1887

Abstract

is the dominant pathogen causing chronic lung infections in patients with cystic fibrosis (CF). After an initial phase characterized by intermittent colonizations, a chronic infection is established upon conversion of from the non-mucoid to the mucoid, alginate-overproducing phenotype. During the chronic infection the isolation of both mucoid and non-mucoid isolates in CF sputum samples is very common. The purpose of the present study was to establish, by sequence analysis, the types of mutations present in the operon in a large number of mucoid and non-mucoid isolates from Scandinavian CF patients and in -derived non-mucoid revertants. Mucoid (83) and non-mucoid isolates (103) from 91 Scandinavian patients with chronic infection and 24 non-mucoid isolates from intermittently colonized CF patients were investigated. In addition, 88 spontaneous non-mucoid revertants obtained from nine mucoid CF isolates were also included in the study. Mutations in were found in 92 % of the mucoid and in up to 70 % of the non-mucoid isolates from chronically infected patients, indicating that the majority of non-mucoid isolates are revertants. None of the non-mucoid isolates from intermittently colonized CF patients harboured mutations. Although has been considered an important gene for secondary-site mutations responsible for reversion to non-mucoidy, only 30 % of the -mutated non-mucoid CF isolates had mutations in . In contrast, 83 % of the -derived spontaneous non-mucoid revertants had mutations in , showing that in the CF lung there is a selection for non-mucoid revertants with secondary-site mutations in genes other than . In addition, we report, to our knowledge for the first time, loss-of-function mutations in the negative regulators and in CF clinical isolates. In some of the CF isolates these mutations are associated with moderate alginate production. In conclusion, most non-mucoid isolates from chronically infected CF patients are revertants and the mechanism of revertance is -independent in the CF lung.

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2008-01-01
2024-04-24
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References

  1. Anthony M., Rose B., Pegler M. B., Elkins M., Service H., Thamotharampillai K., Watson J., Robinson M., Bye P. other authors 2002; Genetic analysis of Pseudomonas aeruginosa isolates from the sputa of Australian adult cystic fibrosis patients. J Clin Microbiol 40:2772–2778
    [Google Scholar]
  2. Bagge N., Schuster M., Hentzer M., Ciofu O., Givskov M., Greenberg E. P., Høiby N. 2004; Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production. Antimicrob Agents Chemother 48:1175–1187
    [Google Scholar]
  3. Bayer A. S., Eftekhar F., Tu J., Nast C. C., Speert D. P. 1990; Oxygen-dependent up-regulation of mucoid exopolysaccharide (alginate) production in Pseudomonas aeruginosa . Infect Immun 58:1344–1349
    [Google Scholar]
  4. Baynham P. J., Brown A. L., Hall L. L., Wozniak D. J. 1999; Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation. Mol Microbiol 33:1069–1080
    [Google Scholar]
  5. Berry A., DeVault J. D., Chakrabarty A. M. 1989; High osmolarity is a signal for enhanced algD transcription in mucoid and nonmucoid Pseudomonas aeruginosa strains. J Bacteriol 171:2312–2317
    [Google Scholar]
  6. Boucher J. C., Martinez-Salazar J., Schurr M. J., Mudd M. H., Yu H., Deretic V. 1996; Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA. J Bacteriol 178:511–523
    [Google Scholar]
  7. Boucher J. C., Schurr M. J., Yu H., Rowen D. W., Deretic V. 1997a; Pseudomonas aeruginosa in cystic fibrosis: role of mucC in the regulation of alginate production and stress sensitivity. Microbiology 143:3473–3480
    [Google Scholar]
