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

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.

Figure 1. 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.

Figure 2. Spatial pH distribution within an intact microcolony.

(A) Orthogonal and (B) cross-sectional sections within the microcolony, which were divided as follows: upper-, middle- and bottom-layer as well as center-, mid-center- and peripheral-region. (C) pH distribution at each orthogonal sections before and (D) after incubation in neutral pH buffer. (E) pH values at each layer and each section of the microcolony. This figure indicates persistence of acidic pH between the bottom and lower-middle layers of the microcolony despite exposure to neutral pH buffer. Double asterisk indicates that the values are significantly different from each other (P < 0.01).

Figure 3. 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.

Figure 4. Representative images of cross-sectional and orthogonal pH distribution within the microcolony.

(A) Before and (B) after neutralization, and (C) after reacidification of EPS-microcolony. When the biofilms were exposed to sucrose, acidic microenvironments within the microcolony were almost completely reinstated, showing pH distributions similar to those prior to neutralization.

Figure 5. Presence of an inner acidic core within the microcolony

. (A) Schematic diagram depicting the acidic core in the interior of the microcolony 3D structure. Representative orthogonal images of microcolony with varying sizes: (B) 100, (C) 60 and (D) 30 μm thicknesses. “Fire” lookup table (LUT) color scheme was used for enhanced contrast to differentiate pH ranges. Red-to-yellow colors indicate the area with pH >5 and dark purple-to-pink colors indicate the area with pH <5. The acidic region (under dotted lines) is clearly visible within large microcolonies (100 μm height), while a relatively smaller acidic core was observed in a microcolony with 60 μm height, and it was not detected in microcolonies with less than 30 μm thickness.

Figure 6. Influence of EPS matrix-degrading enzyme on the pH distribution within the microcolony

. (A) A sequential montage of cross-sectional images of degraded EPS-matrix from top to bottom areas of the microcolony; the images of degraded EPS-matrix were obtained by determining the differences between the images before and after dextranase treatment via the fluorescence image subtraction function of ImageJ; representative orthogonal pH distribution images of (B) intact and (C) dextranase-treated microcolonies. The profile of pH values across the diameter of (D) intact and (E) dextranase-treated microcolonies. Dextranase treatment digested the upper and peripheral EPS layers, and resulted in minimal EPS degradation in the interior or bottom layers of the microcolony. Degradation of the surrounding EPS facilitates neutralization of the microcolony interior environment.

Figure 7. Relationship of spatial pH distribution and atpB gene expression within the microcolony

. Cross-sectional distributions of pH and atpB expression levels at each selected section (red bars) within (A) intact and (B) dextranase (Dex)-treated microcolonies. We measured pH values and atpB expression level before (0 min) and after (60 min) exposure to neutral buffer. The results were presented as ΔpH (C) and fold-change atpB expression (D) (between 60 min and 0 min). The symmetric (‘mirror-image’) plots of pH (C) and atpB (D) from intact and Dex-treated microcolonies indicate that S. mutans residing within the 3D structure is actively responding to local microenvironmental changes. 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.