title: A proteomic characterization of NTHi lysates.
abstract: BACKGROUND
  Non-typeable Haemophilus influenzae (NTHi) is a ubiquitous bacterial pathogen which accounts for a majority of human upper respiratory tract infections. Laboratory lysate preparations from this bacterium are commonly utilized to investigate the promulgation of inflammatory responses in respiratory and middle ear epithelium both in vivo and in vitro. We undertook an unbiased proteomics based analysis of NTHi lysate preps to: (a) identify abundant bacterial proteins present in these lysates that could play a role in NTHi biological effects and (b) determine the protein content variability in different lysate prep batches from the same NTHI strain.
  
  
  METHODS
  Proteomic analysis of laboratory NTHi lysate preparations from clinical strain 12.
  
  
  METHODS
  NTHi lysates were denatured, gel-fractionated, digested by trypsin and the generated peptides were identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS). Western blot analyses for the important proinflammatory enhancer, outer membrane protein 6 (OMP6), was performed to validate the MS findings. Luciferase assays for NF-kB activation were used to measure the pro-inflammatory biologic effects from each NTHi lysate preparation.
  
  
  RESULTS
  The MS identified 793 unique NTHi proteins. Most common and abundant proteins found have been described to either contribute to biofilm formation, elude the innate immune system, or activate epithelial pro-inflammatory pathways such as Toll Like Receptor 2 (TLR-2) signaling and NF-kB transcription factor. Strong positive signal for OMP6 was found in each of the NTHi lysate preparations. Significant NF-kB promoter response activation as expected with NTHi stimulation over control was also noted for each NTHi lysate preparation.
  
  
  CONCLUSIONS
  Proteomics was a successful technique to broadly define the protein content of NTHi lysates. This is the first report of the proteome of NTHi lysates widely used in laboratories to study the biological effect of NTHi. Despite the variability of the protein composition from different preps, all the batches of NTHi lysates induced similar NFκB activation.
  
  
  METHODS
  NA.
authors.affiliation: Sheihk Zayed Institute, Washington, DC, United States; Division of Pediatric Otolaryngology-Head and Neck Surgery, Washington, DC, United States. Electronic address: dpreciad@cnmc.org.
authors.forename: Diego
authors.initials: D
authors.lastname: Preciado
authors.affiliation: Sheihk Zayed Institute, Washington, DC, United States; Division of Pediatric Otolaryngology-Head and Neck Surgery, Washington, DC, United States; Center for Genetic Medicine at Children's National Medical Center, Washington, DC, United States. Electronic address: mpoley@cnmc.org.
authors.forename: Marian
authors.initials: M
authors.lastname: Poley
authors.affiliation: Sheihk Zayed Institute, Washington, DC, United States. Electronic address: st8@princeton.edu.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Tsai
authors.affiliation: Sheihk Zayed Institute, Washington, DC, United States; Division of Pediatric Otolaryngology-Head and Neck Surgery, Washington, DC, United States; Center for Genetic Medicine at Children's National Medical Center, Washington, DC, United States. Electronic address: amarel.saieg@gmail.com.
authors.forename: Amarel
authors.initials: A
authors.lastname: Tomney
authors.affiliation: Center for Genetic Medicine at Children's National Medical Center, Washington, DC, United States. Electronic address: kristy.brown@cnmc.org.
authors.forename: Kristy
authors.initials: K
authors.lastname: Brown
authors.affiliation: Sheihk Zayed Institute, Washington, DC, United States. Electronic address: sval@cnmc.org.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Val
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D006193:Haemophilus influenzae / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D051195:Toll-Like Receptor 2 / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D016328:NF-kappa B; D020543:Proteome; D051195:Toll-Like Receptor 2
keywords: NF-kappaB; NTHi lysates; OMP6; Proteomics