Figure 1.
S. pneumoniae serotype 14 day 1, 3, 5 and 7 biofilms imaged using (a) scanning electron microscopy with Alcian Blue staining (8,000x magnification; scale bar: 5 µm), and (b) confocal microscopy using Live/Dead staining, with sideviews (YZ – right) and (XZ – bottom) sagittal sections of the biofilm.
Scale bar beside YZ sagittal sections represents average biofilm thickness. Scale bar in xy pane: 30 µm. (c) Bar chart comparing the total number of cells between day 1, 3, 5 and 7 biofilms. (d) Bar chart comparing number of viable cells between day 1, 3, 5 and 7 biofilms through CFU cm−2 measurement.
Figure 2.
Volcano plot of the complete iTRAQ proteomic dataset comparing the fold change in protein expression between mid-exponential planktonic S. pneumoniae and day 1 and day 7 biofilms.
Proteins represented met inclusion criteria of ≥3 peptide matches, ≥50 protein score, and ≥5% sequence coverage (p<0.05). Comparative protein data with >1.3 and <0.77 ratios (marked by horizontal lines) were identified as having differential expression.
Figure 3.
Comparative iTRAQ analyses of S. pneumoniae serotype 14 1-day and 7-day old in vitro biofilms to mid-exponential planktonic population protein expression.
Inclusion criteria: ≥3 peptide matches, ≥50 protein score, ≥5% sequence coverage (p<0.05). Comparative protein data with >1.3 and <0.77 ratios identified as having differential expression.
Figure 4.
Qualitative data listing proteins identified with 2 peptide matches using comparative iTRAQ analysis of S. pneumoniae serotype 14 1-day and 7-day old in vitro biofilms to planktonic population protein expression.
Comparative protein data with >1.3 and <0.77 ratios identified as having differential expression.
Figure 5.
Change in expression of enzymes involved in the glycolysis/gluconeogenesis pathway and pyruvate metabolism of S. pneumoniae serotype 14 during biofilm formation.
Based on iTRAQ expression data comparing mid-exponential planktonic cultures to day 1 and 7 biofilms. Proteins with potential moonlighting capability highlighted with their putative alternative functions. Pgm - phosphoglucomutase; pgi – glucose-6-phosphate isomerase; pfk–6-phosphofructokinase; fba – fructose biphosphate aldolase; gapA – glyceraldehyde-3-phosphate dehydrogenase; pgk – phosphoglycerate kinase; gpmA – phosphoglyceromutase; eno – enolase; pyk – pyruvate kinase, ldh – lactate dehydrogenase; adh – alcohol dehydrogenase, pfl – formate acetyltransferase.
Figure 6.
Change in expression of enzymes involved in amino-acid metabolism of day 7 S. pneumoniae serotype 14 biofilms.
Based on iTRAQ expression data comparing mid-exponential planktonic cultures to day 7 biofilms. Highlighting the arginine deiminase (ADI) pathway consisting of arginine deiminase (arcA), ornithine carbamoyltransferase (arcB) and carbamate kinase (arcC), and components of L-aspartate metabolism consisting of adenylosuccinate synthetase (purA), aspartate aminotransferase (aspC), asparagine synthetase (asnA), and glutamate dehydrogenase (gdhA).