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investigating acid production by Streptococcus mutans with a surface-displayed pH-sensitive green fluorescent protein.

Guo L, Hu W, He X, Lux R, McLean J, Shi W - PLoS ONE (2013)

Bottom Line: Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP.Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity.Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, University of California Los Angeles, Los Angeles, California, USA.

ABSTRACT
Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

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Fluorescence signals of pHluorin-Spap fusion proteinin dual- and multispecies oral biofilms. Time-course of surface-expressed pHluorin fluorescence signal in 16 h S. mutans O87 and S. sanguinis dual-species biofilm (A) and saliva-derived biofilms (B) after challenged with 2% sucrose or 50 mM arginine-HCl (pH 7.6). At different time points (0 and 30 min), biofilms were stained with the anti-S. mutans monoclonal Ab SWLA1, the secondary Ab Alexo 633 conjugated goat anti-mouse IgG and CellTracker Orange. SWLA1 (red), CellTracker Orange (blue), GFP (green) and the corresponding overlay images are shown in the four small panels.
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pone-0057182-g005: Fluorescence signals of pHluorin-Spap fusion proteinin dual- and multispecies oral biofilms. Time-course of surface-expressed pHluorin fluorescence signal in 16 h S. mutans O87 and S. sanguinis dual-species biofilm (A) and saliva-derived biofilms (B) after challenged with 2% sucrose or 50 mM arginine-HCl (pH 7.6). At different time points (0 and 30 min), biofilms were stained with the anti-S. mutans monoclonal Ab SWLA1, the secondary Ab Alexo 633 conjugated goat anti-mouse IgG and CellTracker Orange. SWLA1 (red), CellTracker Orange (blue), GFP (green) and the corresponding overlay images are shown in the four small panels.

Mentions: Next we explored the application of the surface-displayed (O87) pHlourins as “pH-meters” to study acid production by S. mutans under biofilm conditions in the presence of other microorganisms. For pHlourin-independent identification of O87 within dual-species biofilms with S. sanguinis or S. gordonii as well as a multi-species environment, we employed a previously described S. mutans-specific monoclonal antibody (MAb) together with a corresponding red-fluorescent labeled secondary antibody. This approach allowed simultaneous monitoring of the spatial localization of S. mutans and its acid production in situ in dual-species (Fig. 5A) and multi-species (Fig. 5B) biofilms. In the presence of sucrose, acids generated by glycolysis lowered the pH in the microcolonies microenvironment of S. mutans as indicated by the corresponding reduction in pHluorin fluorescence intensity. For the dual-species biofilms, the pHluorin fluorescence intensity decreased more significantly after addition of sucrose for 30 min in comparison with addition of arginine (20.0±9.33% vs 6.14±12.86% in fluorescence reduction). Similar reduction rates in fluorescence intensity were observed for the saliva-derived multi-species biofilms (16.14±7.16% vs 5.52±6.23% in fluorescence reduction).


investigating acid production by Streptococcus mutans with a surface-displayed pH-sensitive green fluorescent protein.

Guo L, Hu W, He X, Lux R, McLean J, Shi W - PLoS ONE (2013)

Fluorescence signals of pHluorin-Spap fusion proteinin dual- and multispecies oral biofilms. Time-course of surface-expressed pHluorin fluorescence signal in 16 h S. mutans O87 and S. sanguinis dual-species biofilm (A) and saliva-derived biofilms (B) after challenged with 2% sucrose or 50 mM arginine-HCl (pH 7.6). At different time points (0 and 30 min), biofilms were stained with the anti-S. mutans monoclonal Ab SWLA1, the secondary Ab Alexo 633 conjugated goat anti-mouse IgG and CellTracker Orange. SWLA1 (red), CellTracker Orange (blue), GFP (green) and the corresponding overlay images are shown in the four small panels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585301&req=5

pone-0057182-g005: Fluorescence signals of pHluorin-Spap fusion proteinin dual- and multispecies oral biofilms. Time-course of surface-expressed pHluorin fluorescence signal in 16 h S. mutans O87 and S. sanguinis dual-species biofilm (A) and saliva-derived biofilms (B) after challenged with 2% sucrose or 50 mM arginine-HCl (pH 7.6). At different time points (0 and 30 min), biofilms were stained with the anti-S. mutans monoclonal Ab SWLA1, the secondary Ab Alexo 633 conjugated goat anti-mouse IgG and CellTracker Orange. SWLA1 (red), CellTracker Orange (blue), GFP (green) and the corresponding overlay images are shown in the four small panels.
Mentions: Next we explored the application of the surface-displayed (O87) pHlourins as “pH-meters” to study acid production by S. mutans under biofilm conditions in the presence of other microorganisms. For pHlourin-independent identification of O87 within dual-species biofilms with S. sanguinis or S. gordonii as well as a multi-species environment, we employed a previously described S. mutans-specific monoclonal antibody (MAb) together with a corresponding red-fluorescent labeled secondary antibody. This approach allowed simultaneous monitoring of the spatial localization of S. mutans and its acid production in situ in dual-species (Fig. 5A) and multi-species (Fig. 5B) biofilms. In the presence of sucrose, acids generated by glycolysis lowered the pH in the microcolonies microenvironment of S. mutans as indicated by the corresponding reduction in pHluorin fluorescence intensity. For the dual-species biofilms, the pHluorin fluorescence intensity decreased more significantly after addition of sucrose for 30 min in comparison with addition of arginine (20.0±9.33% vs 6.14±12.86% in fluorescence reduction). Similar reduction rates in fluorescence intensity were observed for the saliva-derived multi-species biofilms (16.14±7.16% vs 5.52±6.23% in fluorescence reduction).

Bottom Line: Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP.Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity.Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, University of California Los Angeles, Los Angeles, California, USA.

ABSTRACT
Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

Show MeSH
Related in: MedlinePlus