Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometryReportar como inadecuado

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BMC Microbiology

, 14:329

Microbial genetics, genomics and proteomics


BackgroundNon-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 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.

ResultsComparisons 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.

ConclusionsThe 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.

KeywordsNon-typeable Haemophilus influenzae Metabolic labeling Biofilms Mass spectrometry AbbreviationsNTHiNon-typeableHaemophilus influenzae

SILACStable isotope labeling by amino acids in cell culture

LC-MSLiquid chromatography-mass spectrometry

SRM-MSSelected reaction monitoring-mass spectrometry

COPDChronic obstructive pulmonary disease


MALDI-TOFMatrix-assisted laser desorption ionization time-of-flight

ESI-MSElectrospray ionization-mass spectrometry

Electronic supplementary materialThe online version of this article doi:10.1186-s12866-014-0329-9 contains supplementary material, which is available to authorized users.

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Autor: Deborah MB Post - Jason M Held - Margaret R Ketterer - Nancy J Phillips - Alexandria Sahu - Michael A Apicella - Bradfo


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