title: Gene expression changes linked to antimicrobial resistance, oxidative stress, iron depletion and retained motility are observed when Burkholderia cenocepacia grows in cystic fibrosis sputum.
abstract: BACKGROUND
  Bacteria from the Burkholderia cepacia complex (Bcc) are the only group of cystic fibrosis (CF) respiratory pathogens that may cause death by an invasive infection known as cepacia syndrome. Their large genome (> 7000 genes) and multiple pathways encoding the same putative functions make virulence factor identification difficult in these bacteria.
  
  
  METHODS
  A novel microarray was designed to the genome of Burkholderia cenocepacia J2315 and transcriptomics used to identify genes that were differentially regulated when the pathogen was grown in a CF sputum-based infection model. Sputum samples from CF individuals infected with the same B. cenocepacia strain as genome isolate were used, hence, other than a dilution into a minimal growth medium (used as the control condition), no further treatment of the sputum was carried out.
  
  
  RESULTS
  A total of 723 coding sequences were significantly altered, with 287 upregulated and 436 downregulated; the microarray-observed expression was validated by quantitative PCR on five selected genes. B. cenocepacia genes with putative functions in antimicrobial resistance, iron uptake, protection against reactive oxygen and nitrogen species, secretion and motility were among the most altered in sputum. Novel upregulated genes included: a transmembrane ferric reductase (BCAL0270) implicated in iron metabolism, a novel protease (BCAL0849) that may play a role in host tissue destruction, an organic hydroperoxide resistance gene (BCAM2753), an oxidoreductase (BCAL1107) and a nitrite/sulfite reductase (BCAM1676) that may play roles in resistance to the host defenses. The assumptions of growth under iron-depletion and oxidative stress formulated from the microarray data were tested and confirmed by independent growth of B. cenocepacia under each respective environmental condition.
  
