Investigating Acid Production by Streptococcus mutans with a Surface-Displayed pH-Sensitive Green Fluorescent Protein

Guo, Lihong; Hu, Wei; He, Xuesong; Lux, Renate; McLean, Jeffrey S.; Shi, Wenyuan

doi:10.1371/journal.pone.0057182

Cited by 45 publications

(47 citation statements)

References 55 publications

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“…Recently, we reported the presence of compartmentalized acidic pH microenvironments in the interior of EPS-enmeshed microcolonies13. Using a surface-displayed pH sensitive fluorescent protein (pHluorins), Guo et al 45 also detected low pH values confined near the cell clusters linking our previous findings with acid accumulation within microcolonies. Here, we demonstrated that S. mutans EPS-matrix facilitates the generation of an acidic core within a 3D microcolony structure despite external exposure to a neutralizing buffer, which in turn activates bacterial gene expression in situ, mediating the biofilm microenvironment and bacterial activity locally.…”

Section: Discussionsupporting

confidence: 75%

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

Hwang

Liu

Kim

et al. 2016

Sci Rep

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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.

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Section: Discussionsupporting

confidence: 75%

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

Hwang

Liu

Kim

et al. 2016

Sci Rep

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“…The results of our previous studies have shown that the synthesis of Gtf-derived glucans leads to the formation of an insoluble EPS-rich matrix scaffold that acts as a diffusion-limiting barrier (15). In parallel, the metabolic activity of S. mutans clustered within the microcolony can produce copious amounts of acids that accumulate locally (15,74). It is conceivable that the alterations in the extracellular matrix containing a dense population of bacterial cells help to prevent acid within the biofilm from diffusing outward, thus prolonging and intensifying the acid attack.…”

Section: Discussionmentioning

confidence: 99%

Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque Biofilms In Vivo

et al. 2014

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dStreptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived ␤1,3-glucans contribute to the EPS matrix structure, while fungal mannan and ␤-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

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“…Our data reveal that defective assembly of EPS matrix and microcolony formation as a result of L-arginine exposure may also contribute to elevated in situ pH at the biofilm-sHA interface. Previous studies have shown that EPS-enmeshed microcolonies could trap acid at the sHA surface and/or restrict the access of neutralizing buffer to generate acidic microenvironments locally due to metabolic activity of the densely packed bacteria enmeshed within a diffusionlimiting EPS matrix, particularly at the deeper layers of the biofilm (6,57,58). Altogether, it appears that the combination of ADS activity and favorable ecological changes with disruption of EPS/ microcolony development may offer a more comprehensive explanation for the biofilm pH-related effects associated with L-arginine exposure.…”

Section: Discussionmentioning

confidence: 99%

l -Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms

et al. 2016

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L-Arginine, a ubiquitous amino acid in human saliva, serves as a substrate for alkali production by arginolytic bacteria. Recently, exogenous L-arginine has been shown to enhance the alkalinogenic potential of oral biofilm and destabilize its microbial community, which might help control dental caries. However, L-arginine exposure may inflict additional changes in the biofilm milieu when bacteria are growing under cariogenic conditions. Here, we investigated how exogenous L-arginine modulates biofilm development using a mixed-species model containing both cariogenic (Streptococcus mutans) and arginolytic (Streptococcus gordonii) bacteria in the presence of sucrose. We observed that 1.5% (wt/vol) L-arginine (also a clinically effective concentration) exposure suppressed the outgrowth of S. mutans, favored S. gordonii dominance, and maintained Actinomyces naeslundii growth within biofilms (versus vehicle control). In parallel, topical L-arginine treatments substantially reduced the amounts of insoluble exopolysaccharides (EPS) by >3-fold, which significantly altered the three-dimensional (3D) architecture of the biofilm. Intriguingly, L-arginine repressed S. mutans genes associated with insoluble EPS (gtfB) and bacteriocin (SMU.150) production, while spxB expression (H 2 O 2 production) by S. gordonii increased sharply during biofilm development, which resulted in higher H 2 O 2 levels in arginine-treated biofilms. These modifications resulted in a markedly defective EPS matrix and areas devoid of any bacterial clusters (microcolonies) on the apatitic surface, while the in situ pH values at the biofilm-apatite interface were nearly one unit higher in arginine-treated biofilms (versus the vehicle control). Our data reveal new biological properties of L-arginine that impact biofilm matrix assembly and the dynamic microbial interactions associated with pathogenic biofilm development, indicating the multiaction potency of this promising biofilm disruptor. IMPORTANCEDental caries is one of the most prevalent and costly infectious diseases worldwide, caused by a biofilm formed on tooth surfaces. Novel strategies that compromise the ability of virulent species to assemble and maintain pathogenic biofilms could be an effective alternative to conventional antimicrobials that indiscriminately kill other oral species, including commensal bacteria. L-Arginine at 1.5% has been shown to be clinically effective in modulating cariogenic biofilms via alkali production by arginolytic bacteria. Using a mixed-species ecological model, we show new mechanisms by which L-arginine disrupts the process of biofilm matrix assembly and the dynamic microbial interactions that are associated with cariogenic biofilm development, without impacting the bacterial viability. These results may aid in the development of enhanced methods to control biofilms using L-arginine. Biofilms formed on surfaces are highly organized and structured microbial communities enmeshed in an extracellular matrix composed of polymeric substances, such as exopoly...

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Investigating Acid Production by Streptococcus mutans with a Surface-Displayed pH-Sensitive Green Fluorescent Protein

Cited by 45 publications

References 55 publications

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

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

Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque Biofilms In Vivo

l -Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms

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