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Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry.

Post DM, Held JM, Ketterer MR, Phillips NJ, Sahu A, Apicella MA, Gibson BW - BMC Microbiol. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Buck Institute for Research on Aging, Novato, CA, 94945, USA. dpost@buckinstitute.org.

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.

No MeSH data available.


Related in: MedlinePlus

Log scale plots of the peptide ratios of the Biofilm:Planktonic (B:P) data observed in the SRM-MS analyses. Each dot represents the average of the B:P ratio for a distinct peptide. The line shows the overall average of the B:P data for each protein. Proteins whose average ratio showed downregulation in the biofilm appear in the grey shaded space of the plot. Proteins whose average ratio showed upregulation in the biofilm are plotted in the non-shaded area of the graph.
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Fig4: Log scale plots of the peptide ratios of the Biofilm:Planktonic (B:P) data observed in the SRM-MS analyses. Each dot represents the average of the B:P ratio for a distinct peptide. The line shows the overall average of the B:P data for each protein. Proteins whose average ratio showed downregulation in the biofilm appear in the grey shaded space of the plot. Proteins whose average ratio showed upregulation in the biofilm are plotted in the non-shaded area of the graph.

Mentions: To obtain better sensitivity and accuracy in the relative quantitation of some of the more important proteins identified in the global SILAC analysis, SRM-MS was performed. Proteins were selected from the list of differentially expressed proteins observed in the ESI-MS/MS analyses; additionally proteins of potential biological significance were also targeted in these analyses. Peptides observed from this set of proteins were refined to include only peptides which contained no methionine or cysteine residues, no missed cleavages, no N-terminal glutamine or glutamic acid, and no adjacent tryptic sites. Nineteen proteins with at least one peptide which met the above criteria were analyzed by SRM-MS to examine whether the biofilm and planktonic samples were differentially expressed. Data for both the “heavy” and “light” populations was collected for 54 of the targeted 61 peptides (Additional file 15). Figure 4, Table 2, and Additional file 16 show the data from 10 of the 19 proteins that we obtained consistent quantitative results to those observed in our initial Protein Pilot analysis of the MS/MS data. The 9 remaining proteins had data that was either inconclusive or was inconsistent with our initial MS/MS dataset and thus were not confirmed by SRM analyses. Of the 10 proteins that were confirmed by SRM analyses, four showed downregulation and six showed upregulation in the biofilm samples compared to the planktonic samples, with all six of the upregulated proteins showing an average peptide ratio of >1.5 and one of the downregulated proteins showing an average peptide ratio of <0.66. The cysteinyl-tRNA synthetase protein, aerobic respiration control protein ArcA, the predicted regulator of cell mophogenesis and NO signaling protein, and the molybdate-binding periplasmic protein were all seen as downregulated in the biofilm. The NAD nucleotidase protein, the heme-binding protein A, the glutamine synthetase protein, the protective surface antigen D15 protein, the probable acyl carrier protein phosphodiesterase, and the DNA gyrase protein subunit A were all found by SRM to be upregulated in the biofilm. One to five distinct peptides were used to target each protein in our multiplexed SRM-MS assays (Additional file 16). Of all of the proteins quantified by SRM, the aerobic respiration control protein ArcA showed the highest level of downregulation in the biofilm and the NAD nucleotidase protein and the heme-binding protein A showed the highest levels of upregulation in the biofilm.Figure 4


Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry.

Post DM, Held JM, Ketterer MR, Phillips NJ, Sahu A, Apicella MA, Gibson BW - BMC Microbiol. (2014)

Log scale plots of the peptide ratios of the Biofilm:Planktonic (B:P) data observed in the SRM-MS analyses. Each dot represents the average of the B:P ratio for a distinct peptide. The line shows the overall average of the B:P data for each protein. Proteins whose average ratio showed downregulation in the biofilm appear in the grey shaded space of the plot. Proteins whose average ratio showed upregulation in the biofilm are plotted in the non-shaded area of the graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302520&req=5

Fig4: Log scale plots of the peptide ratios of the Biofilm:Planktonic (B:P) data observed in the SRM-MS analyses. Each dot represents the average of the B:P ratio for a distinct peptide. The line shows the overall average of the B:P data for each protein. Proteins whose average ratio showed downregulation in the biofilm appear in the grey shaded space of the plot. Proteins whose average ratio showed upregulation in the biofilm are plotted in the non-shaded area of the graph.
Mentions: To obtain better sensitivity and accuracy in the relative quantitation of some of the more important proteins identified in the global SILAC analysis, SRM-MS was performed. Proteins were selected from the list of differentially expressed proteins observed in the ESI-MS/MS analyses; additionally proteins of potential biological significance were also targeted in these analyses. Peptides observed from this set of proteins were refined to include only peptides which contained no methionine or cysteine residues, no missed cleavages, no N-terminal glutamine or glutamic acid, and no adjacent tryptic sites. Nineteen proteins with at least one peptide which met the above criteria were analyzed by SRM-MS to examine whether the biofilm and planktonic samples were differentially expressed. Data for both the “heavy” and “light” populations was collected for 54 of the targeted 61 peptides (Additional file 15). Figure 4, Table 2, and Additional file 16 show the data from 10 of the 19 proteins that we obtained consistent quantitative results to those observed in our initial Protein Pilot analysis of the MS/MS data. The 9 remaining proteins had data that was either inconclusive or was inconsistent with our initial MS/MS dataset and thus were not confirmed by SRM analyses. Of the 10 proteins that were confirmed by SRM analyses, four showed downregulation and six showed upregulation in the biofilm samples compared to the planktonic samples, with all six of the upregulated proteins showing an average peptide ratio of >1.5 and one of the downregulated proteins showing an average peptide ratio of <0.66. The cysteinyl-tRNA synthetase protein, aerobic respiration control protein ArcA, the predicted regulator of cell mophogenesis and NO signaling protein, and the molybdate-binding periplasmic protein were all seen as downregulated in the biofilm. The NAD nucleotidase protein, the heme-binding protein A, the glutamine synthetase protein, the protective surface antigen D15 protein, the probable acyl carrier protein phosphodiesterase, and the DNA gyrase protein subunit A were all found by SRM to be upregulated in the biofilm. One to five distinct peptides were used to target each protein in our multiplexed SRM-MS assays (Additional file 16). Of all of the proteins quantified by SRM, the aerobic respiration control protein ArcA showed the highest level of downregulation in the biofilm and the NAD nucleotidase protein and the heme-binding protein A showed the highest levels of upregulation in the biofilm.Figure 4

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Buck Institute for Research on Aging, Novato, CA, 94945, USA. dpost@buckinstitute.org.

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.

No MeSH data available.


Related in: MedlinePlus