The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms

Streptococcus mutans protein synthesis during mixed-species biofilm development by high-throughput quantitative proteomics.

Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other organisms. Our data provide insights about how S. mutans optimizes its metabolism and adapts/survives within the mixed-species community in response to a dynamically changing environment. This reflects the intricate physiological processes linked to expression of virulence by this bacterium within complex biofilms

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Proteins related to IPS and lipoteichoic acid (LTA) metabolism.

<p>The protein abundance is represented by spectral counting (n  = 2).</p>*<p>Normalized by the numbers of <i>S. mutans</i> detected in each biofilm.</p

Distribution of <i>S. mutans</i> UA159 proteins into specific gene ontology (GO) of biological processes.

<p>The organization was performed via Proteome Center Software.</p

Proteins related to EPS synthesis and remodeling.

<p>The protein abundance is represented by spectral counting (n = 2).</p>*<p>Normalized by the numbers of <i>S. mutans</i> detected in each biofilm.</p>**<p>Data for mixed-species at both time points and single-species biofilms at 67 h were published by Xiao et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045795#pone.0045795-Xiao2" target="_blank">[13]</a>.</p

Number of <i>S. mutans</i> UA159 proteins detected in the biofilms.

<p>From 1966 proteins encoded by this organism. A) Mixed-species biofilms <i>(S. mutans, A. naeslundii</i> and <i>S. oralis</i>) and B) Single-species biofilms (<i>S. mutans</i> alone). Among the common proteins at 67 and 115 h, 817 were detected in both single and mixed-species biofilms. Number of proteins in mixed- U single-species at 67 h is 87 proteins, whereas mixed- U single-species at 115 h are 12 proteins. The number of proteins detected exclusively in mixed-species biofilms was 70 and 19 at 67 and 115 h, respectively; and in single-species biofilms was 51 and 25 at 67 and 115 h, respectively.</p

Dynamics of <i>S. mutans</i> gene expression during mixed-species biofilm development.

<p>Depicts selected <i>S. mutans</i> genes based on the proteome data. Comparison of gene expression data acquired at each time point for mixed-species biofilms (43 h vs. 67 h vs. 91 h vs.115 h) are shown; values connected by line are not significantly different from each other (<i>P</i><0.05; n = 12).</p