  8. Boucher J. C., Yu H., Mudd M. H., Deretic V. 1997b; Mucoid Pseudomonas aeruginosa in cystic fibrosis: characterization of muc mutations in clinical isolates and analysis of clearance in a mouse model of respiratory infection. Infect Immun 65:3838–3846
    [Google Scholar]
  9. Bragonzi A., Wiehlmann L., Klockgether J., Cramer N., Worlitzsch D., Doring G., Tummler B. 2006; Sequence diversity of the mucABD locus in Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Microbiology 152:3261–3269
    [Google Scholar]
  10. Deretic V., Dikshit R., Konyecsni W. M., Chakrabarty A. M., Misra T. K. 1989; The algR gene, which regulates mucoidy in Pseudomonas aeruginosa , belongs to a class of environmentally responsive genes. J Bacteriol 171:1278–1283
    [Google Scholar]
  11. DeVault J. D., Kimbara K., Chakrabarty A. M. 1990; Pulmonary dehydration and infection in cystic fibrosis: evidence that ethanol activates alginate gene expression and induction of mucoidy in Pseudomonas aeruginosa . Mol Microbiol 4:737–745
    [Google Scholar]
  12. DeVries C. A., Ohman D. E. 1994; Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT , encoding a putative alternate sigma factor, and shows evidence for autoregulation. J Bacteriol 176:6677–6687
    [Google Scholar]
  13. Doggett R. G. 1969; Incidence of mucoid Pseudomonas aeruginosa from clinical sources. Appl Microbiol 18:936–937
    [Google Scholar]
  14. Doggett R. G., Harrison G. M., Stillwell R. N., Wallis E. S. 1966; An atypical Pseudomonas aeruginosa associated with cystic fibrosis of the pancreas. J Pediatr 68:215–221
    [Google Scholar]
  15. Drenkard E., Ausubel F. M. 2002; Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416:740–743
    [Google Scholar]
  16. Evans L. R., Linker A. 1973; Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa . J Bacteriol 116:915–924
    [Google Scholar]
  17. Firoved A. M., Boucher J. C., Deretic V. 2002; Global genomic analysis of AlgU ( σ E)-dependent promoters (sigmulon) in Pseudomonas aeruginosa and implications for inflammatory processes in cystic fibrosis. J Bacteriol 184:1057–1064
    [Google Scholar]
  18. Fluge G., Ojeniyi B., Høiby N., Digranes A., Ciofu O., Hunstad E., Haanaes O. C., Storrosten O. T. 2001; Typing of Pseudomonas aeruginosa strains in Norwegian cystic fibrosis patients. Clin Microbiol Infect 7:238–243
    [Google Scholar]
  19. Frederiksen B., Koch C., Høiby N. 1997; Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis. Pediatr Pulmonol 23:330–335
    [Google Scholar]
  20. Fyfe J. A., Govan J. R. 1980; Alginate synthesis in mucoid Pseudomonas aeruginosa : a chromosomal locus involved in control. J Gen Microbiol 119:443–450
    [Google Scholar]
  21. Goldberg J. B., Dahnke T. 1992; Pseudomonas aeruginosa AlgB, which modulates the expression of alginate, is a member of the NtrC subclass of prokaryotic regulators. Mol Microbiol 6:59–66
    [Google Scholar]
  22. Goldberg J. B., Gorman W. L., Flynn J. L., Ohman D. E. 1993; A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species. J Bacteriol 175:1303–1308
    [Google Scholar]
  23. Govan J. R., Deretic V. 1996; Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia . Microbiol Rev 60:539–574
    [Google Scholar]
  24. Govan J. R., Fyfe J. A., McMillan C. 1979; The instability of mucoid Pseudomonas aeruginosa : fluctuation test and improved stability of the mucoid form in shaken culture. J Gen Microbiol 110:229–232
    [Google Scholar]
  25. Govan J. R., Fyfe J. A., Baker N. R. 1983; Heterogeneity and reduction in pulmonary clearance of mucoid Pseudomonas aeruginosa . Rev Infect Dis 5 :Suppl. 5S874–S879
    [Google Scholar]
  26. Haussler S. 2004; Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa . Environ Microbiol 6:546–551
    [Google Scholar]
