiol Lett 242, 287–295.[CrossRef]
    [Google Scholar]
  21. Klausen, M., Heydorn, A., Ragas, P., Lambertsen, L., Aaes-Jorgensen, A., Molin, S. & Tolker-Nielsen, T. ( 2003; ). Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol 48, 1511–1524.[CrossRef]
    [Google Scholar]
  22. Landry, R. M., An, D., Hupp, J. T., Singh, P. K. & Parsek, M. R. ( 2006; ). Mucin–Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance. Mol Microbiol 59, 142–151.[CrossRef]
    [Google Scholar]
  23. Lequette, Y. & Greenberg, E. P. ( 2005; ). Timing and localization of rhamnolipid synthesis gene expression in Pseudomonas aeruginosa biofilms. J Bacteriol 187, 37–44.[CrossRef]
    [Google Scholar]
  24. Lin, H. H., Huang, S. P., Teng, H. C., Ji, D. D., Chen, Y. S. & Chen, Y. L. ( 2006; ). Presence of the exoU gene of Pseudomonas aeruginosa is correlated with cytotoxicity in MDCK cells but not with colonization in BALB/c mice. J Clin Microbiol 44, 4596–4597.[CrossRef]
    [Google Scholar]
  25. Manos, J., Arthur, J., Rose, B., Tingpej, P., Fung, C., Curtis, M., Webb, J. S., Hu, H., Kjelleberg, S. & other authors ( 2008; ). Transcriptome analyses and biofilm-forming characteristics of a clonal Pseudomonas aeruginosa from the cystic fibrosis lung. J Med Microbiol 57, 1454–1465.[CrossRef]
    [Google Scholar]
  26. Manos, J., Arthur, J., Rose, B., Bell, S., Tingpej, P., Hu, H., Webb, J., Kjelleberg, S., Gorrell, M. D. & other authors ( 2009; ). Gene expression characteristics of a cystic fibrosis epidemic strain of Pseudomonas aeruginosa during biofilm and planktonic growth. FEMS Microbiol Lett 292, 107–114.[CrossRef]
    [Google Scholar]
  27. Palmer, K. L., Mashburn, L. M., Singh, P. K. & Whiteley, M. ( 2005; ). Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology. J Bacteriol 187, 5267–5277.[CrossRef]
    [Google Scholar]
  28. Palmer, K. L., Aye, L. M. & Whiteley, M. ( 2007a; ). Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum. J Bacteriol 189, 8079–8087.[CrossRef]
    [Google Scholar]
  29. Palmer, K. L., Brown, S. A. & Whiteley, M. ( 2007b; ). Membrane-bound nitrate reductase is required for anaerobic growth in cystic fibrosis sputum. J Bacteriol 189, 4449–4455.[CrossRef]
    [Google Scholar]
  30. Rahme, L. G., Stevens, E. J., Wolfort, S. F., Shao, J., Tompkins, R. G. & Ausubel, F. M. ( 1995; ). Common virulence factors for bacterial pathogenicity in plants and animals. Science 268, 1899–1902.[CrossRef]
    [Google Scholar]
  31. Reid, D. W., Lam, Q. T., Schneider, H. & Walters, E. H. ( 2004; ). Airway iron and iron-regulatory cytokines in cystic fibrosis. Eur Respir J 24, 286–291.[CrossRef]
    [Google Scholar]
  32. Roy-Burman, A., Savel, R. H., Racine, S., Swanson, B. L., Revadigar, N. S., Fujimoto, J., Sawa, T., Frank, D. W. & Wiener-Kronish, J. P. ( 2001; ). Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J Infect Dis 183, 1767–1774.[CrossRef]
    [Google Scholar]
  33. Sagel, S. D., Kapsner, R., Osberg, I., Sontag, M. K. & Accurso, F. J. ( 2001; ). Airway inflammation in children with cystic fibrosis and healthy children assessed by sputum induction. Am J Respir Crit Care Med 164, 1425–1431.[CrossRef]
    [Google Scholar]
  34. Sarkisova, S., Patrauchan, M. A., Berglund, D., Nivens, D. E. & Franklin, M. J. ( 2005; ). Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. J Bacteriol 187, 4327–4337.[CrossRef]
