title: The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP.
abstract: The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels.
  
  
  OBJECTIVE
  Our work suggests that cross talk between c-di-GMP and cAMP signaling pathways results in downregulation of acute virulence factors in P. aeruginosa biofilm infections. Knowledge about this cross-regulation adds to our understanding of virulence traits and immune evasion by P. aeruginosa in chronic infections and may provide new approaches to eradicate biofilm infections.
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Almblad
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, AB, Canada.
authors.forename: Joe J
authors.initials: JJ
authors.lastname: Harrison
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Rybtke
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Groizeleau
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore.
authors.forename: Michael
authors.initials: M
authors.lastname: Givskov
authors.affiliation: Department of Microbiology, University of Washington, Seattle, Washington, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Parsek
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark ttn@sund.ku.dk.
authors.forename: Tim
authors.initials: T
authors.lastname: Tolker-Nielsen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; C091090:Vfr protein, Pseudomonas aeruginosa; C062025:bis(3',5')-cyclic diguanylic acid; D000242:Cyclic AMP; D006152:Cyclic GMP