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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.


Orthogonal pH distribution within the microcolony.(A) Representative images of orthogonal distribution of pH at each region. (B) Spatial pH profiles (top-to-bottom) at each region. This figure indicates that the periphery of the microcolony structure can be more effectively neutralized by the pH 7.0 buffer (vs center or mid-center region). Asterisk (P < 0.05) and double asterisk (P < 0.01) indicate that the values for the different experimental groups are significantly different from each other.
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f3: Orthogonal pH distribution within the microcolony.(A) Representative images of orthogonal distribution of pH at each region. (B) Spatial pH profiles (top-to-bottom) at each region. This figure indicates that the periphery of the microcolony structure can be more effectively neutralized by the pH 7.0 buffer (vs center or mid-center region). Asterisk (P < 0.05) and double asterisk (P < 0.01) indicate that the values for the different experimental groups are significantly different from each other.

Mentions: The presence of highly acidic pH values within biofilms despite of the neutral-pH environment found in the oral cavity serves as a major virulence attribute for development of dental caries3536. Thus, we analyzed in detail the spatial pH distribution throughout the 3D microcolony structure following incubation in pH 7 buffer (for 60 min). To achieve this, we utilized our pH mapping technique using pH-responsive fluorophore and multi-photon confocal fluorescence microscopy13. Although only one representative image is presented, these analyses were performed in quadruplicate and at least 10 images were recorded under confocal microscopy (Figs 2, 3, 4). We initially ‘trisected’ the microcolony orthogonally as upper/middle/bottom layers and cross-sectionally as center/mid-center/peripheral regions (Fig. 2A,B). Then, we profiled the average pH values distributed across the height of intact microcolony before (Fig. 2C) and after neutralization (Fig. 2D). Before neutralization, the majority of microcolony interior exhibited an acidic microenvironment (pH 4 to 5) across the thickness (height) of the microcolony (Fig. 2C and black columns in 2E). Following incubation in neutral pH buffer, we observed that the upper layer of the microcolony was mostly neutralized from center to peripheral regions (Fig. 2D and gray columns in 2E). In contrast, the bottom layer of microcolony (close to the surface of attachment) remained mostly acidic regardless of cross-sectional regions (center or periphery) even after 60 min of incubation in neutral pH buffer. The middle layer was partially neutralized, particularly at the peripheral region (Fig. 2D,E). These observations indicated persistence of acidic pH values from the bottom to lower-middle layers of the microcolony despite exposure to pH 7 buffer.


Simultaneous spatiotemporal mapping of in situ pH and bacterial activity within an intact 3D microcolony structure
Orthogonal pH distribution within the microcolony.(A) Representative images of orthogonal distribution of pH at each region. (B) Spatial pH profiles (top-to-bottom) at each region. This figure indicates that the periphery of the microcolony structure can be more effectively neutralized by the pH 7.0 buffer (vs center or mid-center region). Asterisk (P < 0.05) and double asterisk (P < 0.01) indicate that the values for the different experimental groups are significantly different from each other.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Orthogonal pH distribution within the microcolony.(A) Representative images of orthogonal distribution of pH at each region. (B) Spatial pH profiles (top-to-bottom) at each region. This figure indicates that the periphery of the microcolony structure can be more effectively neutralized by the pH 7.0 buffer (vs center or mid-center region). Asterisk (P < 0.05) and double asterisk (P < 0.01) indicate that the values for the different experimental groups are significantly different from each other.
Mentions: The presence of highly acidic pH values within biofilms despite of the neutral-pH environment found in the oral cavity serves as a major virulence attribute for development of dental caries3536. Thus, we analyzed in detail the spatial pH distribution throughout the 3D microcolony structure following incubation in pH 7 buffer (for 60 min). To achieve this, we utilized our pH mapping technique using pH-responsive fluorophore and multi-photon confocal fluorescence microscopy13. Although only one representative image is presented, these analyses were performed in quadruplicate and at least 10 images were recorded under confocal microscopy (Figs 2, 3, 4). We initially ‘trisected’ the microcolony orthogonally as upper/middle/bottom layers and cross-sectionally as center/mid-center/peripheral regions (Fig. 2A,B). Then, we profiled the average pH values distributed across the height of intact microcolony before (Fig. 2C) and after neutralization (Fig. 2D). Before neutralization, the majority of microcolony interior exhibited an acidic microenvironment (pH 4 to 5) across the thickness (height) of the microcolony (Fig. 2C and black columns in 2E). Following incubation in neutral pH buffer, we observed that the upper layer of the microcolony was mostly neutralized from center to peripheral regions (Fig. 2D and gray columns in 2E). In contrast, the bottom layer of microcolony (close to the surface of attachment) remained mostly acidic regardless of cross-sectional regions (center or periphery) even after 60 min of incubation in neutral pH buffer. The middle layer was partially neutralized, particularly at the peripheral region (Fig. 2D,E). These observations indicated persistence of acidic pH values from the bottom to lower-middle layers of the microcolony despite exposure to pH 7 buffer.

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.