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Simultaneous spatiotemporal mapping of in situ pH and bacterial activity within an intact 3D microcolony structure

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ABSTRACT

Biofilms are comprised of bacterial-clusters (microcolonies) enmeshed in an extracellular matrix. Streptococcus mutans can produce exopolysaccharides (EPS)-matrix and assemble microcolonies with acidic microenvironments that can cause tooth-decay despite the surrounding neutral-pH found in oral cavity. How the matrix influences the pH and bacterial activity locally remains unclear. Here, we simultaneously analyzed in situ pH and gene expression within intact biofilms and measured the impact of damage to the surrounding EPS-matrix. The spatiotemporal changes of these properties were characterized at a single-microcolony level following incubation in neutral-pH buffer. The middle and bottom-regions as well as inner-section within the microcolony 3D structure were resistant to neutralization (vs. upper and peripheral-region), forming an acidic core. Concomitantly, we used a green fluorescent protein (GFP) reporter to monitor expression of the pH-responsive atpB (PatpB::gfp) by S. mutans within microcolonies. The atpB expression was induced in the acidic core, but sharply decreased at peripheral/upper microcolony regions, congruent with local pH microenvironment. Enzymatic digestion of the surrounding matrix resulted in nearly complete neutralization of microcolony interior and down-regulation of atpB. Altogether, our data reveal that biofilm matrix facilitates formation of an acidic core within microcolonies which in turn activates S. mutans acid-stress response, mediating both the local environment and bacterial activity in situ.

No MeSH data available.


Tri-dimensional (3D) architecture of Streptococcus mutans biofilm.(A) A representative image of S. mutans biofilm comprised of bacterial cell-clusters or microcolonies (green) enmeshed in EPS (red). (B) Orthogonal view of the biofilm. (C) Magnified single microcolony structure (depicted in white dashed-line box). (D) 3D schematic diagram of a single microcolony containing densely-packed bacterial cells.
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f1: Tri-dimensional (3D) architecture of Streptococcus mutans biofilm.(A) A representative image of S. mutans biofilm comprised of bacterial cell-clusters or microcolonies (green) enmeshed in EPS (red). (B) Orthogonal view of the biofilm. (C) Magnified single microcolony structure (depicted in white dashed-line box). (D) 3D schematic diagram of a single microcolony containing densely-packed bacterial cells.

Mentions: S. mutans cells form well-defined 3D microcolonies while producing an exopolysaccharides (EPS)-rich matrix1213. A representative S. mutans biofilm 3D architecture (Fig. 1A) and orthogonal images (Fig. 1B) show the microcolony structure (box with white dashed line; Fig. 1A,C) comprised of bacterial cell-clusters (in green; Fig. 1C) enmeshed with EPS (in red; Fig. 1C). 3D schematic diagram of single microcolony containing bacterial cells (in green) is depicted in Fig. 1D. Here, we focused on the microcolonies with a circular-like shape.


Simultaneous spatiotemporal mapping of in situ pH and bacterial activity within an intact 3D microcolony structure
Tri-dimensional (3D) architecture of Streptococcus mutans biofilm.(A) A representative image of S. mutans biofilm comprised of bacterial cell-clusters or microcolonies (green) enmeshed in EPS (red). (B) Orthogonal view of the biofilm. (C) Magnified single microcolony structure (depicted in white dashed-line box). (D) 3D schematic diagram of a single microcolony containing densely-packed bacterial cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015094&req=5

f1: Tri-dimensional (3D) architecture of Streptococcus mutans biofilm.(A) A representative image of S. mutans biofilm comprised of bacterial cell-clusters or microcolonies (green) enmeshed in EPS (red). (B) Orthogonal view of the biofilm. (C) Magnified single microcolony structure (depicted in white dashed-line box). (D) 3D schematic diagram of a single microcolony containing densely-packed bacterial cells.
Mentions: S. mutans cells form well-defined 3D microcolonies while producing an exopolysaccharides (EPS)-rich matrix1213. A representative S. mutans biofilm 3D architecture (Fig. 1A) and orthogonal images (Fig. 1B) show the microcolony structure (box with white dashed line; Fig. 1A,C) comprised of bacterial cell-clusters (in green; Fig. 1C) enmeshed with EPS (in red; Fig. 1C). 3D schematic diagram of single microcolony containing bacterial cells (in green) is depicted in Fig. 1D. Here, we focused on the microcolonies with a circular-like shape.

View Article: PubMed Central - PubMed

ABSTRACT

Biofilms are comprised of bacterial-clusters (microcolonies) enmeshed in an extracellular matrix. Streptococcus mutans can produce exopolysaccharides (EPS)-matrix and assemble microcolonies with acidic microenvironments that can cause tooth-decay despite the surrounding neutral-pH found in oral cavity. How the matrix influences the pH and bacterial activity locally remains unclear. Here, we simultaneously analyzed in situ pH and gene expression within intact biofilms and measured the impact of damage to the surrounding EPS-matrix. The spatiotemporal changes of these properties were characterized at a single-microcolony level following incubation in neutral-pH buffer. The middle and bottom-regions as well as inner-section within the microcolony 3D structure were resistant to neutralization (vs. upper and peripheral-region), forming an acidic core. Concomitantly, we used a green fluorescent protein (GFP) reporter to monitor expression of the pH-responsive atpB (PatpB::gfp) by S. mutans within microcolonies. The atpB expression was induced in the acidic core, but sharply decreased at peripheral/upper microcolony regions, congruent with local pH microenvironment. Enzymatic digestion of the surrounding matrix resulted in nearly complete neutralization of microcolony interior and down-regulation of atpB. Altogether, our data reveal that biofilm matrix facilitates formation of an acidic core within microcolonies which in turn activates S. mutans acid-stress response, mediating both the local environment and bacterial activity in situ.

No MeSH data available.