Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Abstract

Streptococcus mutans -derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ΔlytS and ΔlytT; LTA – ΔdltA and ΔdltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.

Extracellular matrix influence in Streptococcus mutans gene expression in a cariogenic biofilm

“…In addition, GtfC synthesizes both soluble and insoluble glucans, and the deletion of the gtfB gene dramatically affected the magnitude of gtfC expression; the decrease in fold for gtfC ranged from 4.9 to more than 300 (depending on the developmental phase, the highest difference being at 43 hours, and the lowest at 29 hours; Table ), reflecting the lower quantification of ASP and WSP in Δ gtfB biofilms (vs UA159 biofilms; Figure ). In fact, the difference between the amounts ASP and WSP that exist between the two biofilm types agreed with the difference in the expression of this gene group and also with the confocal microscopy images, proving that biofilms formed by the strain Δ gtfB had a simpler topography and a limited ECM three‐dimensional architecture (Figure ), as observed in previous studies using distinct growth conditions …”

“…Moreover, cell density can regulate gene expression patterns. The populations of S. mutans , S. gordonii and A. naeslundii during the formation of mixed‐species biofilms by UA159 and Δ gtfB strains were verified previously by plating biofilm suspensions at 29, 43, 53, 67, 77, 91, 101 and 115 hours (Figure ). There were variations in the proportions of the three species, with S. mutans being higher after 67 hours, and both S. mutans UA159 and Δ gtfB outcompeted A. naeslundii but not S. gordonii at 115 hours.…”

“…The ASP quantities were distinct for UA159 and Δ gtfB biofilms, specifically, lower amounts were observed in the Δ gtfB biofilm at 67 and 115 hours (Figure ). Moreover, GtfD is responsible for synthesis of water‐soluble polysaccharides (WSP); once again the gene expression of gtfD was lower in Δ gtfB biofilm, which coincides with WSP quantification (Figure ). In addition, GtfC synthesizes both soluble and insoluble glucans, and the deletion of the gtfB gene dramatically affected the magnitude of gtfC expression; the decrease in fold for gtfC ranged from 4.9 to more than 300 (depending on the developmental phase, the highest difference being at 43 hours, and the lowest at 29 hours; Table ), reflecting the lower quantification of ASP and WSP in Δ gtfB biofilms (vs UA159 biofilms; Figure ).…”

“…It is still necessary to prove that SMU _775c is involved with LTA synthesis, but it is influenced similarly to the dltABCD operon when exposed to environmental changes. A significant increase in the amount of LTA in the ECM was observed between 67 and 115 hours for both UA159 and Δ gtfB biofilms (Figure ).…”

“…While the biofilms developed over time, an increase in the quantities of the accumulated metabolic products (ASP, WSP and LTA) was observed in both UA159 and Δ gtfB biofilms (Figure ), having a paradoxical behavior when compared with the decrease of expression levels of related genes (Table ). This apparent contradiction between gene expression and resulting products of metabolism may have occurred because (i) Gtf turnover is slow at the later stages of biofilm development, as the proteins can be still active, whereas the gene expression has decreased; and/or (ii) as ECM matures and becomes more stable, the diffusion inside the biofilm environment becomes more difficult, then nutrients cannot enter well, allowing bacterial growth and cell division primarily in the biofilm external layer .…”