    [Google Scholar]
  35. Schreiber, K., Krieger, R., Benkert, B., Eschbach, M., Arai, H., Schobert, M. & Jahn, D. ( 2007; ). The anaerobic regulatory network required for Pseudomonas aeruginosa nitrate respiration. J Bacteriol 189, 4310–4314.[CrossRef]
    [Google Scholar]
  36. Schuster, M., Lostroh, C. P., Ogi, T. & Greenberg, E. P. ( 2003; ). Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185, 2066–2079.[CrossRef]
    [Google Scholar]
  37. Shen, D. K., Filopon, D., Chaker, H., Boullanger, S., Derouazi, M., Polack, B. & Toussaint, B. ( 2008; ). High-cell-density regulation of the Pseudomonas aeruginosa type III secretion system: implications for tryptophan catabolites. Microbiology 154, 2195–2208.[CrossRef]
    [Google Scholar]
  38. Shrout, J. D., Chopp, D. L., Just, C. L., Hentzer, M., Givskov, M. & Parsek, M. R. ( 2006; ). The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Mol Microbiol 62, 1264–1277.[CrossRef]
    [Google Scholar]
  39. Singh, P. K., Schaefer, A. L., Parsek, M. R., Moninger, T. O., Welsh, M. J. & Greenberg, E. P. ( 2000; ). Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407, 762–764.[CrossRef]
    [Google Scholar]
  40. Smyth, G. ( 2003; ). Statistical issues in cDNA microarray data analysis. In Functional Genomics: Methods and Protocols, pp. 111–136. Edited by M. J. Brownstein & A. B. Khodursky. NJ: Juman Press.
  41. Sriramulu, D. D., Lunsdorf, H., Lam, J. S. & Romling, U. ( 2005; ). Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung. J Med Microbiol 54, 667–676.[CrossRef]
    [Google Scholar]
  42. Tetz, G. V., Artemenko, N. K. & Tetz, V. V. ( 2009; ). Effect of DNase and antibiotics on biofilm characteristics. Antimicrob Agents Chemother 53, 1204–1209.[CrossRef]
    [Google Scholar]
  43. Van Alst, N. E., Wellington, M., Clark, V. L., Haidaris, C. G. & Iglewski, B. H. ( 2009; ). Nitrite reductase NirS is required for type III secretion system expression and virulence in the human monocyte cell line THP-1 by Pseudomonas aeruginosa. Infect Immun 77, 4446–4454.[CrossRef]
    [Google Scholar]
  44. Wagner, V. E., Bushnell, D., Passador, L., Brooks, A. I. & Iglewski, B. H. ( 2003; ). Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J Bacteriol 185, 2080–2095.[CrossRef]
    [Google Scholar]
  45. Waite, R. D., Papakonstantinopoulou, A., Littler, E. & Curtis, M. A. ( 2005; ). Transcriptome analysis of Pseudomonas aeruginosa growth: comparison of gene expression in planktonic cultures and developing and mature biofilms. J Bacteriol 187, 6571–6576.[CrossRef]
    [Google Scholar]
  46. Wang, J., Lory, S., Ramphal, R. & Jin, S. ( 1996; ). Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients. Mol Microbiol 22, 1005–1012.[CrossRef]
    [Google Scholar]
  47. Worlitzsch, D., Tarran, R., Ulrich, M., Schwab, U., Cekici, A., Meyer, K. C., Birrer, P., Bellon, G., Berger, J. & other authors ( 2002; ). Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J Clin Invest 109, 317–325.[CrossRef]
    [Google Scholar]
  48. Yoon, S. S., Coakley, R., Lau, G. W., Lymar, S. V., Gaston, B., Karabulut, A. C., Hennigan, R. F., Hwang, S. H., Buettner, G. & other authors ( 2006; ). Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions. J Clin Invest 116, 436–446.[CrossRef]
    [Google Scholar]
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