Pronounced metabolic changes in adaptation to biofilm growth by Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae accounts for a significant global burden of morbidity and mortality and biofilm development is increasingly recognised as important for colonization and infection. Analysis of protein expression patterns during biofilm development may therefore provide valuable insights to the understanding of pneumococcal persistence strategies and to improve vaccines. iTRAQ (isobaric tagging for relative and absolute quantification), a high-throughput gel-free proteomic approach which allows high resolution quantitative comparisons of protein profiles between multiple phenotypes, was used to interrogate planktonic and biofilm growth in a clinical serotype 14 strain. Comparative analyses of protein expression between log-phase planktonic and 1-day and 7-day biofilm cultures representing nascent and late phase biofilm growth were carried out. Overall, 244 proteins were identified, of which >80% were differentially expressed during biofilm development. Quantitatively and qualitatively, metabolic regulation appeared to play a central role in the adaptation from the planktonic to biofilm phenotype. Pneumococci adapted to biofilm growth by decreasing enzymes involved in the glycolytic pathway, as well as proteins involved in translation, transcription, and virulence. In contrast, proteins with a role in pyruvate, carbohydrate, and arginine metabolism were significantly increased during biofilm development. Downregulation of glycolytic and translational proteins suggests that pneumococcus adopts a covert phenotype whilst adapting to an adherent lifestyle, while utilization of alternative metabolic pathways highlights the resourcefulness of pneumococcus to facilitate survival in diverse environmental conditions. These metabolic proteins, conserved across both the planktonic and biofilm phenotypes, may also represent target candidates for future vaccine development and treatment strategies. Data are available via ProteomeXchange with identifier PXD001182.

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).