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

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.


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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.
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f7: 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.

Mentions: In an acidic environment, S. mutans increases the production of F-ATPase (H+-translocating enzyme) via up-regulation of atp genes to pump protons out of the cells that prevent acidification of the cytoplasm, helping the bacterium to tolerate and survive the low pH conditions42. In S. mutans, the atpB gene encodes the beta subunit of the F-ATPase and its expression is highly induced at pH 5 while attenuated at pH 7.033. Thus, to explore the localized expression of an acidic pH responsive gene within an intact microcolony, we generated a strain of S. mutans expressing green fluorescent protein (GFP) under the control of the atpB promoter (PatpB::gfp). We first compared in situ atpB gene expression and pH within an intact microcolony structure as shown in Fig. 7. We determined pH and atpB expression level before (0 min) and after (60 min) exposure to neutral buffer at three different layers across the center of the microcolony (see location depicted by red bars; Fig. 7A). The results were presented as ΔpH and fold-change atpB expression (between 60 min and 0 min).


Simultaneous spatiotemporal mapping of in situ pH and bacterial activity within an intact 3D microcolony structure
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: 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.
Mentions: In an acidic environment, S. mutans increases the production of F-ATPase (H+-translocating enzyme) via up-regulation of atp genes to pump protons out of the cells that prevent acidification of the cytoplasm, helping the bacterium to tolerate and survive the low pH conditions42. In S. mutans, the atpB gene encodes the beta subunit of the F-ATPase and its expression is highly induced at pH 5 while attenuated at pH 7.033. Thus, to explore the localized expression of an acidic pH responsive gene within an intact microcolony, we generated a strain of S. mutans expressing green fluorescent protein (GFP) under the control of the atpB promoter (PatpB::gfp). We first compared in situ atpB gene expression and pH within an intact microcolony structure as shown in Fig. 7. We determined pH and atpB expression level before (0 min) and after (60 min) exposure to neutral buffer at three different layers across the center of the microcolony (see location depicted by red bars; Fig. 7A). The results were presented as ΔpH and fold-change atpB expression (between 60 min and 0 min).

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.


Related in: MedlinePlus