  
  CONCLUSIONS
  Overall, our first full transcriptomic analysis of B. cenocepacia demonstrated the pathogen alters expression of over 10% of the 7176 genes within its genome when it grows in CF sputum. Novel genetic pathways involved in responses to antimicrobial resistance, oxidative stress, and iron metabolism were revealed by the microarray analysis. Virulence factors such as the cable pilus and Cenocepacia Pathogenicity Island were unaltered in expression. However, B. cenocepacia sustained or increased expression of motility-associated genes in sputum, maintaining a potentially invasive phenotype associated with cepacia syndrome.
authors.affiliation: Cardiff School of Biosciences, Cardiff University, Cardiff, UK. pavel.drevinek@lfmotol.cuni.cz
authors.forename: Pavel
authors.initials: P
authors.lastname: Drevinek
authors.forename: Matthew T G
authors.initials: MT
authors.lastname: Holden
authors.forename: Zhaoping
authors.initials: Z
authors.lastname: Ge
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Jones
authors.forename: Ian
authors.initials: I
authors.lastname: Ketchell
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
authors.forename: Eshwar
authors.initials: E
authors.lastname: Mahenthiralingam
mesh_terms: D000328:Adult; D001426:Bacterial Proteins / Q000235:genetics; D042602:Burkholderia cepacia complex / Q000254:growth & development / Q000502:physiology*; D003550:Cystic Fibrosis; D024881:Drug Resistance, Bacterial; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007501:Iron / Q000378:metabolism; D008124:Locomotion; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013183:Sputum / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D007501:Iron
references.citation: Lancet. 1993 Jul 3;342(8862):15-9
references.pmid: 7686239
references.citation: Antimicrob Agents Chemother. 2004 Apr;48(4):1145-50
references.pmid: 15047514
references.citation: Nat Rev Microbiol. 2004 Oct;2(10):820-32
references.pmid: 15378046
references.citation: Proc Natl Acad Sci U S A. 2004 Sep 28;101(39):14240-5
references.pmid: 15377794
references.citation: Infect Immun. 1993 Aug;61(8):3157-63
references.pmid: 7687582
references.citation: Infect Immun. 1994 Feb;62(2):596-605
references.pmid: 8300217
references.citation: Mol Microbiol. 1993 Nov;10(3):529-44
references.pmid: 7968531
references.citation: J Bacteriol. 1998 May;180(10):2636-43
references.pmid: 9573147
references.citation: J Bacteriol. 1998 Jun;180(11):2936-42
references.pmid: 9603885
references.citation: Am J Respir Crit Care Med. 1999 Sep;160(3):796-801
references.pmid: 10471599
references.citation: Thorax. 2004 Nov;59(11):948-51
references.pmid: 15516469
references.citation: Nat Rev Microbiol. 2005 Feb;3(2):144-56
references.pmid: 15643431
references.citation: J Med Microbiol. 2005 Jul;54(Pt 7):655-9
references.pmid: 15947430
references.citation: J Bacteriol. 2005 Aug;187(15):5267-77
references.pmid: 16030221
references.citation: Infect Immun. 2005 Aug;73(8):4982-92
references.pmid: 16041013
references.citation: J Clin Microbiol. 2005 Sep;43(9):4665-73
references.pmid: 16145124
references.citation: BMC Genomics. 2006;7:72
references.pmid: 16595014
references.citation: J Biol Chem. 2006 Jun 16;281(24):16256-63
references.pmid: 16603772
references.citation: Infect Immun. 2006 Jul;74(7):4083-93
references.pmid: 16790782
references.citation: BMC Microbiol. 2006;6:66
references.pmid: 16857052
references.citation: Environ Microbiol. 2007 Apr;9(4):1017-34
references.pmid: 17359273
references.citation: Biometals. 2007 Jun;20(3-4):431-52
references.pmid: 17295049
references.citation: Future Microbiol. 2007 Apr;2(2):153-64
references.pmid: 17661652
references.citation: Cell. 2007 Sep 7;130(5):797-810
references.pmid: 17803904
references.citation: FEMS Microbiol Lett. 2007 Nov;276(1):83-92
references.pmid: 17937666
references.citation: J Bacteriol. 2000 Apr;182(8):2077-87
references.pmid: 10735848
references.citation: Am J Respir Crit Care Med. 2000 Apr;161(4 Pt 1):1206-12
references.pmid: 10764313
references.citation: Infect Immun. 2000 Dec;68(12):6554-60
references.pmid: 11083764
references.citation: Microbiology. 2001 Jan;147(Pt 1):97-109
references.pmid: 11160804
references.citation: Res Microbiol. 2001 Jan-Feb;152(1):17-26
references.pmid: 11281321
references.citation: Mol Microbiol. 2001 May;40(3):542-54
references.pmid: 11359562
references.citation: Am J Respir Crit Care Med. 2001 Jul 1;164(1):92-6
references.pmid: 11435245
references.citation: FEMS Microbiol Lett. 2001 Aug 21;202(2):157-64
references.pmid: 11520608
references.citation: Clin Infect Dis. 2001 Nov 1;33(9):1469-75
references.pmid: 11588691
references.citation: Infect Immun. 2002 Apr;70(4):1799-806
references.pmid: 11895941
references.citation: J Biol Chem. 2002 Jun 28;277(26):23664-9
references.pmid: 11960983
references.citation: Paediatr Respir Rev. 2002 Sep;3(3):230-5
references.pmid: 12376059
references.citation: Nucleic Acids Res. 2001 May 1;29(9):e45
references.pmid: 11328886
references.citation: EMBO J. 2002 Dec 16;21(24):6649-59
references.pmid: 12485986
references.citation: Res Microbiol. 2003 Mar;154(2):91-6
references.pmid: 12648723
references.citation: Microbiology. 2003 Apr;149(Pt 4):961-71
references.pmid: 12686638
references.citation: Microbiology. 2003 Aug;149(Pt 8):2263-71
references.pmid: 12904566
references.citation: Microbiology. 2003 Nov;149(Pt 11):3051-72
references.pmid: 14600218
references.citation: FEMS Microbiol Lett. 2003 Nov 21;228(2):287-97
references.pmid: 14638436
references.citation: Microbiology. 2003 Dec;149(Pt 12):3649-58
references.pmid: 14663096
references.citation: J Clin Invest. 2004 Feb;113(3):464-73
references.pmid: 14755343
references.citation: Infect Immun. 2004 Mar;72(3):1537-47
references.pmid: 14977960
references.citation: Microbiology. 2004 Mar;150(Pt 3):516; discussion 516-8
references.pmid: 14993297
references.citation: Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6664-8
references.pmid: 15084751