  27. Hoffmann N., Rasmussen T. B., Jensen P. O., Stub C., Hentzer M., Molin S., Ciofu O., Givskov M., Johansen H. K., Høiby N. 2005; Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis. Infect Immun 73:2504–2514
    [Google Scholar]
  28. Høiby N. 1975; Prevalence of mucoid strains of Pseudomonas aeruginosa in bacteriological specimens from patients with cystic fibrosis and patients with other diseases. Acta Pathol Microbiol Scand Suppl 83:549–552
    [Google Scholar]
  29. Høiby N. 1977; Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis. A survey. Acta Pathol Microbiol Scand Suppl 262:1–96
    [Google Scholar]
  30. Høiby N., Giwercman B., Jensen E. T., Johansen H. K., Kronborg G., Pressler T., Kharazmi A. 1993; Immune response in cystic fibrosis – helpful or harmful?. In 18th European CF Conference pp 133–139 Edited by Escobar H. Baquero F., Suarez. Madrid: Elsevier;
    [Google Scholar]
  31. Høiby N., Frederiksen B., Pressler T. 2005; Eradication of early Pseudomonas aeruginosa infection. J Cyst Fibros 4 :Suppl. 249–54
    [Google Scholar]
  32. Jelsbak L., Johansen H. K., Frost A. L., Thøgersen R., Thomsen L. E., Ciofu O., Yang L., Haagensen J. A., Høiby N., Molin S. 2007; Molecular epidemiology and dynamics of Pseudomonas aeruginosa populations in lungs of cystic fibrosis patients. Infect Immun 75:2214–2224
    [Google Scholar]
  33. Kessler B., de Lorenzo V., Timmis K. N. 1992; A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet 233:293–301
    [Google Scholar]
  34. Knutson C. A., Jeanes A. 1968; A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem 24:470–481
    [Google Scholar]
  35. Lee B., Haagensen J. A., Ciofu O., Andersen J. B., Høiby N., Molin S. 2005; Heterogeneity of biofilms formed by nonmucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J Clin Microbiol 43:5247–5255
    [Google Scholar]
  36. Martin D. W., Schurr M. J., Mudd M. H., Deretic V. 1993a; Differentiation of Pseudomonas aeruginosa into the alginate-producing form: inactivation of mucB causes conversion to mucoidy. Mol Microbiol 9:497–506
    [Google Scholar]
  37. Martin D. W., Schurr M. J., Mudd M. H., Govan J. R., Holloway B. W., Deretic V. 1993b; Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients. Proc Natl Acad Sci U S A 90:8377–8381
    [Google Scholar]
  38. Martin D. W., Schurr M. J., Yu H., Deretic V. 1994; Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa : relationship to σ E and stress response. J Bacteriol 176:6688–6696
    [Google Scholar]
  39. Mathee K., McPherson C. J., Ohman D. E. 1997; Posttranslational control of the algT ( algU )-encoded σ 22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN). J Bacteriol 179:3711–3720
    [Google Scholar]
  40. Mathee K., Ciofu O., Sternberg C., Lindum P. W., Campbell J. L., Jensen P., Johnsen A. H., Givskov M., Ohman D. E. other authors 1999; Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145:1349–1357
    [Google Scholar]
  41. Nikolskaya A. N., Galperin M. Y. 2002; A novel type of conserved DNA-binding domain in the transcriptional regulators of the AlgR/AgrA/LytR family. Nucleic Acids Res 30:2453–2459
    [Google Scholar]
  42. Ohman D. E. 1981; Genetic mapping of chromosomal determinants for the production of exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun 33:142–148
    [Google Scholar]
  43. Ojeniyi B., Høiby N., Rosdahl V. T. 1991; Genome fingerprinting as a typing method used on polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients. APMIS 99:492–498
    [Google Scholar]
  44. Ojeniyi B., Petersen U. S., Høiby N. 1993; Comparison of genome fingerprinting with conventional typing methods used on Pseudomonas aeruginosa isolates from cystic fibrosis patients. APMIS 101:168–175
    [Google Scholar]
  45. Palma M., DeLuca D., Worgall S., Quadri L. E. 2004; Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide. J Bacteriol 186:248–252
    [Google Scholar]
