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.
Highlights
Biofilms are comprised of dense, highly hydrated clusters of microbial cells that are embedded with and surrounded by an extracellular matrix of polymeric substances, such as exopolysaccharides (EPS), proteins, and nucleic acids[1,2]
Using micro-scale maps of in situ pH and gene expression via an atpB::green fluorescent protein (GFP) reporter, we demonstrated that changes in the pH microenvironment modulate atpB activity throughout the 3D structure following incubation in neutral pH buffer
Streptococcus mutans can orchestrate the assembly of caries-causing biofilms by producing EPS-matrix, while its acidogenic and aciduric properties help this microbe to create an acidic environment within plaque
Summary
Biofilms are comprised of dense, highly hydrated clusters of microbial cells that are embedded with and surrounded by an extracellular matrix of polymeric substances, such as exopolysaccharides (EPS), proteins, and nucleic acids[1,2]. Previous in situ gene expression studies using fluorescent protein reporters with non-invasive imaging technique such as confocal microscopy has advanced the ability to detect localized gene expression[22,23,24,25] Emerging technologies such as microfluidic and 3D printing approaches have provided additional opportunities to create microenvironments to mimic the spatial confinement and chemical heterogeneity of microcolonies and study their impact on microbial behavior[5,7,26,27,28,29,30,31]. Recent studies combining high-resolution microscopy and genetic techniques have facilitated microanatomical and spatial order of physiological differentiation and EPS deposition during biofilm formation[32], which can help to elucidate the influence that bacterial activity and the matrix have on the local microenvironment. This study provides new insights into how the matrix can modulate both the local pH microenvironment and bacterial activity in situ within the microcolony 3D structure
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