title: Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry.
abstract: BACKGROUND
  Non-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either "light" (L) isoleucine or "heavy" (H) (13)C6-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence.
  
  
  RESULTS
  Comparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A.
  
  
  CONCLUSIONS
  The present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival.
authors.affiliation: The Buck Institute for Research on Aging, Novato, CA, 94945, USA. dpost@buckinstitute.org.
authors.forename: Deborah M B
authors.initials: DM
authors.lastname: Post
authors.affiliation: Washington University School of Medicine, St. Louis, MO, 63110, USA. jheld@DOM.wustl.edu.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Held
authors.affiliation: The University of Iowa, Iowa City, IA, 52242, USA. margaret-ketterer@uiowa.edu.
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Ketterer
authors.affiliation: The University of California San Francisco, San Francisco, CA, 94143, USA. Nancy.Phillips@ucsf.edu.
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Phillips
authors.affiliation: The Buck Institute for Research on Aging, Novato, CA, 94945, USA. asahu@buckinstitute.org.
authors.forename: Alexandria
authors.initials: A
authors.lastname: Sahu
authors.affiliation: The University of Iowa, Iowa City, IA, 52242, USA. michael-apicella@uiowa.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Apicella
authors.affiliation: The Buck Institute for Research on Aging, Novato, CA, 94945, USA. bgibson@buckinstitute.org.|The University of California San Francisco, San Francisco, CA, 94143, USA. bgibson@buckinstitute.org.
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D018441:Biofilms / Q000254:growth & development*; D002853:Chromatography, Liquid; D006193:Haemophilus influenzae / Q000737:chemistry* / Q000502:physiology*; D007553:Isotope Labeling; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*
chemical_list: D001426:Bacterial Proteins; D020543:Proteome