  46. Pedersen S. S. 1992; Lung infection with alginate-producing, mucoid Pseudomonas aeruginosa in cystic fibrosis. APMIS Suppl 28:1–79
    [Google Scholar]
  47. Pedersen S. S., Espersen F., Høiby N., Shand G. H. 1989; Purification, characterization, and immunological cross-reactivity of alginates produced by mucoid Pseudomonas aeruginosa from patients with cystic fibrosis. J Clin Microbiol 27:691–699
    [Google Scholar]
  48. Pugashetti B. K., Metzger H. M. Jr, Vadas L., Feingold D. S. 1982; Phenotypic differences among clinically isolated mucoid Pseudomonas aeruginosa strains. J Clin Microbiol 16:686–691
    [Google Scholar]
  49. Römling U., Tümmler B. 2000; Achieving 100 % typeability of Pseudomonas aeruginosa by pulsed-field gel electrophoresis. J Clin Microbiol 38:464–465
    [Google Scholar]
  50. Rowen D. W., Deretic V. 2000; Membrane-to-cytosol redistribution of ECF sigma factor AlgU and conversion to mucoidy in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Mol Microbiol 36:314–327
    [Google Scholar]
  51. Schurr M. J., Martin D. W., Mudd M. H., Deretic V. 1994; Gene cluster controlling conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa : functional analysis in a heterologous host and role in the instability of mucoidy. J Bacteriol 176:3375–3382
    [Google Scholar]
  52. Schurr M. J., Yu H., Martinez-Salazar J. M., Boucher J. C., Deretic V. 1996; Control of AlgU, a member of the σ E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis. J Bacteriol 178:4997–5004
    [Google Scholar]
  53. Speert D. P., Farmer S. W., Campbell M. E., Musser J. M., Selander R. K., Kuo S. 1990; Conversion of Pseudomonas aeruginosa to the phenotype characteristic of strains from patients with cystic fibrosis. J Clin Microbiol 28:188–194
    [Google Scholar]
  54. Tart A. H., Blanks M. J., Wozniak D. J. 2006; The AlgT-dependent transcriptional regulator AmrZ (AlgZ) inhibits flagellum biosynthesis in mucoid, nonmotile Pseudomonas aeruginosa cystic fibrosis isolates. J Bacteriol 188:6483–6489
    [Google Scholar]
  55. Tenover F. C., Arbeit R. D., Goering R. V., Mickelsen P. A., Murray B. E., Persing D. H., Swaminathan B. 1995; Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233–2239
    [Google Scholar]
  56. Terry J. M., Pina S. E., Mattingly S. J. 1991; Environmental conditions which influence mucoid conversion Pseudomonas aeruginosa PAO1. Infect Immun 59:471–477
    [Google Scholar]
  57. Wood L. F., Ohman D. E. 2006; Independent regulation of MucD, an HtrA-like protease in Pseudomonas aeruginosa , and the role of its proteolytic motif in alginate gene regulation. J Bacteriol 188:3134–3137
    [Google Scholar]
  58. Wood L. F., Leech A. J., Ohman D. E. 2006; Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa : roles of sigma (AlgT) and the AlgW and Prc proteases. Mol Microbiol 62:412–426
    [Google Scholar]
  59. Wozniak D. J., Ohman D. E. 1991; Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription. J Bacteriol 173:1406–1413
    [Google Scholar]
  60. Wyckoff T. J., Thomas B., Hassett D. J., Wozniak D. J. 2002; Static growth of mucoid Pseudomonas aeruginosa selects for non-mucoid variants that have acquired flagellum-dependent motility. Microbiology 148:3423–3430
    [Google Scholar]
  61. 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
    [Google Scholar]
  62. Yu H., Schurr M. J., Deretic V. 1995; Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E. coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P. aeruginosa . J Bacteriol 177:3259–3268
    [Google Scholar]
  63. Yu H., Boucher J. C., Hibler N. S., Deretic V. 1996; Virulence properties of Pseudomonas aeruginosa lacking the extreme-stress sigma factor AlgU (sigma E). Infect Immun 64:2774–2781
    [Google Scholar]
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